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CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957 (1958)

Chapter: ABNORMAL HEMOGLOBINS

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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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Suggested Citation:"ABNORMAL HEMOGLOBINS." National Research Council. 1958. CONFERENCE ON HEMOGLOBIN: 2-3 MAY 1957. Washington, DC: The National Academies Press. doi: 10.17226/9550.
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PART III. ABNORMAL HEMOGLOBINS ELECTROPHORETIC ANALYSES OF THE ABNORMAL HUMAN HEMOGLOBINS HARVEY A. ITANO Although other methods have been introduced in recent years for the separation of hemoglobin components, electrophoretic analysis remains the most effective and widely used technique. The moving boundary method, which was used exclusively in the early studies of the abnormal forms of human hemoglobin, 223 retains its advantage for certain purposes, for example, electrophoresis in acid buffers, determination of absolute mobilities and isoelectric points, and separation of components of similar mobilities. However, zone electrophoresis is more readily available and is better adapted for the screening of large populations. The accelerated rate of discovery of new forms in recent years is largely the result of the widespread use of zone electrophoresis. This method is also a more effective tool for the separation and purification of components in useful quantities. Other investigators will discuss the application of zone electrophoresis elsewhere in these Proceedings, and I shall confine my remarks to practical applications of the moving boundary method in the study of human hemoglobin. In order to facilitate further discussion and clarify the notation on the figures which accompany this presentation, I shall review briefly the nomen- clature of the human hemoglobins as agreed upon by interested investi- gators.4 5 Normal adult hemoglobin is the form found in most humans and is called hemoglobin A. Since minor electrophoretic components are present in the hemoglobin prepared from the red cells of normal adults,6 the designa- tion Al has been given to the major electrophoretic component, which com- prises 90-95 per cent of the total and which is electrophoretically homog- eneous.7 The minor components will be discussed elsewhere in these Pro- ceedings. The component designated hemoglobin A in the present discussion and figures is, strictly speaking, the Al component of the most recent nomen- clature.' Fetal hemoglobin is hemoglobin F. and the abnormal hemoglobin characteristic of sickling cells is hemoglobin S. Since sickle cell hemoglobin was also designated hemoglobin ~ at one time,3 the letter B has been omitted in naming the other abnormal forms, which are called hemoglobins C, D, E, G. etc., in the order of their discovery. In the initial studies of hemoglobins A and S,~ it was shown that hemo- globin A migrates more rapidly at alkaline pH and that hemoglobin S migrates more rapidly at acid pH (fig. 1~. At pH 6.9 the two forms migrate in opposite directions (fig. 2~. These observations led to the conclusion that · ~ 1 · 1 A 144

ELECTROPHORETIC ANALYSES ITANO 145 the difference in the molecules is in their net charge, for if one of the mole- cules has a greater frictional resistance to transport than the other, its mo- bility would be lower on both sides of the isoelectric point. X-ray diffraction studies supported this conclusion by show-in" that molecules of hemoglobins A and S are identical in shape.S 43.0 o -1.0 _ -2.0 -3.0 =50 ~ _~ ~ , __- ~C~. ~v I ] - emle I'm\\ 8.0 TO S.0 6.0 70 pH FIG. 2. ( below ) Electrophoretic pat- terns of carbonmonoxyhemoglobins A and S in nhosohate huger of nH ~S.9. ionic strength 0.1. Hemoglobins A and S migrate in opposite directions as anion and cation, respectively, in this buffer. r----r~ rip 7 From Pauling, L., Itano, H. A., Singer, S. J., and ~'ells, I. C.: Science 110: 54~, 1949. by Sickle Anemia FIG. 1. ( above ) Relation- ship of mobility to pH for carbonmonoxyhemoglobins A and S in phosphate buf- fers of 0.1 ionic strength. From Pauling, L., Itano, H. A., Singer, S. J., and Wells, I. C.: Science 110: 543, 1949. a) Normal Sicftis Trait d) Mixture crf a) and b) The net charge of a protein molecule depends, not only on the number and identity of ionizable groups, but also on the pK of the groups and on the affinity of the molecules for ions other than hydrogen ion. Since the relative mobilities of the various forms of human hemoglobin at a given pH do not depend upon their ionic environment, the actual basis of their mobility dif- ferences in all probability lies in the numbers and relative positions of their acidic and basic amino acid residues. Although the pK of each type of ion- izable group lies within a characteristic range, the exact value varies with structure, charge, number, and proximity of neighboring groups. It is there- fore conceivable that two forms of human hemoglobin have the same amino acid composition and yet have different net charges at the same pH. It is also possible for two hemoglobins to differ in their content of charged

146 PART III. ABNORMAL HEMOGLOBINS amino acid residues and have the same electrophoretic mobility at a given pH if their net charges are equal. Hemoglobins which have the same electro- phoretic behavior may be regarded as belonging; to an "electrophoretic pheno- type," and the detection of genetically-determined alterations that do not affect net charge requires the use of an independent method. Hemoglobins S and D, for example, are indistinguishable by electrophoresis (fig. 3 ); how- ever, solubility measurements and sickling tests indicate that they are dif- ferent molecular species. 1 3, 9 Normal (A/A) Hi_ In_ Sickle Trait (A/S) Sickle Cell Anemia (S/S) Slekle Cell Anem i a plus Fetal tS/S,f) M. H. (A/O) R. H. (S/D) B. H. (S./ D, F) FIG. 3. Electrophoretic pat- terns showing the similarity of carbonmonoxyhemoglobins S and D in cacodylate buffer of pH 6.5, ionic strength 0.1. From Stu rgeon, P., Itano, H. A., and Bergren, W. R.: Blood 10: 389, 1955. Hemoglobins A and S were resolved in phosphate buffers of ionic strength 0.1, and their charge difference was estimated to be two to four units from titration and electrophoretic data.t These hemoglobins are also well resolved in cacodylate buffer of pH 6.5 and ionic strength 0.1. Hemoglobin S and C are also well resolved in the cacodylate buffer although their mobility dif- ference is less than that between A and S.' However, a mixture of hemo- globins A and F or of hemoglobins S and E does not resolve in this buffer.~° The components in the respective mixtures are so closely similar in mobility that separation of their boundaries is obscured by diffusion. The absolute mobility of each component can be determined separately; however, it is a difficult task technically to obtain sufficient accuracy to establish the small differences in mobility encountered in the study of the

ELECTROPHORETIC ANALYSES ITANO 147 human hemoglobins. Such small differences can be demonstrated in other ways. If the pH-mobility curves of the two components are not parallel, a different pH which results in better separation can be found. This is true of hemoglobins S and E (Eg. 4), which differ more in mobility in alkaline buffers than in acid buffers.9 ii Components of very similar mobility may be H E M O G LO B I N E Acid ~ , ti Sicl~lc Trait (AJS) L ~ I ~ a I I n 0 Norma ~ (~/A) Control Slel~l. Ccil-~/omogiobin C Disease (S/C) Sicille Call-~halesesmia Dieso.e (A,f/S) ad, H. (A,f/E) FIG. 4. Comparison of moving boundary patterns of carbonmonoxyhemoglobin in cacodylate buffer of pH 6.5, ionic strength 0.1, with the results of paper electro- phoresis in barbital buffer of pH 9.2, ionic strength 0.01. The close similarity of hemoglobins S and E in the acid louder is contrasted with their difference in the alkaline buyer. The preponderance of hemoglobin E and the presence of hemoglobin associated with the thalassemia gene is also shown. From Sturgeon, P., Itano, H. A., and Bergren, W. R.: Blood 10: 389, 1955. resolved with use of very dilute buffers,7 it in which continuous sharpening of the boundaries of one limb overcomes the effect of diffusion ~ fig. j ~ . Finally, each of the two similar components can be mixed with a known hemo- globin which differs appreciably in mobility from the components to be com- pared. The mixtures are analyzed under identical conditions, and the rela-

log PART. III. ABNORMAL HEMOGLOBINS Modification ~ pH 65 pil 8.8 1 Sickle Cell si F Anemia Sickle Cell st A F S Thalassemia Sickle Cell CtS sit Hb-C Disease ~ _i Sickle Cell l Hb-D Disease si A Ei ~ IF (a) sl A,F - (e) (b) (f ) Si ~ jA F (C) (d) . (h) FIG. 5. ( left) Comparison of the components of carbonmonoxyhemoglobin in three types of sickle cell disease. To the left, analyses in cacodylate buffer of pH 6.5, ionic strength 0.1. To the right, analyses in 0.01 M Na.,HPO4. Hemoglobins A and F are resolved in the dilute phosphate buffer and are not resolved in the cacodylate buffer. Hemoglobins S and D are not resolved in either buffer and are distinguished from each other by solubility measurements.3 FIG. 6. ( right) Ascending boundary patterns of various mixtures of human car- bonmonoxyhemoglobin in cacodylate buffer of plI 6.5, ionic strength 0.1. These ex- periments show the slight but discernible differences in mobility between hemoglobins A and F and between hemoglobins S and E at this pH. Hemoglobin F is the reference component in the comparison of hemoglobins S and E. Hemoglobin S is the reference component in the comparison of hemoglobins ~ and F. The difference between E and F is virtually the same as that between A and S. From Itano, PI. A., Bergren, W. R., and Sturgeon, P.: Medicine 35: 121, 1956. tive mobilities of the two components in each mixture is observed (fig. 6~. Comparison of the patterns obtained in this manner will permit the detection of very small differences in mobility. A reference mixture that contains two known hemoglobins which migrate more rapidly and more slowly, respectively, than the two similar hemoglobins can also be used, and the relative position of the boundary of each of the two hemoglobins can be compared with those of the reference components (fig. 7~. The proportion of the reference compo- nent or components should be the same in both mixtures of a pair of compara- tive analyses since the apparent relative Nobilities vary with composition. Ci. F Go S F FIG. 7.—Ascending boundary patterns of two mixtures of car- bonmonoxyhemoglobin in caco- dylate buffer of pH 6.5, ionic strength 0.~. Hemoglobins F and C were used as reference com- ponents to compare hemoglob- ins E and S. The difference in net charge between hemoglob- ins E and S in this buffer ap- pears to be less than one.

ELECTROPHORETIC ANALYSES ITANO 149 The greater reliability of the mixture method over the determination of absolute mobilities rests upon the fact that differences in mobility are less sensitive to slight changes in pH than are absolute mobilities. Electrophoretic analyses not only detect the components in a mixture but also show the relative amounts of each of the components. Because of the diagnostic and genetic significance of the composition of the hemoglobin mixture in an individual, samples used in electrophoretic analyses of hemo- globin are not pooled. In order to avoid alteration of composition that might result from crystallization or other procedures for fractionation, samples are analyzed as obtained from hemolyzates of washed red cells, and preparative procedures other than removal of insoluble stroma material by centrifugation is avoided. Aside from the disappearance of fetal hemoglobin in infants and its appearance in some forms of chronic anemia,~3 the composition in a given individual probably remains constant with time.~4 At 0.1 ionic strength the apparent composition of a mixture indicated by the schlieren pattern is close to the true composition,~4 but at low ionic strength the disparity between apparent and true composition may become large. Although it is possible to compute the deviation from true composition,7 it is usually more practicable to obtain an empirical relationship between apparent and true compositions by analyzing known mixtures in the same buffer. Both inherited and acquired characteristics of the hemoglobin molecule are demonstrable by electrophoresis. Inherited characteristics are presumably in- corporated into the structure of the molecule at the time of its biosynthesis under genetic controls Acquired characteristics result from alterations that take place after completion of biosynthesis and include denaturation of A X~ (a) fib Fib ~ SEA (c) HbCf~b: | (e) HbAHbs (b) fib sHbs I (d) Nb5Ht: I (f) Nba~bC FIG. 8. Hemoglobins A, S. and C are synthesized under the control of allelic genes at the locus designated Hb.'3 24 The electrophoretic patterns of carbonmonoxyhemo- globin in cacodylate buffer of pH 6.5, ionic strength 0.1 corresponding to the six possible combinations of the three genes are shown. The X component is probably the same as hemoglobin A.,.5' 6 The presence of fetal in homozygosity for the sickle cell gene is shown. From Itano, H. A., and Pauling, L.: Svensk. Kem. Tidskr., in press.

150 PART III. ABNORMAL HEMOGLOBINS globin and both reversible and irreversible reactions involving the heme groups. In general, an individual has one or two major components in his red cells, depending upon his genotype (fig. 8~. These are the adult hemo- globins, normal (A) and abnormal ~ S. C, D, E, etc. ~ . In addition, fetal hemoglobin (F) occurs in severe anemia,: and minor components are also present in anemic as well as non-anemic individuals.6 The proportion of the major components as well as the type appears to be an inherited characteristic. i' In most cases of heterozygosity for the genes for hemoglobin A and for an abnormal hemoglobin, hemoglobin A comprises more than 50 per cent of the total. However, in the simultaneous presence of one gene for thalassemia and one gene for an abnormal hemoglobin,9 i& the abnormal hemoglobin is usually the preponderant species (figs. 4, 94. In other words, the net effect of the thalassemia gene, according to the results of electrophoretic studies, is the relative suppression of the synthesis of hemo- globin A or of an electrophoretic phenotype of hemoglobin A. (a) Normal . ~ c ) (e) Sickle Cell ~ ~ Father BG Trait (b) Sickle Cel' Anemia (d) l Patient LO(F) _ (f) Mother E FG ~ F`IG. 9. Electrophoretic patterns of carbonmonoxyhemoglobin in cacodylate buffer of pH 6.5, ionic strength 0.1, showing the effect of a thalassemia gene on electrophor- etic composition. Since the mother is heterozygous for the sickle cell gene and the father does not have this gene, the patient cannot be homozygous in the sickle cell gene However, the pattern closely resembles that seen in sickle cell anemia. The relative increase in hemoglobin S can be ascribed to the thalassemia gene, which was inherited from the father and which inhibits the synthesis of hemoglobin A. The slow boundary in the patient's pattern represents both hemoglobins A and F. which are not resolved in the buffer used. Art elevated proportion of hemoglobin A., is evident in the pattern of thalassemia minor. From Sturgeon, P., Itano, H. A., and Valentine, W. N.: Blood 7: 350, 1952. _, ~ Inherited differences are demonstrated on preparations . in which all or nearly all of the molecules of hemoglobin are present in the native state and as the same compound, such as carbonmonoxyhemoglobin or ferrohemoglobin. Each of the inherited hemoglobin components in a preparation may become heterogeneous if the molecules are altered after synthesis, either within the circulating red cell or during preparation, storage, or analysis. If the degree of heterogeneity changes during storage or in the course of an analytical

ELECTROPHORETIC ANALYSES ITANO 151 procedure, it is likely that alterations are taking, place in the state of the molecule. An example of a reaction which changes the net charge of a molecule of hemoglobin is the oxidation of ferrohemoglobin or one of its compounds to ferrihemoglobin, which results in an increase of one unit in the net positive charge of each heme iron. In order to study the effect of this reaction on electrophoretic homogeneity, partially oxidized mixtures of carbonmonoxy- hemc~globin were used since the components of such mixtures, carbonmonoxy- hemoglobin and ferrihemoglobin, are stable compounds. Analyses of partially oxidized preparations of carbonmonoxyhemoglobin in phosphate buffer of pH 6.85 and ionic strength 0.01 revealed a mixture of components, including some with mobilities between those of carbonmonoxyLemoglobin and ferri- hemoglobin ~ fig. 10 ~ . Comparison of the composition of such mixtures as determined by electrophoretic arid spectrophotometric analyses indicated the intermediate components are molecular species in which one to three of the four heme irons are oxidized. FIG. TO.—Separation of intermediate compounds of carbonmonoxyhemoglobin and ferrihemoglobin by electrophoresis in phosphate buffer of pH 6.85, ionic strength 0.01. The major components correspond to molecules in which 0, 1, and 2 of the four hemes of carbonmonoxyhemoglobin have dissociated carbon monoxide and acquired r positive charges by oxidation lo rom ltano, H. A., and Robinson, E.: J. Am. Chem. Soc. 78: 6415, 1956. terrihemoglobin combines with cyanide ion at each of the hemes to form ferrihemoglobin cyanide, a stable compound which dissociates very slowly. The cyanide ion neutralizes The positive charge on the heme iron of ferri- hemoglobin and diminishes the electrophoretic mobility of the latter com- pound at acid plI. Partial saturation of ferrihemoglobin with cyanide re- sulted in the appearance of components identifiable as molecules in which one to three of the ferrihemes were combined with cyanide.~9 Ferribemoglobin cyanide, produced by the complete saturation of the ferriDemes with cyanide, was found to have the same mobility as carbonmonoxybemoglobin. Other alterations that can affect the electrophoretic mobility of hemo- globin no doubt occur. Loss of amide groups from the globin, configurational changes of globin that alter the pK of dissociating groups, and degradation o the hemes are possible examples. The occurrence of an alteration of this type is to be suspected if electrophoretic data cannot be related to genetic data. Or if the degree of heterogeneity in a given sample changes with time. The buffer used in the separation of intermediate compounds can be applied to other separations. Since intermediate compounds that differ by a

152 PART III. ABNORMAL HEMOGLOBINS single charge can be separated, these components can be used as markers to estimate the charge difference between the normal and abnormal he~no- globins. Results to date indicate that difference in charge between hemo- globins S and C in phosphate buffer of pH 6.85 is less than that between hemoglobins A and S. As noted earlier, a similar observation was made in cacodylate buffer of pH 6.5. A problem that interests both biochemists and physical chemists is the homogeneity of a protein synthesized under the control of a single gene. If random errors occur in the incorporation of charged amino acids during the biosynthetic process, components that differ from the principal component by multiples of a single charge would be produced, and such components would be distributed symmetrically about the major component. While minor com- ponents do occur, these are not distributed symmetrically and do not appear as discrete components that differ in multiples of a single charge. Therefore such minor components do not arise from random errors in incorporation and their presence suggests either the action of a different gene or alterations that occur after synthesis. The results summarized above indicate the reliability and relative sim- plicity of the electrophoretic method for the detection of small differences in the net charge of hemoglobin molecules. The fact that all of the known abnormal forms of human hemoglobin are electrophoretically abnormal is partially a consequence of the almost exclusive reliance of investigators on eliectrophoresis for the initial detection of abnormal forms. However, it is also true that no unambiguous physical method is yet available for the de- tection of slight alterations that do not affect the net charge of a protein. Other methods that have been successful in the separation of similar proteins also appear to depend upon differences in net charge. The chromatographic separation of the human hemoglobins is one example of such a separation,''° and the countercurrent distribution of insulin components that differ by an amide group is another.' ''- On the other hand, insulin molecules that have the same net charge and differ only in their content of uncharged amino acid residues are indistinguishable by physical methods, including countercurrent distribution.22 Thus, while it is likely that mutations which result in the alteration of content of uncharged residues occur, there exists no satisfactory physical method for the detection of such events. The electrophoretically normal hemoglobin associated with the thalassemia allele may indeed be an abnormal hemoglobin in which the net charge is normal and which is synthesized at a subnormal rate. A complete amino acid analysis may provide evidence for a net change in composition but does not detect differences in position of residues. An alteration such as the transposition of two similar uncharged amino acid residues within a polypeptide chain, for example, can be detected only by complete sequential analyses. Until less laborious procedures for the

ELECTROPHORETIC ANALYSES ITANO 153 detection of abnormal molecules with normal reset charge are developed, experimental studies of the effect of mutations on the structure of hemo- globin will depend on the use of electrophoretically abnormal forms. REFERENCES 1. Pauling, L., Itano, H. A., Singer, S. J., and Wells, I. C.: Sickle cell anemia, a molecular disease, Science 110: 543 (25 Nov.) 1949. 2. Itano, H. A., and Neel, J. V.: A new inherited abnormality of human hemoglobin, Proc. Nat'l. Acad. Sci., IJ. S. 36: 613 (Nov.) 1950. 3. Itano, H. A.: A third abnormal hemoglobin associated with hereditary hemo- lytic anemia, Proc. Nat'l. Acad. Sci., U. S. 37: 775 (Dec.) 1951. . Chernoff, A. I., et al.: Statement concerning a system of nomenclature for the varieties of human hemoglobin, Blood 8: 386, April 1953; Science ll8: 116 (July) 1953. 5. Lehmann, H.: International Society of Hematology: The hemoglobinopathies, Blood I2: 90 (Jan.) 1957. 6. Kunkel, H. G., and Wallenius, G.: New hemoglobin in normal adult blood, Sci- ence 122: 288 (12 Aug.) 1955. 7. Hoch, H.: The steady state, a test for electrophoretic homogeneity, B~ochem. I. (London) 46: 199 (Feb.) 1950. 8. Perutz, M. F., Liquori, A. M., and Eirich, F.: X-ray and solubility studies of haemoglobin of sickle-cell anemia patients, Nature 167: 929 (9 June) 1951. 9. Sturgeon, P., Itano, H. A., and Bergren, W. R.: Clinical manifestations of in- herited abnormal hemoglobins. I. The interaction of hemoglobin S with hemo- globin D. II. Interaction of hemoglobin E and thalassemia trait, Blood 10: 389 (May) 1955. 10. Itano, H. A., Bergren, W. R., and Sturgeon, P.: The abnormal human hemo- globins, Medicine 35: 121 (May) 1956. 11. Itano, H. A., Bergren, W. R., and Sturgeon, P.: Identification of a fourth ab- normal human hemoglobin, J. Am. Chem. Soc. 76: 2278 (20 April) 1954. 12. Itano, H. A., and Robinson, E.: Demonstration of intermediate forms of carbon- monoxy- and ferrihemoglobin by moving boundary electrophoresis, J. Am. Chem. Soc. 78: 6415 (20 Dec.) 1956. 13. Singer, K., Chernoff, A. I., and Singer, L.: Studies on abnormal hemoglobins. I. Their demonstration in sickle cell anemia and other hematolo'~ric disorders by means of alkali denaturation. II. Their identification by means of the method of fractional denaturation, Blood 6: 413, 429 (May) 1951. 14. Wells, I. C., and Itano, lI A.: Ratio of sickle-cell anemia hemoglobin to normal hemoglobin in sicklemics, J. Biol. Chem. 188: 65 (Jan.) 1951. 1 5. Itano, PI. A.: The human hemoglobins: Their properties and genetic control, Advances in Protein Chem. 12) in press. 16. Neel, l. V., Wells, I. C., and Itano, H. A.: Familial differences in the propor- tion of abnormal hemoglobin present in the sickle cell trait, J. Clin. Invest. 30: 1120 (Oct.) 1951. 17. Itano, H. A.: Qualitative and quantitative control of adult hemoglobin syn- thesis a multiple allele hypothesis, Am. J. Human Genetics 5: 34 (March) 1953. 18. Sturgeon, P., Itano, H. A., and Valentine, W. N.: Chronic hemolytic anemia associated with thalassemia and sickling traits, Blood 7: 350 (March) 1952.

154 PART III. ABNORMAL HEMOGLOBINS 19. Itano, H. A., and Robinson, E.: Hemoglobin intermediates, Fed. Proc. 16: 199 (March) 1957. 20. Huisman, T. H. J., and Prins, H. K.: Chromatographic estimation of four dif- ferent human hemoglobins, J. Lab. Clin. Med. 46: 255 (Aug.) 1955. 21. Harfenist, E. J., and Craig, L. C.: Countercurrent distribution studies with in- sulin, J. Am. Chem. Soc. 74: 3083 (20 June) 1952. 22. Harfenist, E. J.: The amino acid compositions of insulins isolated from beef, pork, and sheep glands, l. Am. Chem. Soc. 75: 5528 (20 Nov.) 1953. 23. Ranney, H. M.: Observations on the inheritance of sickle-cell hemoglobin and hemoglobin C, J. Clin. Invest. 33: 1634 (Dec.) 1954. 24. Allison, A. C.: Notation for hemoglobin types and genes controlling their syn- thesis, Science 122: 640 (7 Oct.) 1955. DISCUSSION Dr. J. Steinhardt: It is worth mentioning that in our work with ferro- hemoglobin we have never been able to separate ferrocyanide ion from meth- emoglobin prepared with ferricyanide, no matter how long we dialyzed. I suppose this does have some effect on the net charge and mobility. Dr. Itano: I think the facts that the boundaries in the methemoglobin- methemoglobin cyanide solutions are not as sharp as those in the methemo- globin-carbonmonoxyhemoglobin solutions and that the proportions appear to change with time indicate that there is some dissociation of the cyanide from the me/hemoglobin. Actually, there has to be a finite rate of dissocia- tion since the equilibrium constant of the reaction is measurable. The other consideration is that the spectrophotometric method is not as sensitive as electrophoresis in the detection of these minor changes in heme. To be more specific, say that a preparation contains after storage 1 or 2 per cent me/hemoglobin. It is rather difficult to pick up this change by spectro- photometry except by the most careful work. However, at this low level of oxidation, probably only one heme out of four will be affected in any one molecule so that a orate to two per cent heterogeneity in methemoglobin corresponds to two to eight per cent heterogeneity by electrophoresis. Dr. F. Do. J. Roughton: I was greatly interested to read Dr. Itano's pre- liminary communication last December, and now to hear this fuller evidence - direct evidence of intermediates in the case of ferri compounds and of CO ferrohemoglobin-ferrihemoglobin mixtures. Yesterday I mentioned how slowly nitric oxide hemoglobin dissociates thousands of times more slowly than carbon monoxide hemoglobin. I wondered whether there might be a possibility of Dr. Itano and Miss Robinson demonstrating something with completely ferrous hemoglobin compounds if they used nitric oxide hemo- globin as one of the members. Dr. Itano: Since we are doing electrophoretic experiments one of the important considerations was a difference in charge. That is the reason for our using ferric compounds. Although ferrohemoglobin and its compounds

DISCUSSION 155 differ in the pK values of their heme-linked groups, I am not sure whether the fractional differences in net charge that these heme-linked groups cause would be detectable by electrophoresis. The reaction between oxygen and hemoglobin is too fast. Unless the nitric oxide hemoglobin differs significantly in net charge from hemoglobin, I do not think we can do it by electrophoresis. Dr. Roughfo~z: But if you had a mixture of nitric oxide and hemoglobin and plain hemoglobin, the dissociation of the nitric oxide hemoglobin inter- mediates would be so frightfully slow. esoeciallv at 0°. it might take actually days to occur. D'. R. Ber~esch: Dr. Itano, you said that ferricyanide is known only to affect the heme and leaves the globin unaffected. There exists the possibility that ferricyanide also oxidizes -SH groups of the globin, and Dr. Remmer, working in Dr. Shemin's laboratory, a few years ago told me privately that much more ferricyanide was reduced by hemoglobin than could be accounted for on the basis of iron. Would you like to comment on this? Dr. Itano: I investigated this matter quite thoroughly before beginning the experiments. Anson and Mirsky studied this very phenomenon years ago and found that if one carried out the oxidation below about pH 6.8, which we did, no sulThydryl groups are oxidized. Also, I think the studies of Conant and of Wyman indicate a virtually stoichiometric reaction between ferricya- nide and the iron of hemoglobin. On the other hand, if the reaction is carried out at higher pH, some sulfhydryls are oxidized. I think also that sulihydryls in globin may be more susceptible to oxidation than those in hemoglobin. The other factor is that unless oxidation of the sulfhydryl groups pro- duces isomers or some aggregates, one would not expect an intramolecular oxidation of two sulibydryls to disulfide to affect electrophoretic mobility. I am not aware of any oxidation of sulfLydryl that would increase the positive charge of the molecule, which is the type of charge alteration we have observed. Dr. V. 211. Ingram: I would like to confirm what Dr. Itano said. If you oxidize oxyhemoglobin with ferricyanide very carefully at pH 6 and at low temperatures, there is no change of available -SH groups in the native horse or bovine hemoglobin. If you use alkaline pH, then you do lose -SH groups. Dr. John H. Taylor: Our results confirm and extend what has been brought out already. We have used the reaction with p-chloromercuribenzoate ~ PCMI3 ~ to measure available -SH groups in several native mammalian hemoglobins before and after treatment with ferricyanide, as well as with some other reagents. Oxyhemoglobin samples freshly prepared in the presence of EDTA show a definite number of PCMB-reactive sites, depending upon the species, e.g., human 2, bovine 2 and canine 4 -SH per mole (68,000~. If you oxidize any of these hemoglobins cautiously with ferricyanide at pH 7 you do not change the number of titratable -SH groups. If you oxidize with excess ferricyanide at pH 9, much as described by Ansor~ and Mirsky, ~ J ~ ~ A _ ~ J

156 PART III. ABNORMAL HEMOGLOBINS you do abolish a definite number of -SH groups two being removed in all three instances mentioned and you do increase the negative charge on the molecule as shown by suitable electrophoretic measurements. We should like to suggest that the increased charge may arise from oxidation of -SH to the sulfinate or sulfonate level, rather than through dimerization or other structural change, but we have not yet proved this to be so. Samples of methemoglobin in which the number of PCMB-reactive groups has been decreased by alkaline ferricyanide treatment are less homogeneous than "normal" methemoglobin with unchanged -SH, as you can readily demonstrate by means of paper strip electrophoresis. The heterogeneity in- creases slowly as the material is kept in the cold and the visible absorption spectrum also changes. The faster moving fraction, isolated from a starch slab, shows the greatest difference in spectrum from normal me/hemoglobin. It would be of interest to compare this material with the heterogeneous fraction of human hemoglobin, described by Dr. Kunkel, which also appears to increase with the age of the preparation.

ZONE ELECTROPHORESIS AND THE MINOR HEMOGLOBIN COMPONENTS OF NORMAL HUMAN BLOOD HENRY G. KUNKEL Filter paper electrophoresis because of its simplicity and widespread avail- ability has been of great value in elucidating the various abnormal hemo- globins. For population screening a simple technique of this type was essen- tial. However, a number of limitations have gradually become apparent; adsorption in the path of migration, inequality of migration in different parts of the paper and poor adaptability to preparative isolation represent a few of these limitations. As an alternative procedure we have been interested in the use of other supporting media such as potato starch and polyvinyl chloride particles, pare ticularly for the isolation of various hemoglobin fractions. Both of these media, employed in the form of a thin slab, although possessing inherent limitations of their own, have been of some value for these purposes. The starch block technique has the disadvantage that extraneous materials fro ~ the starch itself frequently contaminate the purified hemoglobin fractions. The polyvinyl chloride supporting medium avoids this limitation. However the ease of handling the starch, the homogeneity of migration, the low elect troosmotic flow and the ready visibility of minor colored components against the white starch background have made this procedure more generally useful Fig. 1 illustrates a photograph taken with transmitted light on orthochro~ matic film of the red components of various normal and~abnormal hemo- globins separated on a thin starch block. The separation is in general very ~7 .~ ~.:-~ .. ~ , . FIG. 1.—Comparison of different Hbs separated on the same starch slab. Line at left is site of application. Anode.-to the right. Barbital buffer pH 8.6 I/2 0.05. a. Normal Hbs b. Homozygous C c. Sickle cell anemia d. Umbilical cord e. Sickle cell trait

158 PART III. ABNORMAL HEMOGLOBINS similar to that observed with filter paper. The use of broader starch slabs permitted the separation of as many as thirty specimens in a single experiment with resolution similar to that seen in fig. 1. For detecting the abnormal hemoglobins, the hemoglobin solutions were usually diluted with buffer to a protein concentration of approximately 3 per cent. Quantitative elusion of hemoglobin from starch segments could be carried out by displacement filtra- tion over ground glass filters, thus permitting direct determination of rela- tive concentration from hemoglobin color. Quantitation was most accurate when the CO or cyanmethemoglobin derivatives were employed. c c ........ _: _ _ r _ _4 ............... FIG. 2. Photograph of multiple normal Hb specimens separated on a broad starch slab. Two specimens from indi- viduals with sickle cell trait are included for comparison. The minor Hb A`, is visible in all specimens. . _ ~ _ ~ ~ ~ ~ ~ ~~ ~ ~~ ~ a ................ ' ~ I"'"''' '' .~ ~'~,~' ' I, ,~ ''' ''"''''"'. ........ ... . . ~ ~ ~ ~~ ~ ~ ~ ~ ~ ~ .~ ~ ~ . ~,~.~., ~.~ .~ ~ ~ ~.~ All ~ ................................................ ~ :~.~.~ ~~ it . ~ ~ ~T .. ~~ ~ ~ . .~. .. ~ ................................................. - ... , ~.~ ~ ~ ~ ~ ~ i ~ ~ ,~ ...... ~ i ~ .. ~ ~ ..... , ... . i I.~.~ ..... ............ .. . ~ T ~ ~ i. ~ ............ I .. ~~ i. ~ ~: ~ ~ ~ C ~ i ... ~~ .~ .... i .~.. .. ... ~ ~.~ ~ g. :::::::: i::: FIG. 3. Comparison of the mobilities of the isolated normal Hb components separated at equal concentrations. a. Slow An b. Main A c. Fast fraction The normal hemoglobin sample shown at the top of fig. 1 shows two dis- tinct components. A minor fraction with a mobility just slightly greater than Hb C was apparent with all specimens from normal individuals. This frac- tioni 2 (termed Any was best visualized when the hemoglobin solution was separated at a 10 per cent protein concentration. Fig. 2 shows multiple normal specimens on a broad starch block. Two specimens from individuals with sickle cell trait are also included. All of these showed the As component at approximately equal concentration. In addition to the A2 fraction, the main A fraction always showed faster

ZONE ELECTROPHORESIS OF HEMOGLOBIN A KUNKEL 159 migrating material projecting in front of the round A spot;) this is visible in both figs. ~ and 2. Some of this material migrated markedly faster than Hb A. Isolation and re-running showed that it kept its original fast mobility. Fig. 3 shows the result of one such experiment where the isolated Hb A2, Hb A from the main A peak, and fast material were examined at equal concentrations on the same starch block. The A2 Hb kept its original slow mobility. The fIb A no longer showed projecting faster hemoglobin and moved as a sharp band. The fast fraction was broad and retained the original mobility. The same components were observed with the polyvinyl supporting medium. Ordinary filter paper strips did not show the A., clearly but thicker paper, which permitted more hemoglobin to be applied, brought out this component. Derivatives of oxyhemoglobin, carbonmonoxyhemoglobin, me/hemoglobin, cyanmethemcglobin and reduced hemoglobin all showed a similar relative distribution for the minor components despite differences in over-all mo- bility. Different anticoagulants and different procedures of faking the red blood cells gave similar results. Numerous observations with the isolated A component indicated that it did not give rise to the A, fraction. However, con- siderable evidence was obtained that the fast fraction or material like it could be produced from Hb A. This transformation occurred slowly with oxy- and carbonmonoxyhemoglobin but was particularly rapid when methe- moglobin or cyanmethemoglobin was used. Old hemoglobin samples also were found to contain considerably more of the fast fraction. Although the fast fraction could not be quantitated, it appeared to be present at a con- centration somewhere between 4 and 12 per cent in fresh hemoglobin speci- mens. The accumulated evidence strongly suggested that the A2 component was a well-defined entity but that the fast fraction might be derived from Hb A. Some degree of quantitation of the A2 component was possible. Replicate analysis of one normal hemoglobin sample (separated 16 times) showed a mean value of 2.51 per cent with a standard deviation of + 0.31. Examina- tion of the blood of 65 normal individuals showed that the As level was 2.54 per cent + 0.35. In a larger series of normal individuals studied sub- sequently, no A' level above 3.3 per cent was encountered. However, in pa- tients with thalassemia considerable elevation was frequently found. The highest level observed was 11 per cent in an adult with an intermediate type of thalassemia. Four individuals classified as intermediate thalassemia were studied and all showed unusually high levels. The over-all results in patients with thalassemia excluding the Cooley's type (34 cases) was 5.11 per cent + 1.36. The infants with Cooley's anemia frequently had relatively normal levels. Two patients with thalassemia minor also showed levels in the normal range. The significance of the elevation in thalassemia was not apparent. No elevated values were obtained in any other condition studied.2 One possible

160 PART III. ABNORMAL HEMOGLOBINS explanation is that a compensatory increase of lIb A2 similar to that found for fetal hemoglobin occurred in the presence of defective Hb A synthesis. However, the frequency of levels just twice the normal in thalassemia minor remains unexplained. To further determine the significance of the A2 arid fast hemoglobin fractions, comparative specific activities were obtained after administration of F'e59. Observations by Schapira and associates3 had indicated that this method gave evidence for the heterogeneity of human hemoglobin. The possibility also arose that such experiments would provide evidence regarding the presence of the minor fractions in all red blood cells or just in selected cells at high concentration. Table I shows representative results obtained in two essentially normal individuals at different times after the intravenous in- jection of approximately 32 tic of Few (0.024 ma. Fe). In the experiments with patient ELF. the separated hemoglobin was divided into three fractions designated A2, A and fast. With the hemoglobin of patient Y.R. further subdivision was carried out. The fast material was divided into two parts, a very fast fraction, designated fasts and a relatively slower fast fraction designated fasts. Hb A was also divided into a fast and slow part. In each case, all of the separated hemoglobin fell into one of these fractions and the relative percentage of each in the different experiments is indicated in table I. The most striking finding was the low specific activity of the fast fraction compared to that of Hb A. This was evident in all experiments and can be seen best from the relative specific activity calculated by assigning the value 1.0 to either the whole of Hb A, or to its least contaminated slower portion. No essential differences were observed whether the radioactivity was meas- ured in terms of hemin or as the whole hemoglobin solution. The experi- ments with the second patient are the more informative because the fasts fraction was completely free of Hb A. The fasts fraction as well as the whole fast fraction in the experiments with the first patient did not represent pure samples but probably contained some Hb A. FIb As showed a specific activity similar to but always slightly below Hb A. However, it was somewhat di~- cult to make an exact comparison because of the range in specific activities of the A and the overlapping fast fractions. These results appeared to indicate that the fast hemoglobin had special significance despite the fact that it could be produced in vitro from Hb A. The exact reason for the low specific activity of this fraction compared to the bulk of the Hb A is not clear. One possible explanation is that the fast component was more concentrated in old red blood cells and was formed in vivo from Hb A under the stress of survival in the blood stream. References to fractions which may have corresponded to these minor hemo- globins have previously appeared in the literature. The As component prob- ably had been observed by earlier investigators employing the classical Tiselius procedure in occasional specimens of blood from normal persons,3 4

ZONE ELECTROPHORESIS OF HEMOGLOBIN A KUNKEL 161 TABLE I COMPARISON BETWEEN THE SPECIFIC ACTIVITY OF VARIOUS IEMCGLOBIN FRACTIONS AT DIFFERENT TIMES AFTER ADMINISTRATION OF FE59 Days after Hb % of Spec. Act. Ratio of Subj. Feo9 Fract. Total cts/100 fly F`e Spec. Act. . fast 5.2 152. .584 4~/2 A 92.3 2g6. 1.0 A, 2 5 282. .95 f ast 4.8 208. .49 M.F. 11 A 93.1 425. 1.0 A2 2.1 390. .92 fast 4.2 198. .56 21 A 93.2 354. 1.0 A2 2.6 297. .84 fast' 3.1 145. .32 fasts 13.6 397. .87 7 Af . ; 1.4 420. .92 Ash. 30.4 455. 1.0 A`, 1.6 446. .9 . fasts 1.2 178. .43 fasts 3.7 232. .56 Y.R. 14 Af . 59.4 353. .85 Asl. 33.6 417. 1.0 AS 2.3 333. .80 fast' 2.2 127. .64 fast., 4.5 179. .89 37 Af . 51.1 220. 1.1 Ash. 40.8 200. 1.0 An 1.4 180. .9 _ sickle cell patients and thalassemia patients6 but had not been defined as a hemoglobin. It also resembles one of the chromatographic subfractions ob- tained by Morrison and Cook: from normal human blood. Recently additional information concerning Hb A2 in thalassemia has been obtained by Gerald,8 Aksoy and co-workers9 and Josephson and Singer.~° The fast fraction, since it is impossible to quantitate, is not easily related to other fractions described in the literature. It seems possible that the heterogeneity noted by Derrien and his associates, Shavit and Breuer~2 and Hochi3 may be caused by this fraction. Also the recently described component

162 PART III. ABNORMAL HEMOGLOBINS of Berry and Chanutin14 would appear to be similar although these workers observed a disappearance of their second component in stored cells rather than an increase as in the present study. Considerable further work is neces- sary to resolve these results obtained in different laboratories with different techniques. Summary. Evidence has been presented for the presence of two minor hemoglobin fractions in normal human blood. Tile one designated Fib As is a clearly defined entity which is characteristically elevated in thalassemia. The other represents a heterogeneous fast fraction which may be produced from Hb A. Studies with Few indicate that it has biological significance. REFERENCES 1. Kunkel, H. G., arid Wallenius, G.: New hemoglobin in normal adult blood. Science, 122: 288, 1957. 2. Kunkel, H. G., Ceppellini, R., Muller-Eberhard, U., and Wolf, J.: Observations on the minor basic hemoglobin component in the blood of normal individuals and patients with thalassemia, J. Clin. Invest. (in press). 3. Schapira, G., Dreyfus, I.-C., and Kruh, J.: Heterogeneous metabolism of haemo- globins, Ciba Foundation Symposium on Porphyrin Biosynthesis and Metab- olism. Churchill Ltd., London, and Little Brown Boston 1955. . . . . . 4. Stern, K. G., Reiner, M., and Silber, R. PI.: On the electrophoretic pattern of red blood cell proteins, J. Biol. Chem. 161: 731, 1945. 5. Pauling, L., Itano, H. A., Singer, S. J., and Wells, I. C.: Sickle cell anemia, a molecular disease, Science, 110: 543, 1949. 6. Singer, K., Chapman, A. Z.. Goldberg, S. R., and Rubenstein, H. M.: Studies on abnormal hemoglobins. X. A new syndrome: hemoglobin C-thalassemia disease, Blood, 9: 1032, 1954. 7. Morrison, M., and Cool;, J. L.: Chromatographic fractionation of normal adult oxyhemoglobin, Science, 122: 920, 1955. 8. Gerald, P.: to be published. 9. Aksoy, M., Lehman, H., and Luan Eng, L. I.: The recognition of haemoglobins A2 and E, Lancet, 272: 792, 1957. 10. Josephson, A. M., and Singer, E.: to be published. 11. Derrien, Y., and Reynaud, J., Sur l'heterogeneite electrophoretique de l'hemo- globine humaine (so jets adultes normaux), Compt. rend. soc. biol. 147: 660, 1953. 12. Shavit, N., and Breuer, M.: Electrophoretic heterogeneity of normal adult human hemoglobin at low ionic strengths and higher temperatures, Biochim. et Biophys. Acta, 18: 241, 1955. 13. Hoch, H.: The steady state, a test for electrophoretic homogeneity, ]3iochem. I. 46: 199, 1950. 14. Berry, E. R., and Chanutin, A.: Electrophoretic studies on red cell extracts of stored blood, J. Clin. Invest. ]6: 225, 1957. DISCUSSION Dr. Yves Derrien: In connection with Dr. Kunkel's very interesting talk, I should like to comment briefly about researches in the same field, partly

DISCUSSION 163 published in 1955 and described in more detail in my paper later in this Conference. ~.~ ~ :::: I.,< .. :::~:~::~:::~:~:::~:~:~::::::::~:~:~:~:~:~::: · ,. ,,, f . ..H,.~,,..h,.,.,. Act... FIG. 1. I;lectrophoresis of normal adult hemoglobin (I) and "protein X" (II). As can be seen in the upper part of the illustration (fig. 1), paper elec- trophoresis of normal adult hemoglobin in veronal buffer of pH 8.8 and ionic strength 0.025 always shows a small component in front of the major spot of Hb A and another one slightly slower than Hb C. This slow minor component, only visible after staining and corresponding to a colorless pro- tein we called "protein X" in 1955, is not removed by a single crystallization of the hemoglobin preparation nor by passing it through absorbents like celite. It is resistant to denaturation by bases and passes into the all~ali-resistant fraction of hemoglobin. Paper electrophoresis of this fraction (II) shows a large spot of protein X, a small spot of a slightly faster nonchromogenic com- ponent called X~ and the alkali-resistant hemoglobin. Protein X can be iso- lated by elusion of its spot and contains from 15 to 20 per cent hemoglobin, as determined by iron/nitrogen ratio and spectrophotometric analysis. I should like to ask Dr. Kunkel if it is to be excluded that his As minor component corresponds to hemoglobin A adsorbed on the so-called proteins X. This hypothesis is suggested by the additional fact that, as is A2, proteins X are always present in the hemoglobin of adults and of patients with Cooley's anemia but are never detected or only in extremely small amounts in newborn hemoglobin. For normal subjects proteins X appear from 11 to 12 months after birth. Dr. Keel: I did not have time to mention the non-hemoglobin compo- nents that orate observes following electrophoresis of red cell hemolysates. There are at least three that are constantly present and can be observed as clear bands or peaks. One of these which migrates slightly slower than Hb As at pH 8.6 is present at a concentration of 1 to 3 per cent of the total protein. This may well be Dr. Derrien's ~ protein. It has no relation to Hb A2 and can be separated from it by electrophoresis. We know this protein rather well because it has caused contamination or the Hb As when separation was not completely optimal. There is no possibility that fIb As represents a complex between Hb A and some other protein because it has the same sedimentation

164 PART III. ABNORMAL HEMOGLOBINS coefficient as Hb A and also has a very similar heme protein and iron protein ratio. Dr. Jerome Vinograd: I Irish to ask Dr. Kunkel whether his results were obtained with recrystallized material or with the material as it came from the red cells. Dr. Kunkel: We have had the opportunity of studying two crystalline specimens and both of these showed the As component to be present at the same 2.5 per cent concentration that was in the original hemoglobin solution. However, most of our work was done with the whole hemoglobin solution.

A METHOD FOR THE CHARACTERIZATION OF ABNORMAL HUMAN HEMOGLOBINS BASED UPON DIFFERENCES IN CHROMATOGRAPHIC BEHAVIOR ON AMBERLITE IRC 50* T. H. J. HUISMAN Next to the electrophoretic techniques for the identification of abnormal hemoglobins, the new chromatographic method using the cation exchanger Amberlite IRC 50 may be most valuable. This simple routine method, which was described in detail in 1955,1 is found to be useful not only for the identi- fication of the hemoglobins A, S. C, E and IF 2 but also for some rare types of human hemoglobin. In figure 1 the chromatographic behavior of nine dif- ferent types of human hemoglobin (A, S. C, D, E, F. H. I and J) is given. of.. ~ ~ . :::: :::::: FIG. 1. The separation of different abnormal human hemoglobins on Amberlite IRC-50. Citrate builder pH 6.0, sodium ion concentration 0.15. 1. C + S ~ A + F 4. A + H 2. D + A 5. A + I 3. E + A + Fit 6. A + J (unseparated) Next to the well known separation of the hemoglobins C, S. A and Fit it was found: a. Fib D has the same mobility as sickle-cell hemoglobin. b. Especially when a small amount of hemoglobin ~ about 5 ma. ~ chromatographed, a complete separation of a mixture of the hemo- globins A, E and Fit into the three components is possible. c. Hb H shows a high mobility and is easily distinguished from fetal hemoglobin. ~ Dr. Huisman was unable to attend the Conference and this paper was read and the illustrations shown by Dr. London. 165 . IS

166 PART III. ABNORMAL HEMOGLOBINS d. The mobility of the rare Hb I is between those of Hb A and Hb F. e. It seems impossible to separate lIb ~ from the adult component. A comparison of the relative Nobilities of the different hemoglobins in paper electropl~oresis and in chromatography is given in figure 2. In general the same sequence exists. For the hemoglobins ~ and H important differences C ~ ' E o - ' , 5 ~ , D ~ ' F ~ _ ,. A q J l 1 H , O _ 1 ' I | ,_~ paper electrophoresis I I chromatography _ Flu. 2. Comparison of the relative mobilities of different types of human hemoglobin in paper electrophoresis (open blocks) and chromatography ( filled blocks ) . were found, as the fetal pigment moves faster than the normal adult hemo- globin in chromatography and Hb H still much faster than Hb F. With the other hemoglobin types, smaller differences are present for the hemoglobins E, I and I. Each method has some advantages. The identification of hemoglobins E and I, for instance, is much easier with paper electrophoretic techniques. The chromatographic estimation of Hb A, on the other hand, may be important; the characterization of this hemoglobin by paper electrophoresis is difficult. Hemoglobins H and I can also be distinguished by using the chromatographic method. The use of both methods at the same time is of importance for the definite identification of an abnormal hemoglobin type. REFERENCES 1. Huisman, T. H. J., and Prins, H. K.: J. Lab. Clin. NIed. 46: 255, 1955. 2. Prins, H. K., and Huisman, T. H. J.: Nature 177: 820, 1956. DISCUSSION Dr. Martin ILlorriso?z: I would like to outline briefly our procedure for the column chromatographic separation of human hemoglobins. The tech-

DISCUSSION 167 nique eve employ is to adsorb the hemoglobin on a resin bed of XE 97 (IRC- 50 ~ which is in equilibrium with a phosphate-citrate buffer, pH 6.3, and which has a sodium ion concentration of 65 mEq. per liter. The hemoglobin solution itself has been previously dialyzed against the same buffer. The pro- tein is then eluted from a 1 ~ 50 cm. column by gradually increasing the sodium ion concentration. When the oxyhemoglobin obtained from a hemolysate of the red cells of a normal adult is investigated in this way, a chromatogram such as is shown in the figure 1 is obtained. Note that the hemoglobin is eluted in three fractions. too o 080 a' Us 060 A a: a: o 0.40 020 ,~ _ 1 0 20 30 co 50 TUBE NUMBER 60 70 FIG. I.- Chromatogram of hemo- lysates of normal adult red cells. The most rapidly eluted of these components represents approximately 10 per cent of the total hemoglobin contained in the l~emolysate. The second, or major, component represents about 86 per cent of the total hemoglobin, while the third and most slowly eluted component comprises 4 to 6 per cent of the hemoglobin and, as subsequent illustrations will show. moves chromato- graphically as Fib E. ~ . . . . More detailed studies of each fraction were undertaken. By employing dilute buffers, eve found that the first, or most rapidly eluted component, could be further separated into two colored components, the first of these being a protein fraction ravish methemoglobin reductase properties. When the major component, representing 86 per cent of the hemoglobin of the hemo- lysate of the normal adult, was purified by column chromatography or starch block electrophoresis, a single peak, shown in figure 2, was obtained. This demonstrates that tile fractions were neither column artifacts nor conversion products of the protein and further verifies that when a single hemoglobin is present, a single peak is obtained. Our efforts then naturally focused upon identification of the components which we found in the normal adult. Since there was substantial evidence i the literature that fetal hemoglobin occurs in the normal adult in small per- centages, she began with pooled cord hemoglobin, the chromatogram of which is shown in figure 3. The first component to come o* the column was re- sistant to alkaline denaturation. This, as well as spectral analysis, definitely

168 60r ~50 E o ~ 4 0 z 30 c lo: ° 20 On q 10 PART III. ABNORMAL HEMOGLOBINS FIG. 2. Single peak obtained by chroma- tography of major fraction in figure 1. 1 ~ 1 !~1 20 30 40 50 60 70 TUBE NUMBER established it as fetal hemoglobin, which is in sharp contrast to the results obtained from the normal adult component which came off the column in the same region. The adult component is not resistant to alkaline denaturation and on electrophoresis at pH 8.6 moves more rapidly than Hb A, while fetal hemoglobin, which is resistant, moves behind fIb A on electrophoresis. 1 800 1600 ha. 10m to - Z ,200 - ~ 1000 be z 800 o A 600 o 0 4 00 200 1 C ) ! ! ~———~ 1 10 20 30 40 50 60 TUBE NUMBER I POOLED CORD HEMOGLOBIN (5 ~ am pies) FIG. 3. Chromatogram of pooled cord blood hemo- globin. so so 6 o 501 40 SO 20 °T :~> Z C3O o <t X Q O 51 CHILE CALL GANDHI A . ~ -I ~ ~ , , , I ~ 1 1 1 0 20 30 So 50 60 70 80 90 100 Tuse NUMBER FIG. 4. Chromatogram of hemoglobin of homozygous sickle cell individual. Another of the human hemoglobins, one which could be labeled as homozy- gous S by electrophoresis, produced a chromatogram such as is shown on figure 4. It is apparent that Hb S moves as a homogeneous compound behind any of the components of the normal adult. This actually demonstrates two things: first, the column produced no multiple components, further confirming our data that a single protein gives but a single peak in this method, and second, that the genetic apparatus which gives rise to the multiple components in normal adult hemoglobin is completely altered in the homozygous sickle cell individual. After a number of abnormal hemoglobins had been investigated, a composite chromatogram such as is shown on figure j could be drawn. Hb F (fetal) moves off the column in the region of tube 10, Hb A at tube 30, Hb E at

D1SCL; SSIO3N 1G9 ~ . A Zen i ! ~ ~ ~ FIG. 5.—Composite chromato- 0 ~ :~ \, ~ ~ gram Of several types of hemo- 0 10 20 30 40 50 60 70 80 90 TUBE NUMBER tube 55, Hb S at tube 65 and Hb C at tube 7j. Of the hemoglobins investi- gated, Hb E moves chromatographically as the third component of normal adult. The movement of the hemoglobins in column chromatography is directly related to the charge carried by the protein.' This relationship is demonstrated as follows: Relative Mobilities of Human Hemoglobins on electrophoresis at pH 8.6 pH 6.5 On chromatography at pH 6.3 A ~ S E C C S E A F A E S C Below the iso-electric point, hemoglobin carries a positive charge. On elec- trophoresis, the compound with the lowest mobility has the smallest charge. This means it will be least strongly adsorbed on the column and will, there- fore, move off the column most readily. Conversely, the compound with the greatest positive charge will be most strongly adsorbed to the column and will be eluted last. It follows, then, that electrophoretic mobility and column chromatographic mobility are inversely related, and that this relationship is a direct consequence of the charge carried by the protein. Essentially, no anomaly has been found to this situation. (See, however, bibliographic ref- erence No. 3~. Dr. Itano, in his earlier talk, described the separation of hemoglobin mole- cules in which the iron atoms have been oxidized.4 If the major component of normal adult hemoglobin is oxidized into any of these types between methe- moglobin and CO-hemoglobin, their separation can be achieved. Boardman and Partridge,5 in their first study of the chromatography of hemoglobins by ion exchange resins shorted that they could separate bovine methemoglobin from bovine CO-hemoglobin. We have found that we car separate and spec-

170 PART III. ABNOR'VIAL HEMOGLOBINS trophotometrically demonstrate the separation of the various intermediates achieved by the partial oxidation of the iron atoms of hemoglobin. Table I shows the type of separation achieved, which is very good and has the ad- vantage of making possible spectrophotometric inspection of each f faction. When this is done, the spectrophotometric analysis shows that the fractions TABLE I COLUMN- CHROMAT~GRAPHIC MOVEMENT OF FORMS OF HEMOGLOBIN A Compound CO-hemoglobin Intermediates Methemoglobin Ratio Fe+'/Fe~ 3 4/0 311 2/2 1/3 0/4 Peak Tube No. 28 54 65 75 90 correspond to hemoglobin ire which the iron atoms were progressively oxidized. As was the case with the abnormal hemoglobins, the forms of Hb A move off the column according to the charge on the molecule. The molecule with all the iron atoms in the ferrous form is least positively charged and is elated snore readily, while the methemoglobin carries the highest positive charge and is the last form to be eluted. Thus, the forms of hemoglobin may lead to erroneous results in the quantitative analysis of mixtures of Hb A and abnormal hemoglobins unless due care is exerted. The procedure was also used to investigate the possible dissociation of Ilb A into components. Employing exactly the same buffers and chromato- graphic system, with the exception that all solutions were made four molar with respect to urea, we investigated the homogeneity of Hb A. It would appear that if Hb A did dissociate under these conditions, we might be able to separate the components from one another. However, under the experi- mental conditions we employed, we could detect no dissociation. I might mention that our results with thalassemia are, qualitatively at least, comparable with the results Dr. Kunkel~ 7 has obtained. However, we find that in thalassemia major there is a higher concentration of our third component which appears to move like Hb E on our column. Atcknowledgment: Figures 1, 2, 4 and 5 appear in Federation Proceedings 16: 764, 1957, and are reproduced by permission of the publishers, the Fed- eration of American Societies for Experimental Biology. REFERENCES 1. Morrison, M. and Cook, J.: Chromatographic fractionation of normal adult oxy- hemoglobin, Science 122: 920, 1955. 2. Morrison, M. and Cook, J.: Column chromatography of human hemoglobins, Fed. Proc. Vol. 16 (September) 1957. 3. Huisman, T. H. I. and Prins, H. K.: Chromatographic estimation of four different human hemoglobins, J. Lab. and Clin. Med. 46: 255, 1955. 4. Itano, H. A. and Robinson, E.: Demonstration of intermediate forms of carbon-

DISCUSSION 171 monoxy- and ferclbemo~lobln by movlDg boundary electropbores~, J. A. C. S. 76: 641i, 1966. 5. Boardman, N. K. and Fartdd~e, S. Hi.: Sep~radoD of neutral proteins OD 1OD excbaDge reslDs, Plocbem. J. SP: 643, 1933. Kunket H. O. and ~allenlus, G.: Bed bemo~loblD ID DOrma1 adult blood, Science 722: 2SS, 19i5. 7. Junket H. O.: D~trlbutloD and sl~nlEcance of the minor bemo~loblD components of DormaI bumaD blood, Fed. Proc. Vol. 16 (September) 1937.

THE ALKALI DENATURATION PROCEDURES* AMOZ I. CHERNOFF All current techniques for the determination of fetal hemoglobin based upon the resistance of the embryonic pigment to denaturation by highly alkaline solutions stem from the observations made almost one hundred years ago by von Korber,~ who demonstrated that hemoglobin solutions prepared from cord blood erythrocytes were resistant to the destructive effects of NaOH whereas hemoglobin preparations obtained from normal adults were rapidly destroyed under the same experimental conditions. Since the original observations of von Korber, relatively little has been learned con- cerning the kinetics of this reaction or about the general phenomenon of . . hemoglobin denaturation. Nevertheless, many procedures are available for the quantitative estimation of the fetal pigment based upon its resistance to alkali denaturation. In general, these techniques utilize two different ap- proaches. One method depends upon the change in light absorption at selected wave lengths as the hemoglobin is converted from the oxy form to the alka- line chromogen. If one follows these minute to minute changes in light ab- sorption, it is possible to determine not only the rate of reaction but also to calculate the percentage of Hb F in a mixture of hemoglobins. The second group of methods involves the precipitation of the denatured protein by 1/3 saturated (NH4~2 SO4 followed by the determination of the per cent of undenatured hemoglobin. If this procedure is carried out at varying intervals of time, the rate of reaction and per cent of alkali-resistant hemoglobin may be determined as in the first technique described. In practice, however, it becomes possible to select a convenient time period during which all alkali- sensitive hemoglobins are denatured whereas those which are alkali-resistant are only minimally affected. In the method described by Singer and Chernoff, . · c> thlS time lIlterVa. . IS one mlnute.~ Although there is general agreement as to the usefulness of these two techniques in the quantitative determination of alkali-resistant hemoglobins, considerable disagreement is present concerning a number of features of the alkali denaturation procedures. These may be listed as follows: 1 ) Does the chemical state of the hemoglobin alter the results of alkali denaturation ? 2) What is the nature of the alkali-resistant fraction left after the re- action is complete when using the precipitation technique? 3) Is the alkali-resistant fraction always identical with fetal hemoglobin? 4) How do the two methods compare in their accuracy of Hb F deter- m~nat~on ~ Most investigators have used oxyhemoglobin as the starting material in the *; Some of the studies reported in this paper were supported by United States Public Health Service Grant No. A-1615. 172

ALKALI DENATURATION PROCEDURES CHERNOFF 173 alkali denaturation procedures. Nevertheless, certain of the discrepancies which show up between the various techniques may be explained by the presence of hemoglobin forms other than oxyhemoglobin. Thus CO-hemo- globin is known to have a slower rate of denaturation than does oxybemo- globin.3 4 The results of the alkali denaturation procedures would therefore be elevated in the presence of significant amounts of this compound, as has been pointed out by [onxis and Huisman.5 The 'denaturation of methemo- globir~ precedes at the same rate, or somewhat more rapidly, than that of oxy- hemoglobin.3 Likewise, cyanhemoglobin demonstrates a reaction rate identical to that of oxyhemoglobin but is felt by Kunzer6 to constitute a better start- ing material because of the lower residual values after one minute of reac- tion an observation which wee will shortly refer to again. Finally, dif- ferences in the results of the alkali denaturation techniques may be caused by the method of preparation of the hemoglobin as, for example, when saponin is utilized as the hemolyzing agent. Baar and co-workers have shown that adult hemoglobin solutions prepared wild saponin often demonstrate two or more components on denaturation, whereas those hemolyzed with water be- have ire a manner suggestive of a monomolecular reaction.3 These observa- tions may explain the reports of a number of investigators that two or more hemoglobin components are present in normal adult hemoglobin solutions when studied by the denaturation technique's. Following what appears to tee ' complete denaturation of normal adult oxy- hemoglobin solutions, a residual spectrophotometric reading of up to 1.8 per cent of the original hemoglobin concentration is noted.~ Although the ma- terial is benzidine positive, the evidence is inconclusive as to whether the com- pour~d causing this residual reading represents fetal hemoglobin, is a particu- larly al'~ali-resistant non-fetal hemoglobin fraction, is a denatured hemo- chromogen which is not precipitated by saturated (NH4~2SO4 or is, indeed, a heme pigment at all. Prolonged reaction times of up to 24 hours reduce the amount of, but do not eliminate, this substance. That part of this material is truly fetal hemoglobin is suggested by a number of studies indicating the presence of small quantities of fetal hemoglobin in the normal adult. These studies have been carried out by immunologic techniques,7 by amino acid determinations on the residual protein,S and by solubility determinations such as described by Roche and Derrien.9 The remaining portion of the residual material has been ascribed to one of two causes. Betke believes that the de- natured hemochromogen unites with albumin carried along with incompletely washed erythrocytes in the preparation of the hemoglobin solution.~° Others have postulated that some of the products of denaturation are not precipi- tated by (NH4~2SO4 and hence enter the filtrate to contribute to the resid- ual reading. Although direct proof of the latter postulate is lacking, the idea seems to provide a logical explanation for the presence of the residual ma- terial noted above.

174 PART III. ABNORMAL HEMOGLOBINS The third problem for consideration is that of the possible identity of the alkali-resistant pigment with fetal hemoglobin. There is considerable evi- dence that small quantities of fetal hemoglobin are present in normal adults, up to approximately 0.5 per cent of the total pigment being of the fetal variety. Tl~e studies suggesting this identity have been alluded to above and involve three entirely different approaches immunologic, amino-acid com- position and solubility studies. In pathologic states, however, there is some disagreement as to the identity of the alkali-resistant compound and fetal hemoglobin. Thus, Larsen and co-workers believe tl-~at the alkali-resistant material in untreated pernicious anemia is probably related to a stromal factor found in the macrocytes of this disease.'' The alkali-resistant pigment in sickle cell anemia is claimed by van der Schaaf and Huisman to be different from fetal hemoglobin on the basis of careful amino-acid analyses of the pro- te~n when separated by ion exchange chromatography.13 On the other hand, there is considerable evidence that the alkali-resistant fraction in sickle cell anemia, as well as in all other hereditary hemoglobin diseases, is identical with the fetal compound. To cite only a few of the techniques used in arriving at this conclusion, one may mention studies of the electrophoretic, spectrophctometric, immunologic, amino-acid composition and solubility char- acteristics of the resistant material. It should seem, therefore, that, in the present stage of our knowledge, the a.lkali-resistant pigment is probab~v identical with fetal hemoglobin. That startle alkali-resistant f factions may however not be Hb F cannot be definitely excluded. Of the various methods for determining Hb r, else alkali denaturation technique is by far the most sensitive practical method. Ultraviolet spectro- photometry cannot be used with less than 10-20 per cent of the fetal com- pound. Electrophoretic techniques cannot determine less than ten per cent of this pigment when in combination with adult hemoglobin. Chromato- graphic procedures are not feasible with less than 5-10 per cent of this com- pound, when Hb A is present. Amino acid determinations, as well as immun- ologic determinations, both of which are sensitive to considerably less than O.3 per cent of Hb ~ in combination with fIb A, are tedious and can only be done in special laboratories. Thus the alkali denaturation technique remains as the single most practical method for Hb F determination. The technique employing the change in light absorption at selected wave lengths is claimed by [onxis and HuismanS to be a more accurate method than the precipitation technique of Singer and Chernoff.'' Nevertheless, the former procedure cannot be utilized adequately with less than ten per cent Hb F and the technique is somewhat more cumbersome. The one minute denaturation pro- cedure is sensitive to at least 2 per cent and as modified by Kunzer to approximately one per cent Hb in any mixture of hemoglobins, is easily performed in clinical laboratories and does not require elaborate apparatus. The difficulties of falsely high values in the lower concentrations of Hb F

ALKALI DENATURATION PROCEDURES CHERNOFF 175 and falsely low values in the higher ranges, as suggested by [onxis and Huisman, are not of sufficient magnitude to detract from the clinical useful- ness of the procedure. Using the one minute alkali denaturation test, we have now examined many thousand blood specimens. Tn~ren~A amount nT Wh F malt he found in almost all the hereditary hemolytic syndromes, particulary in thalassem~a and the hereditary hemoglobin diseases, as well as in a number of acquired hematologic diseases in which the marrow Is infiltrated by malignant or foreign cells. The use of the alkali denaturation procedure may serve, there- fore, as an important adjunct to the investigation of a number of obscure hematologic diseases. REFEREN CES 1. Von Korber, E.: Uber Diderenzen des Blutfarbstoffes, Inaugural dissertation. Dorpat, 1866. Cited by Bischoff, H., Ztschr. f. d. yes. expert Med. 48: 472~89, 1926. 2. Singer, K., Chernoff, A. I., and Singer, L.: Studies on abnormal hemoglobins. I. Their demonstration in sickle cell anemia and other hematologic disorders by means of alkali denaturation, Blood 6: 413~28, 1951. 3. Baar, H. S., and Hickmans, B. M.: Alkali denaturation of oxyhemoglobin, hemo- globin, carbonmonoxybemoglobin, methemoglobin and cyanmethemoglobin, l. Physiol. 100: 3P, 1941. 4. Singer, K., and Fisher, B.: Studies on abnormal hemoglobins. VII. Composition of non-S hemoglobin fraction in sickle-cell anemia bloods: comparative quan- titative study by methods of electrophoresis and alkali denaturation, J. Lab. & Clin. Med. 42: 193 - 204, 195 3. Jonxis, l. H. P., and Huisman, T. H. J.: The detection and estimation of fetal hemoglobin by means of the alkali denaturation test, Blood 11: 1009-1018, 1956. 6. Kunzer, W.: Untersuchungen uber das Vorkommen fetalen Hamoglobins bei Blutl;rankheiten. Zeitschrift fur Kinderheilkunde 76: 58-72. 1955. 7. 8. Chernoff, A. I.: Immunologic studies of hemoglobins. I. Production of antihemo globin sera and their immunologic characteristics, Blood S: 399~12, 1953. Huisman, T. H. J., Jonxis, l. H. P., and Dozy, A.: Is foetal haemoglobin present in the blood of normal human adults ? Biochim. Biophys. Acta 18: 576 - 577, 1955. 9. Roche, J., Derrien, Y., Reynaud, J., Laurent, G., and Roques, M.: Sur l'heteroge- neite des hemo~lobines. I. Technique d'etablissement des courbes de solu- bilite et premiers essais de fractionnement, Bull. Soc. chim. biol. 36: 51 - 63, 19 j4. 10. Betide, K., Greinacher, I., and Leber, E.: fiber die 13indung von Hamatin an Plasmaeiweiss. Zugleich ein Beitrag our Methodik der Alkalidenaturierung von Blutiarbstoff, Biochemische Zeitschrift 326: 1-8, 1954. 11. Chernoff, A. I.: Immunologic studies of hemoglobins. II. Quantitative precipitin test using anti-fetal hemoglobin sera, Blood S: 413~21, 1953. 12. Iversen, O. H., and Larsen, G.: EIemoglobin in pernicious and allied anemias. The significance of an abnormal alkali denaturation curve, Scand. l. of Clin. and Lab. Invest. 8: 159 - 167, 1956. 13. van der Schaaf, P. C., and Huisman, T. H. l.: The estimation of some different kinds of human hemoglobin, Recueil des Travaux Chimiques des Pays-gas 74: 563-570, 1955.

176 PART' III. ABNORMAL HEMOGLOBINS DISCUSSION Mr. Jbuer R. Rol~i~zson: With respect to the problems of separation and quantitation of hemoglobin ~ to which Dr. Chernoff has referred, I should like to describe a technique which has been devised in our laboratory. We found that this method clearly separates hemoglobin F as well as hemo- globins A, S. and C, and when used in conjunction with filter paper electro- phoresis differentiates hemoglobin D from hemoglobin S. The method consists of preparing a 1~2 per cent agar gel using a pH 6.2, citric-citrate buffer of 0.05 molarity. The agar is poured in a 1-2 mm. layer on a 4 in. x 10 in. glass plate and allowed to gel. The hemoglobin solutions are introduced into the gel by placing small pieces of filter paper soaked with hemolysate in slits made with a razor blade. Paper wicks are placed on the ends of the plate and the plate is sprayed with plastic. The plate is then placed in a horizontal type of electrophoresis apparatus and a current of 20 ma. at 350 volts is allowed to flow for 16 hours. The plastic is then stripped off and the plate stained with amino-schwartz 10B. FIG. 1.- Agar gel electrophoresis. Com- parison of two Hb AS and two normal Hb A individuals. Figure 1 demonstrates the type of pattern obtained in two individuals known to be of hemoglobin type AS and two normal individuals with 2.5 per cent alkali-resistant hemoglobin determined chemically. Note the order of separation—hemoglobin F fastest, followed by hemoglobins A and S. At this pH hemoglobin should migrate toward the cathode and it does. How- ever, the order is the reverse of that obtained by free electrophoresis. This reversal clearly indicated to us that the method depended upon absorption on the agar as well as upon electrophoresis and should be called an electro- chromatographic method. Note also the presence of a distinct spot just ahead of hemoglobin A and also the increased density in the position of hemoglobin S in the two normal individuals. The spot between hemoglobin A and F seems to be present in most bloods examined. This minor component could be identical with Dr. Kunkel's "A2." The slight increase in density in the

DISCUSSION FIG. 2. Ag a r gel e l ectrop ho r es is. Comparison of hemoglobins with va ~ ying degrees of alkali resistance. 177 hemoglobin S position, on the other hand, apparently occurs only rarely,- ill normal individuals. Figure 2 illustrates the results obtained with a sample containing 87 gel cent alkali-resistant hemoglobin as determined chemically. Note the spots in the positions of hemoglobins A and C, on sample 1, obtained from an infant possibly representing genotype AC. Sample 2 was a typical SC combination on paper. The method clearly shows additional minor components in the positions of hemoglobins A and F; the alkali resistance was 2 per cent chemi- cally. Sample 3 appeared as only hemoglobin S on paper and had 1.5 per cent alkali resistance. Note the faint spot in the hemoglobin A position. Sample 4 illustrates what happens when too much hemoglobin is placed on the plate. Figure 3 is a paper electrophoretic pattern at pH 8.6 of an interesting Greek family in which the father gave a typical AS pattern, and the mother gave a typical A pattern, but manifested thalassemia minor hematologically. Two of the children, Catherine and George, showed an SF pattern and have 10 and 15 per cent alkali resistance and hematologically are classified as .... ... .~.... FIGS. 3 and 4. Comparative results of paper electrophoresis (left) and agar gel electrophoresis ( right) from same family.

178 PART III. ABNORMAL HEMOGLOBINS sickle cell thalassemia. One other child, Gus, gave a typical hemoglobin A pattern. Figure 4 presents the picture obtained for this family on agar. Note the presence of small amounts of fetal hemoglobin in the mother, Gus, and the father, as well as a hemoglobin-like component in the hemoglobin A position in the two children with sickle cell thalassemia disease. Figure 5 illustrates a typical paper electrophoretic pattern at pH 8.6 of two typical AS individuals and two individuals whose hemolysates had pre- viously been shown to contain hemoglobins A and D, by solubility tests, lack of sickling, and free electrc~phoresis method. Note the distance of separation of hemoglobin S and D from A. Figure 6 shows the agar patterns for the ..~......... ...~..~. a. 2:': f "":"' .,, ~.,....~.............. . ...... .. ....... . .... . . ~ ~ . ~ FIGS. 5 and 6.- Comparative results of paper electrophoresis (left) and agar gel electrophoresis (right) from same individuals. same individuals. Note the failure of hemoglobin D to separate from hemo- globin A. This provides another means of differentiating hemoglobin D from hemoglobin S. The ability of this technique to detect low concentrations of hemoglobin F makes this method a useful tool. It also is able to separate clearly hemo- globins A, S. and C, and detects the presence of small quantities of other hemoglobin-like components. Indeed, in the light of the possible genetic im- plications, these findings of an A-like hemoglobin in the S and C combination and in the S alone may be the method's most valuable aspect. Dr. A. M. Josephson: I would like to make a point with relation to Mr. Robinson's discussion. We have been doing the same thing in our laboratory. We are using a similar technique, however using pH 6.5, which completely separates the S. A, F. and the other hemoglobin fractions, giving a very simple techr~ique to identify this form of the sickle cell thalassemia disease, rather than through the complicated mechanism of Tiselius electrophoresis.

IMMUNOLOGIC ASPECTS OF THE HUMAN HEMOGLOBINS AMOZ I. CHERNOF`F For many years the antigenicity of human hemoglobin was seriously ques- tioned. Most investigators now agree, however, that human hemoglobin, though weakly antigenic, can induce the formation of specific antibodies in a number of animal species. The immunologic specificity of normal adult and fetal hemoglobin precipitins was first described in 1940 by Darrow and associates) who noted striking qualitative differences between the two types of antibody. These studies were confirmed and extended by Vecchio and J 13arbagallo,2 by Campbell and Goodman and in our own laboratory as well as by several more recent studies carried out in this country and in Europe.5 A number of factors have contributed to the production of more specific as well as more potent antisera in recent years. Carefully crystallized hemo- globins, presumably free from contaminating proteins, have been prepared for immunization purposes by utilizing the techniques of DraDkin and others. Purified material separated by column chromatography and by starch electro- phoresis has also been used in the immunologic studies of the human hemo- globins. A wide range of animals has been tested. The rabbit, guinea pig, arid chicken have been found to be most sensitive as anti-hemoglobin anti- body producers. Furthermore, the recent interest in the use of adjuvants of various sorts has permitted the preparation of more potent sera than hereto- fore available. Finally, better techniques for quantitating antibody antigen reactions have permitted a more exact delineation of the activity of these sera. At present, only lIbs A, S and F have been studied exhaustively by im- munologic techniques although eve have in progress the preparation of anti- sera to Hbs C, D and E as well as to a new hemoglobin type tentatively re- ferred to as Durham No. 1. Only Hb F has been demonstrated to have im- rnlunologic specificity, while antisera to Hbs A and S. in the hands of most workers in this field, cross react completely. Furthermore, Hb C, D, E, H. I and Durham No. ~ have been tested against anti-lIb A and anti-Hb S sera and found to give a positive precipitin reaction. These results suggest there- fore that, with the exception of fIb F. all the other varieties of pigment have very similar or identical antigenic sites on the surface of the globin moeity. These observations do not, however, rule out the possibility that specific dif- fcrences in structure may be localized within individual portions of the pro- tein molecule, or that the differences are either too small to be detected, or are masked by a common antigenic structure. The lack of immunologic specificity between such compounds as Hb A and Hb S is somewhat surprising in view of the marked physical-chemical dis- ~ Some of the studies reported in this paper were supported by United States Public Health Service Grant No. A-1615. 179

180 PART III. ABNORMAL HEMOGLOBINS similarities between these two proteins. However, an explanation for this lack of specificity may be found in recent studies using chromatographic techniques which suggest that Hb S and Hb A differ primarily in the charge of one poly- peptide chain of the molecule.6 For some time we have been using Ouchterlony's technique of double dif- fusion in apart in an effort to obtain more specific information concerning the immunologic activity of the antihemoglobin sera. Similar studies have been reported recently by Ruggieri and Marchi.S The technique is as follows: Small agar plates are prepared which contain three wells as may be seen in figure 1. Solutions of the antigens and antibodies are introduced into the wells FIN. 1. Ouchterlony's double dif- fusion in agar technique. Upper wells ntain Hb A solution; lower well anti-Hb A serum. and permitted to diffuse through the agar. If a precipitin reaction occurs, it is manifest by a distinct line of precipitation within the substance of the agar. The number, position and type of lines which develop provide useful informa- tion for the evaluation of the precipitin reaction. Thus the number of lines indicates the minimum number of reactants in the system. The position of the zone of precipitation depends on the relative concentrations of the antibody and antigen. A line appearing about halfway between two wells would suggest that the reactants are present in equivalent amounts or that one is working in the zone of optimal proportions. If two different solutions are tested against the same antiserum, a continuous line arching as in figure 1 would suggest the same immunologic reactants in each of the upper two wells. Lines which in- tersect or are only partially shared indicate different antigens in the two test solutions. The results of our studies with the hemoglobins may be summarized as follows: Anti-Hb A sera react weakly with Hb F and strongly with all other hemoglobin types. In most instances two or three lines of precipitation become apparent after 10-14 days of reaction. At times the line of precipitation with Hb F seems to merge with one of the three lines seen with Hb A. Anti-Hb F sera react most strongly with specimens known to contain Hb F. but also

IMMUNOLOGIC ASPECTS CHERNOFF FLOODED WITH 6~ air ,,J H GB A ~,¢ fit AGAR PLATE S <~ ~> fLOODED WITH HGB f 5/ ;_ HGB SOLUTION fROM NORMAL I I ADULT ERYTHROCYTES it_ HGB f SOLUT10N PREPARED FROM I CORD SLOOD WANDA VS ISEAR~o PFRODUCED 181 FIG. 2.—Ouchterlony's double dif- fusion in agar Hb-anti Hb system. ( Hbg in the illustration is an alter- nate usage of the more familiar Hb for Hemoglobin.) weakly with many normal adult hemoglobin specimens. When the plates are flooded with an excess of Hb A or Hb F which places the reaction with that particular hemoglobin into the zone of antigen excess and thus inhibits pre- cipitation, the patterns diagrammed in figure 2 are seen. It was postulated that, since normal hemoglobin solutions contained some Hb F. the anti-A sera actually had some anti-Hb F antibodies. Hence the weak reaction between anti-A and Hb F. Similarly, anti-F would be expected to react with the small amount of Hb F present in the adult specimens. When the plates are flooded with Hb A the reaction anti-Hb A-Hb A is suppressed but not that of anti- IIb F-Hb 1~. Similarly, when Hb F is used to flood the plates, reactions be- tween anti-F and the corresponding hemoglobin are suppressed. It is interesting to speculate further as to the nature of the multiple lines seen in the reaction between anti-A sera and Hb A. At first these lines were considered as artifacts since they were slow in developing and quite faint, in spite of the fact that such multiple lines should indicate a minimum of at least the same number of reacting compounds. In the light of recent world faith starch electrophoresis and chromatography of adult hemoglobin solutions, it is apparent that as many as three components may be present normally. It will, therefore, be of interest to test aliquots of these fractions against the antisera, especially with the flooding technique, to determine which, if any, of these lines may be produced by the minor adult hemoglobin fractions. Simi- larly, it would seem important to test these antisera by the immunoelectrophor-

182 PART III. ABNORMAL HEMOGLOI3INS esls technique against all abnormal types of hemoglobin in an effort to demon- · . . ~ . strafe antlgenIc speclilclty. In summary, it may be stated that except for Hb F. no immunologic specif- icity exists between the other forms of human hemoglobins examined to date. The lack of specificity may be due to identical surface configurations of the protein moiety, or to the masking of minimal alterations by a larger common antigenic structure. REFEREN CES I. Darrow, R. R., Nowakovsky, S., and Austin, M. H.: Specificity of fetal and of adult human hemoglobin precipitins, Arch. Path. 30: 873-880, 1940. 2. Vecchio, F~., and Barbagallo, E.: Ricerche sierologiche sul potere antigene di taluni tipi di emoglobina umana normal) et patologici. I. L'emoglobina dell'anemia di Cooley e dell'anemia drepanocitica, Pediatria 58: 481~96, 1950. 3. Goodman, M., and Campbell, D. H.: Differences in antigenic specificity of human normal adult, fetal, and sickle cell anemia hemoglobins, Blood 8: 422033, 1953. 4. Chernoff, A. I.: Immunologic studies of hemoglobins. I. Production of anti- hemoglobin sera and their immunologic characteristics, Blood 8: 399012, 1953. Aksoy, M.: Anti-hemoglobin serum production and its relationship to £etal and adult hemoglobin, Acta Haematologica 13: 226-234, 1955. 6. Ingram, V. M.: A specific chemical difference between the globins of normal human and sickle-cell anaemia haemoglobins, Nature 178: 792-794, 1956. 7. Ouchterlony, O.: Antigen-antibody reactions in gels, Acta Path. et Microbiol. Scandinav. 26: 507-515, 1949. 8. lluggieri, P., and Marchi, M.: Ricerche sulfa specificita antigene delle emoglobine umane normal) ( adulta e fetale ) mediante precipitazione in agar, Estratto dalla Rivista dell'Istituto Sieroterapico Italiano 30: 405013, 1955. 5. DISCUSSION Dr. S. J. Singer: I am not entirely familiar with the work of Campbell arid Goodman, but I had the impression that by quantitative cross reaction pre- cipitin testing, some difference was found between sickle and normal hemo- globins. Dr. Chernoff: Very minor. Dr. Si~zger: Small, but distinct.

STUI)IES ON THE HETEROGENEITY Of ADULT AND FETAL HEMOGLOBINS BY SALTING-OUT, ALKALI DENATURATION AND MOVING BOUNDARY ELECTROPHORESIS DIFFERENTIATION OF HEMOGLOBIN FROM NEWBORN CHILD AND FROM PATIENTS WITH COOL1EY'S ANEMIA YVES DERRI1:N During the past ten years we have studied, in collaboration with Roche and other co-workers, the heterogeneity of hemoglobins, using chiefly salting-out, alkali denaturation and electrophoresis techniques. With the salting-out method applied under carefully controlled conditions, three fractions Oft, f2, f3) have been distinguished in Hb ~ and at least two (~ and as groups, the latter in most cases splitting further into a'2 and a',2) ire Hb A, as shown in figure 1.0 3 4 The upper curves express the solubility 1Eo it_ CDH: /1~1~_ 05 ~ \ 2 _ ; \~ ~ - ! \,'., ~ ~OA=6% ~ I O Trove - I- i~ ~o ~_, 80 Is c 90 20E ~A' _] 1SO E 10 05 C - 80 HE Boo 1sO 100 50 - ma COW Novena By °~£ ! ~ , I _ __\ ~ _, _ ~ ~ ~ Nf' ~ \7~ ~~ '.- ,?0~.. B2% ~^ Groupe, A= 69 % ~ I ~ ~~' , 1 ! ' ' : ~ ! ! ! 1 85 SO c 1 ~ ! , FIG. 1. Above Salting-out curves for carbonmonoxy hemo- globins of normal adults and new- born children. Chromoprotein con- centration = about 0.3 per cent; pH—6.7; temperature - 24° C.; R.D.~. = alkali-resistant fraction; C - salt concentration, expressed in per cent by volume of tile stock salt solution (equimolar mixture of 3.5 M mono- and dipotassium phos- phate); E -~ optical density of fil- trates at ~ — 500 my. Below: Derivative curves. /\E — decrease of optical density for each increment ( /\C) of rne salt concentration C. /! C — 1. Ordi- r~ate AE multiplied by 103. E as a function of salt concentration C; the lower curves are derivatives of the corresponding upper curves.' ~ The changing of fetal into adult: fractions has been followed during the embryonic life and after birth, as partly shown in figure 2. Data of this kind have been obtained for man and for cattle.` s Some abnormal human pigments have been studied by the same method and our chief results are summarized in figures 3 and 4. Hemoglobins S or D, identified by electrophoresis and by Itano's test,9 are easily detected by salting- i8:

184 PART III. ABNORMAL HEMOGLOBINS 20 10 o AS x2/3 10 5 GS _ · ~ 80 p1 90 c Ma 90 c I AS Adu/f e ! 1 \ t2 \/j | at fit O ~\V~Ob ~ it. IS 80 90 C 80 90 C 15 10 ( TV _ ._ _ _~ 5 E ~5 JEg 150 1 FIG. 2. Derivative curves for carbonmonoxyhemoglobin of a normal adult, a new- born child and 3 6-, 74-, 80- and 90-day-old children. Conditions of salting-out as in figure 1. _~03 Adulte Normal , ~ Hemoglobinose I) DA (~6O9J,' [% ~ R DA Labor ) ~ 6 % (D D'~D_54 % . \ 1 1 O ~ Aim__ Aim"._ 50 B5 90 C So es So C TOO- - :! _ , , ,. ~ , A' h—\ SCT \ S RDArdhO,J 2% ~ ~ S'- S.,.. 5' ~ ~ ..\ ~ 65 C 90 dS 90 C - - 80 AS YU 1 1 FIG. 3. Salting-out curves and corresponding derivative curves for carbonmonoxy- hemoglobin of normal adult and of subjects with hemoglobin D trait or sickle cell trait Chromoprotein concentration— about 1.5 per cent. E—optical density of fil- trates at ~ = 550 my. Other details as in figure 1.

HETEROGENEITY OF HEMOGLOBINS A AND F—DERRIEN lgS out experiments. Both are salted out in an identical way, at a slightly lower salt concentration than the fraction a~, and yield two components each (S' and S. D' and D), the second-named (S and D) occurring in much larger proportion.~° ii Hemoglobin F in the newborn child' the fetal-like pigment of patients suffering from Cooley's anemia, and hemoglobin C have practically the same solubility but only hemoglobin C is alkali-labile.)'' i3 The indi- viduality of hemoglobins A, F. S. D and C can be defined by salting-out as well as by electrophoresis to j Ha l to I j E ~~: Solved -AYe E, ~ '4ne~m/e E ~ = ffemog/ob/nose: V9 ~ - N~£ ! ~ :, 1- , ~ _:, . . . _ I\ I I N~ 1 ~ t ~ ~ ~ ' ~ t ~ ~ I \ ................. - N , I ~ ~ _ ~ _ At, (5 _ ~ ~~-~ 05 _~,;_ . ~ \~2 1 ~ ~l \~ 1 ~~ . ~ __ [__ ~ ~ I ~ AD A 4% ~C(ft~ ~~. - Grou,oe C. 66? i ~ . 05 _ . I , /'D A /6 % i - G'oa,oef; I'd i 0 . I . ~ I a:, :' , 1 . ! . - ~ 0 65 90 C 9 of; 0 65 0 C 9 i ~ 0 86 90 C ~ i AL ~ it it, Af ~ ~ f, BE ~(,~,1~ I /50 - 1~24.~2 /50 - ~ I50 I ~ fOl 56 700 ~~ 50t - 11 ~— oft ~ 90 c 9 j OS :~: - ~ ~ 9 - C - 1 OBO 55 SO C - s FIG. 4.- Salting-out curves and corresponding derivative curves for carbonmonoxy- hemoglobin of newborn child and subjects with Cooley's anemia or hemoglobin C disease. R.D.A.— all~ali-resistant fraction of oxyhemoglobin. Salting-out conditions as in figure 3. On the other hand, the presence of fractions of different solubilities in hemoglobins A and :F has presented a quite new and still unsolved problem. The objection has been made that such discontinuities in salting-out curves probably indicate changes in the nature of the solid phase, resulting either from the precipitation of different crystalline forms of hemoglobin or from ~ change in the type of aggregation of the same hemoglobin due to interaction with small molecules or ions in the solvent. Our results do not support such interpretations. The arguments in favor of true heterogeneity of hemoglobins A and :F will be considered under the fol- lowing five areas of discussion. 1. The discontinuities in salting-out curves define an identical number of fractions whatever the nature of the neutral salt (ammonium sulphate or potassium phosphates), or the value of pH (between 6.5 and 8.5), or the

186 PART III. ABNORMAL HEMOGLOBINS ~ r 05 ~: 93 (J: _ - l~t ~ - ~N ~ _ . ~ .\ ~1 ~'.E ''~ ~ \,~91 90 1 ', , , 1: . ~ ' . ~' _ ' ~ PO 30 40 ~ ~ 60 (£ . 01 _ 109S 3 '5- , /~- : ~,~ . , t,O- . \ n ~ . ~ :~ ~ \; Fo ' ~o 80 50 40 50 C 60 1.0 . /093 ,_. - . ' ~N -94S ~ -: ~ --03 \ ~s ~2  ~\ ._ . 1 , 1 , ~ 1_ S 20 logi° 40 50 C 60 ~'\\ ~ ~S 2 ,3 /~ PS 20 '~.S ~: - PO 30 40 50 C 60 tO ~5 ~IG. 5. Crystal solubility curves for horse carbonmonoxyhemoglobin (no. 1 ) and for separated fractions (nos. 2, 3, 4). S and log S are both functions of C, salt con- centration. C = per cent saturation in ammonium sulphate; S solubility expressed in optical density of filtrates; pH 6.4; temperature 24° C. ro 0,5 o at 200 15O nn 5D I,0 f 0,' ~ ~o COHb Ad hum f Fractmn P76 \ I N°V-53 ~ ~ de COH~ N° V-53 _ ~ Solub//~te de cr~st.~u~ _ \~~ So/ub//ite ae cr~(~ux ~ a =; Jo/o \ a _ 65 0/o =:ai__ I 05 \ _ \ ' \t _ \\ ~ ~2 ~a ~ ~ _ < ~~, ,[! , , ~) o 75 80 C 85 75 80 C 85 |~ ~ _. _ 2aoE ~d___ ~ ~ _ ;~]1: (~\ 75 80 C 85 75 80 C B5 a, 66 F`IG. 6. Crystal solubility curves and corresponding derivative curves for normal adult carbonmonoxyhemo- globin No. V-53 and for fraction P 76 separated from it. pH— 6.7; temp- erature 24° C. Other details as in figure 1.

HETEROGENEITY OF HEMOGLOBINS A AND ~ DERRIEN 187 nature of the solid phase, whether amorphous (salting-out curves) or crys- talline (solubility curves or crystal suspensions). Neither the combination with oxygen or carbon monoxide nor the partial or total oxidation into ferribemo- globir~ changes the heterogeneity of a preparation.~5 On the basis of these experimental data, it is deemed unlikely that the discontinuities in salting-out curves represent transition points between the regions of stability of different solid phases of the same hemoglobin. 2. It is always possible to isolate, in a more or less pure state, some of the fractions individualized in the salting-out curves of hemoglobins. As is shown in figure 5, the two major components of horse hemoglobin have been isolated.] The overlapping of fractions al and as of human adult hemoglobin made their separation more difficult. However, figure 6 shows that the fraction al may be partly purified by a single fractionation procedure. On the other hand, it was shown by ultracentrifugal studies that, in a 1 M solution of the primary and secondary phosphate mixture of pH 6.7 used for salting-out experiments, normal adult hemoglobin is homogeneous and totally dissociated into half molecules.* All of these data contradict any interpretation that fractions a, or ~2 were formed by reversible molecular dissociation or association or by salt binding. The fact that one of the fractions keeps the same salting-out fea- ture after being isolated as was seen in the initial mixture leads to the same 1,0 100 50 j Dhd/dsse~m/e ~0 demo9/o61i70se C to /iemo>7/ob/nose C- :05 ~05 'me Na. \\ i, ! P- -ma- SKI I . ~ ~\C(f,] PAD `4. 36;/ ~ Grau,ae C.PQ~ :2l_c,f~ con BO B5 90 C 0~ ! I ! , ~~ 5 08 C ~; go: C 9 5 _ ~£ l !\a2 1\ . ~,.~,,,,~C Grau,ae C. PQ~ :2~_C.fs . ' . ! ! ! ~ :1~r l ADA, 6` If, Gro~,oc C. Big ~ j . ~ ! . ^. A A A , 150 ~ ~ 150 ~ a ~ LIC to o . i 1~ ' : tC4If,, · ~ \: _ Ida | I,,, 90 its FIG. 7.- Salting-out curves and corresponding derivative curves for carbonmonoxy- hemoglobin of subj ects with thalassemia minor, hemoglobin C trait and hemoglobin C-thalassemia association. RD.A. ~ alkali-resistant fraction of oxyhemoglobin. Salt- ing-out conditions as in figure 3. Tonnelat, l., and Derrien, Y.: Unpublished studies.

188 PART III. ABNORMAL HEMOGLOBINS 1, o 1 o,s 0 HE 150 inn .sn 0~ leucem. lymphoide ~ card) ~ .__ , Pa ~ I \ >a , ~ 2, ~[ so 85 C 90 1,0 l eucem. myeloide ~ _ ~ ~ /~iJJ ~ _ _ —\ ~ ~1 _ \2 _ I \; O ! ~ ~ ~ ~ ~ 1 ~ ~ >_ 7 , 80 a, 65 ~ 9 AL 150 06 5L 80 85 vi 90 1 0 0,5 0 ,, I,, ,, I ,,: o 80 8s C TV at I ! 50 00 50 l Jocose Pique I {B/S ~ · I to, · _W \ I ' 1 ~ '~-- 1 1 W~ FIG. 8. Salting-out curves and corresponding derivative curves for carbonmonoxy- hemoglobin of patients suffering from lymphoid leukemia, myeloid leukemia and acute leukosis. Salting-out conditions as in figure 1. conclusion. Thus it has been established that normal human adult hemoglobin contains two components: a: and a2 (or a group). The absence or the very low proportion of al during the first two months after birth (fig. 2), in thal- assemia minor, in hemoglobin C trait (fig. 7), and in some cases of acute leukosis or myeloid leukemia (fig. 8) support the concept of the individuality of fractions al and a2. 3. Research on the blood pigment of the newborn child extends to hemo- globin F the evidence of heterogeneity shown by hemoglobin :'i. The per- centage of alkali-resistant pigment and the percentages of fractions f, identified bar salting-out, have been determined and compared in a large series of carbon- monoxyhemoglobin samples prepared from umbilical cord blood collected at 90 MA To ~0 70L ~0 fool 70t 60 \ o/ o . . \ / To Hip / ~ /o a\ / \ To o o o \ ~ Of 50~ J J AS OND J FMA~J JASONDJ F~A~J JASOND . . . ~ t953 t954 /95 5 FIG. 9. Comparative yield in alkali-resistant fraction (R.D.A. COO) and in fractions of the f group (f To ) of carbonmonoxyhemoglob- in from newborn children at dif- ferent times of year.

HETEROGENEITY OF HEMOGLOBINS ~ AND F—DERRIEN 189 various seasons of the year. As figure 9 shows, 80 + 5 per cent of the pigment is alkali-resistant in every season. On the other hard, the level of the fractions I in the salting-out curves reaches a minimal value of 55 per cent in Tanuary- February, and a maximum, nearly identical with the level of the alkali-resist- ant fraction, in summer. Such observations suggest that the alkali-resistant hemoglobin corresponds essentially to fractions f during the summer arid partly to fractions of the as group during the winter. This hypothesis is con- frmed by comparative studies of the total carbonmonoxyhemoglobin of 14 such I,0 ~ 04 ~ . . . ~32 ~ ~ COHbN-N `` ~Q.~.A. ~ - .~,1 ,1°2-S5 f ~ Cat, isolee : ~ I \ ! ~ \2 ~ ~ \jf; os I Sir, 0,5 _ ~ ~ - ~ tip - ~ ~ - | ~ _ ~ ~ off? _~.~.A. B4~o ~r i i ~ ¢roupef 56% 1 AN? Groupe f 70% I $: I: ~ F ~ ~ I! ~ o o 80 85 90 C 95 80 Us 90 c 95 Af I ~ fief ~ f I 150 ~ A, I 150 t ~ ~ at ~ 11 t fl III . I , J0O _ IN ~ tocE - ~f ~~ 50 / ~ \~3- 50 _ - - ?1 ~ ~ O 4_?_l 1 ~ ~ ~ 11111 O 80 35 SO C 95 80 85 FIG. 11. Salting-out curves and ,, . . corresponding c er1vat1ve curves for newborn carbonmonoxyhemo- globin No. 4—55 (child born in June) and for its isolated alkali- resistant fraction (R.D.A.). Salt- ing;-out conditions as in figure 3. so 80 85 90 C SS FIG. 10.—Salting-out curves arid corresponding derivative curves for newborn carbonmonoxyhemo- globin No. 2—55 (child born in January) and for its isolated al- kali-resistant fraction (R.D.A.). Salting-out conditions as in figure 3. 1,0 j 1,G— ~ ~~ COH.b SN H ~ ~ Proof. R D.A. ~~-~ ~ ~ ~ 1~ ~ NO i, \t,I 0~5 . ~ ~ At as ~ \l tiroupef: 73% | `\~5 Groupe ~ 93% ~ \\f3 9S oh do R 4 - ~ , _ \ to I 1 1 ~ 1, 1 ~ 1 1,,,~1 o , 11 1 ~ l l l l 80 85 90 ~ 95 SO . 85 90 C 95 .~^ ~ ~ I Her ton v So 85 90 C 95

190 PART III. ABNORMAL HEMOGLOBINS samples of blood from newborns and of their alkali-resistant fraction isolated by our technique. As shown in figures 10 and 1l, the alkali-resistant pig- ment can yield up to 30 per cent of fractions as in January and may be prac- tically free of fractions other than f in [une. The reliability of the salting-out curves method can be seen from the quantitative data assembled in table I: the ratio of the fractions f to the total alkali-resistant fraction of the pigment is nearly the same whether the fractions f are determined by salting-out of the total pigment or of the isolated alkali- TABLE I Carbonmonoxyhemoglobin of Newborn Children | Isola ted R.D.A^. Fraction Components ~0 No. Components % a', + at | f Group |~.D.A. Fract. f Group % oFf R D A | f GrouP ~ a ~ ~ a 13 18 21 24 27 29 31 1-55 2-55 4-55 8-55 1-56 1-5/ 4-57 47 31 28 17 21 32 27 42 41 24 35 44 43 26 53 69 72 82 73 64 73 56 56 73 63 55 57 74 77 83 80 86 83 75 77 80 84 77 78 79 80 86 69 83 90 95 88 85 95 70 67 95 ~1 70 71 86 70 85 94 93 90 87 93 72 70 93 70 73 87 26 11 6 5 10 13 7 28 30 6 10 28 27 13 resistant fraction. Such data give experimental evidence of the significance of the proportions of components as estimated by the salting-out method and show that the fractions a., of newborn children contains in some cases two kinds of pigments one of the adult type and the other of the fetal type- as determined by their resistance to alkali denaturation. The individuality of the alkali-resistant pigment of the as group can also be tested by experiments whose results are reported in figure 12. A partial fractionation of the isolated alkali-resistant hemoglobin can be achieved by salting-out and leads to an enrichment either in fractions as or in fractions f. Therefore, both types of pigments have to be considered as different. Furthermore, preliminary attempts to separate the components of the f group allowed us to partly purify some of these, especially fir. 4. The alkali-resistant fraction of normal human adults, isolated by a suitable technique,iS is spectrophotometrically identical to the whole pigment

HETEROGENEITY OF HEMOGLOBINS A AND ~—DERRIEN 191 't t o,; u . . . 80 85 90 C 95 ~ __—,'2 ; f88 = )_ ~ ~ X,- _._ V _ 1 i O 1( 1111 1'11 85 90 C -- ~ PS9-92 : I I , . . ~ · I \~? , 85 So C 95 FIG. 12.—Salting-out curves for the alkali-resistant fraction (~.D.A.) iso- lated from newborn carbonmonoxy- hemoglobin No. 2 5 5 and for its fractionation products F 88, P 88 and P 89-92. Salting-out conditions as in figure 3. and contains about 3 per cent isoleucine.iS This fraction is a mixture of colorless protein, called X, and an alkali-resistant hemoglobin, as shown by its content of iron and nitrogen and by paper electrophoresis followed by brom- phenol stainingi9 (Sg. 13~. In veror~al buffer the protein ~ is slightly slower than hemoglobin C while the mobility of the alkali-resistant pigment is similar to the mobility of hemoglobin A. Beginning with the eleventh month after birth, the spot of protein X is visible in the whole hemoglobin of all subjects FIG. 13. Paper electrophoresis, using apparatus of the Grassman and Hannig type, W-hatmann No. 3—MM; veronal buffer of pH 8.8 and ionic strength 0.02S or phos- phate buff en of pH 6.5 and ionic strength 0.02; bromphenol blue staining. I ~ normal adult carbonmonoxyhemoglobin; II—alkali-resistant f raction isolated from I. III - alkali-resistant fraction isolated from the oxy-derivative (O2TIb) of the same pigment; IV - protein X isolated from III by elusion of its spot.

192 PART III. ABNORMAL HEMOGLOBINS ifs either normal or pathological condition, including that of Cooley's anemia. Like the slow hemoglobin A2 discovered by Kunkel and Wallenius,20 protein X is not detected in the hemoglobin of newborn children. The hemoglobin of normal adults contains about 1.5 per cent of protein X, which passes almost totally into the alkali-resistant fraction. This fraction contains about 50 per cent of alkali-resistant pigment when isolated from oxyhemoglobin and about 75 per cent when isolated from carbonmonoxyhemoglobin. Such data are in accord with earlier observations showing that, for a given hemoglobin prepara- tion, the carbonmonoxy-derivative contains a greater proportion of alkali-re- sistant hemoglobin than the oxy-derivative.2i 2~ O3 ~4 By submitting the isolated alkali-resistant fraction to oxidation, and again to fractionation by alkali denaturation, both types of components are separated as follows: alkali resistance of the hemoglobin is lost by oxidation to ferri- hemoglobin~4 and this is removed as alkaline ferrihemochromogen precipitate; protein X remains in solution. As shown in figure 14, the isolated protein X is nearly homogeneous as shown by electrophoresis in the Tiselius-Svensson apparatus. In phosphate buffer of pH 6.5 and ionic strength 0.2, its mobility is 1.7, a value very close to that of -globulin of serum. In cacodylate buffer of pH 6.5 and ionic strength 0.018, protein X shows the same mobility as the component 1 observed in the hemolysates of all red cells (see table II). In ~ x7 PROTf INS X A OUt TE (466, 1 (136) Pompon I phosphor/qua ~ I r/2= 0,2 . dSC./+I ~SC./-J 360 ma 234 ran PROTEINS X +A D UL OF (4 6 5) tampon AX A phosphoriq,~e I x FJ \< r/2= 0,0 3 ~ As c. /- _ ~ 7300mn canyon c~codyl/q(,e r/2 = Otto Id PR O TEI NE X +ADVL TE (464) ~sc.t—J ~ 27s mn ~ am Con c~codyllque [~/2= 0,018 FrG. 14.—Moving boundary electrophoresis, using a Tiselius-Svensson apparatus. No. 466: isolated protein X in phosphate buffer of pH 7.8 and ionic strength 0.2. No. 465: artificial mixture of protein X and normal adult carbonmonoxyhemoglobin in phosphate buffer of pH 8.2 and ionic strength 0.03. No. 138: normal adult carbonmon- oxyhemoglobin in cacodylate buffer of phi 6.5 and ionic strength 0.018. No. 464: arti- ficial mixture of protein X and normal adult carbonmonoxyhemoglobin. Electrophoretic conditions as in experiment No. 138.

HETEROGENEITY OF HEMOGLOBINS A AND F—DERRIEN 193 TABLE II Components Nature 2 2' 3 ) s' ) s' Apparent mobility U' .10 ( Mean value ) Protein "X" Accompanying protein ( ?) Hb R.D.A. (Thalassemia) Hb A and Hb R.D.A. (Thalassemia ) Hb A and Hb R.D.A. (Hb F) Hb R.D.A. (Hb F`) - 1.7 +4.4 t5.0 +5.1 +5.2 alkaline as well as in acid buffer, this protein moves more slowly than hemo- globir1 A either in moving boundary or paper electrophoresis (fig. 13~. The isoleucine content of protein X is of the order of 4 per cent. The isoleucine content of the isolated pigment is very close to one per cent instead of the zero to 0.3 per cent in hemoglobin A and the 1.5 to 1.9 per cent in hemoglobin F'.~5 ~6~7 Therefore, the alkali-resistant fraction of adult hemoglobin must be different from hemoglobin ~ at least for a major por- tion. Such alkal:-resistant fractions contain components of the adult type fat, a'~. and ads)—partly changed into products of lower solubility Gil, be, 63~- C,5 ~'2 a~ . it, 5.p V,J ~ _ ~1 E C0f/6 Ad 3M E ~ ~r~chona/c~/mores/st~ ~ 1~ EN ~ C0~6 N-~. | o Dam , <,2 p~ A_3~N t. Grou,oe by= 9%~6 ~ ..... ..... ...... f O ~ ~ ~ . ~ ~ O ~ ! ! ~ ~ ~ ~ ~ ! ' . ~ ! ! ! —~ O ~ ! ! ' ! ! ! ' ' ! ~ 60 65 C 90 75 BE B5 C 90 80 85 90 C 95 150 d~ . = /501 1 ~ 1 i HE 1\ 5.p as !_ /00 06 7 ! ' ~ ' 'C ' ~ ' 50 ~ ~ _—/so/Be Be ccJ,7t At. ~ ! _ ~51 ~ \a 2 - ~ ~ ) Grope fig 8 % - ~ ( f I ) | ~ Grouse, 73 % ! 1 h' ~ ~ - C'Y To 1 ~~9OC9s 65 C 90 80 B5 90 C 95 FIG. 15. Salting-out curves and corresponding derivative curves for newborn and normal adult carbonmonoxyhemoglobins and for the alkali-resistant fraction isolated from the latter. SaIting-out conditions as in figure 3. ~ This change in solubility is due to the great dilution of the soluble pigment ob- tained by the denaturation method.

194 PART III. ABNORMAL HEMOGLOBINS o,s o 50 HE 50 ~r~c~ior'~/ca/mores~s~ E Croci. ~/ca//r~ores/s~ E is Iso/ee dame/~;e. _ i~o/ee dome/~ge ~ ~ ;, `85 |(C9/lb Ad§S1{ ~ ff:O//b Adders In, ~ ~ w-/1 BY. 05 -~A ~ W-NtO% Q5 I dam, _ 42' | \tai' | ~ Na'£ 1 - ,,,,,: 1 t --\ ~ - ~ - Gyp 'Poe i, 30 % ~ , Groove fop 3& ,~: , Grove fop 60 O O ! ~ I ~ 1 1 . ~ ~ 1 1 1 ! 1 BY 85 90 C B5 90 C 80 85 90 C 95 tOO ~ s30(~,~' 'l lid 150 AL 100 So 1 1 .~ 1,, /5o HE ~50 50 `~a1 ~r~ct/o/7 d/cd/iito~es/s '' _~_ Jso/ee dume/d~7~re. I' (6086 Ad809( ,l ~ WN~x . drama)_ ~:~ Aid Otter: ~ ~ ~ ~59 4~50i ~~ O BE 90 C 65 90 C 85 90 C 95 J FIG. 16. Salting-out curves and corresponding derivative curves of alkali-re- ~istant fractions isolated from mixtures of various proportions of normal adult and newborn carbonmonoxyhemoglobins Salting-out conditions as in figure 3. —and very little, if any, fetal hemoglobin (fig. 15), as checked by experi- ments which show that, after addition of only 5 per cent of newborns' hemo- globin to the whole pigment of an adult, the proportion of components f reaches 30 per cent of the alkali-resistant fraction isolated from the hemoglobin mixture (fig. 16~. This invalidates the initial assumption of Roche, Derrien and Roques3 7 that 5 to 10 per cent of most soluble fractions of the total normal adult hemoglobin corresponds to fetal hemoglobin. Almost the whole of these fractions are alkali-labile and the alkali-resistant hemoglobin of normal adults are mainly salted-out as components of the a type. Studies of the alkali-resistant hemoglobin of subjects suffering from sickle-cell anemia lead to a similar conclusion.~3 2s 5. Electrophoresis of hemoglobins in the Tiselius-Svensson apparatus, in cacodylate buffer of pH 6.5 and very low ionic strength (~2 0.018), provides a new set of arguments supporting the heterogeneity of these pig- ments.29' 30 In a concentration of 1.0 per cent and with a potential gradient of 7 voltsicm., normal adult hemoglobin gradually separates into two major com- ponents named 3 and 4, as shown in the top row of figure 17. Peak 3 is fol- lowed by very small peaks 1 and 2 of slower components, of which the slower has been identified as protein X (fig. 14~. Peak 4 is heterogeneous. As electro- phoresis proceeds, peak 4 separates into 4' and 4, and a small peak named 5, slightly more rapid, appears., T Berry and Chanutin3t have recently confirmed the resolution of two components (A and B) in human adult hemoglobin. According to these authors, a third faster boundary (I), presumably identical to our component 5, represents the concentration gradient of the cacodylate ions.

HETEROGENEITY OF HEMOGLOBINS A AND F—DERRIEN 195 ADVLTE (1387 N-N (444) 3 ~ 3,¢ 3 150mn 2 1 ~ ~ . 234 mn 211 _ _ -~L 24 0 me ~5 ~ 25mn 31 ~5 A.COOlEX il 5 3 (423) 2 Jo 2 me' 1017mn 111 arc I-) 2 41- l330 mn 3 ; 55 a sc.~-' FIG. 17. Moving boundary electrophoresis of human carbonmonoxyhemoglobin No. 138, from normal adult; No. 444, from newborn child; No. 423, from patient with Cooley's anemia. Patterns recorded at different times in the L. K. B. Tiselius-Svensson apparatus. Standardized experimental conditions: concentration of hemoglobin solu- tions ~ 1 per cent; cacodylate buffer of pH 6.5 and ionic strength 0.018; potential gradient = 7 ~roltlcm.; temperature - 2° C. Diagrams obtained with hemoglobin of newborn children (fig. 17, middle row N-N) show only a small amount of component 3 and always record im- portant quantities of a component which often separates into peaks 4 and 5 during long-run experiments. Hemoglobin of subjects suffering from Cooley's anemia has an electrophoretic behavior very different from that of the new- born child, as is shown in the bottom row of figure 17. Component 3 pre- dominates, whereas component 4 is usually absent. Peak 5 splits into 5 and 5' and ~ new component 2' appears. Thus this pathological hemoglobin can Basil be differentiated from both hemoglobin :F and hemoglobin A in dilute cacody- late buffer. Peak 2' had previously been found only in hemoglobin preparations from tl~alassemia blood (thalassemia major or minor). Components 3, 4 and 5 show apparently identical mobilities in the hemoglobin of the adult, the rlew- born child and patients with Cooley's anemia (table Il). Additional evidence of this identity is furnished by electrophoretic analysis of artificial mixtures of normal adult hemoglobin with the hemoglobin of newborn children or of patients suffering from Cooley's anemia (fig. 18~. The components of each hemoglobin are superimposed according to their respective mobilities, without appearance of a new peak and without disappearance of any peaks present in

196 PART III. ABNORMAL HEMOGLOBINS ~ s 1 N-N (444) 133 Omn 3 ADUL HE I ' A.COOLEX (44n (4 5 1) ADUL TE + N-N (455) 1310rnn 3 ADULTE 5 +A.COOLEY 4+s 3 1~0 An s asc.(_)_ FIG. 18.—Moving boundary electrophoresis of human carbonmonoxyhemoglobin No. 444, from newborn child; No. 451, from normal adult; No. 447, from patient with Cooley's anemia; No. 455, a mixture of carbonmonoxyhemoglobins Nos. 444 and 451; No. 449, a mixture of carbonmonoxyhemoglobins Nos. 451 and 447. Standardized experimental conditions as in figure 17. the initial hemoglobin preparations. All these preparations have been sub- mitted to moving boundary electrophoresis in the form of 100 per cent car- bonmonoxyhemoglobin. They remain in this form during the electrophoresis run, as checked spectrophotometrically at the end of the experiment. They were homogeneous in the ultracentrifuge in the same buffer used for the electrophoresis. The observations in the foregoing five sections support the hypothesis of a true heterogeneity of hemoglobins. Furthermore, they present some evidence that hemoglobins of the fetal type in newborn children and in patients with Cooley's anemia are not identical, even though these cannot be differentiated by electrophoresis either in cacodylate buffer of pH 6.5 and ionic strength 0.1 or in phosphate buffer of pH 8.2 and ionic strength 0.03, or by salting-out curves or by chemical methods. Whatever the nature of the difference for which immunological evidence has just been reported* comparative electro- phoretic studies of newborn and Cooley's-anemia hemoglobins of similar yield of an alkali-resistant fraction lead to the conclusion that resistance to denatura- tion by bases can be common to different hemoglobins. On the other hand, the same electrophoretic component can be common to various hemoglobins (table II). For example, the peak 3 includes an alkali- ~ Diacono, fI.: Compt. rend. Soc. de Biol. (in press).

HETEROGENEITY OF HEMOGLOBINS A AND ~ DERRIEN 197 resistant hemoglobin in the blood of Cooley's anemia patients and art alkali- iabile hemoglobin in normal adult blood. According to these data it appears that, as sensitive as is the technique of electrophoresis in dilute buffer, it does not allow a precise definition of the exact number of components of any nor- mal or pathological mixture in red cells. As seen in sections 3 and 4 above, solubility and alkali denaturation experiments lead to a similar conclusion about the sensitivity of the salting-out method by which resolution of alkali- labile and alkali-resistant fraction of the a type are not obtained. However, it is of interest to point out that the degree of heterogeneity shown in normal adult or newborn hemoglobin is of the same order of magnitude whether de- termined by salting-out or by electrophoresis. For example, the resolution of two main components bat and a2 or 3 and 4) in adult hemoglobin is obtained by both methods. Summary. 1. Herx~oglobin A contains at least two components and hemo- globin ~ includes fractions of the a., and f solubility types, the f type in much larger proportion. 2. The alkali-resistant hemoglobins of normal adults and of subjects with sickle cell anemia are of the a solubility type, at least for a considerable por- tion. Their isoleucine content is significantly lower than that of hemoglobin F. 3. Hemoglobins of newborn children and of patients with Cooley's anemia which yield similar amounts of alkali-resistant fraction are definitely different as shown by electrophoretic behavior in cacodylate buffer of very low ionic strength. REFEREN CES 1. Roche, J., Derrien, Y., Reynaud, J., Laurent, G., and Roques, M.: Sur l'heterogene- ite des hemoglobines. I. Technique d'etablissement des courbes de solubilite et premiers essais de fractionnement, Bull. Soc. Chim. biol. 36: 51, 1954. 2. Derrien, Y., and }loche, J.: Etude comparee des hemoglobines du nouveau-ne, de l'enfant et de l'homme adulte par la methode des courbes de relargage (salt- ing-out), First International Congress of Biochemistry, Cambridge 19-25 Au- gust 1949, Abstracts of Communications, p. 368. 3. Roche, J., Derrien, Y., and Roques, M.: Sur l'heterou eneite des hemoglobines . . . . ~ , ~ humaines chez l'adulte et le foetus, Compt. rend. Soc. biol. 146: 689, 1952. 4. Roche, J., and Derrien, Y.: Les hemoglobines humaines et les modifications phy- siologiques et pathologiques de leurs caracteres, Le Sang 21: 97, 1953. 5. Derrien, Y.: Individualisation, characterization and fractionation of the serum proteins by salting-out, Svensk. Kem. Tidsl~r. 59: 139, 1947. 6. Derrien, Y., Laurent, G., and Reynaud, J.: Individualisation et caracterisation des constituents proteiques du serum par la methode des courbes de relargage, J. de chem. phvs. 48: 651, 1951. 7. Roche, J., Derrien, Y., and Roques, M.: Sur le remplacement des hemoglobines de type adulte par celles de type foetal au cours du developpement embryonnaire et apres la naissance, chez l'homme et chez le boenf, Bull. Soc. chim. biol. 35: 933, 1953. 8. Roche, J., Derrien, Y., and Roques' M.: Sur les hemoglobines du boeuf et cur

198 PART III. ABNORMAL HEMOGLOBINS leurs transformations au cours du developpement foetal et apres la naissance, Compt. rend. Soc. biol. 146: 694, 1952. 9. Itano, H. A.: Solubilities of naturally occurring mixtures of human hemoglobin, Arch. Biochem. 47: 148, 1953. 10. Roche, J., Derrien, Y., Gallais, P., and Roques, M.: Sur les hemoglobines des sang a drepanocytes (hematies en faucille ou falciformes), Compt. rend. Soc. biol. 146: 889, 1952. 11. Derrien, Y., Cabannes, R., Laurent, G., and Roche, J.: Sur l'individualisation de l'hemoglobine D, Compt. rend. Soc. biol. 149: 1350, 1955. 12. Derrien, Y., Laurent, G., and Roche, T.: Sur l'indi~ridualisation de l'hemoglobine C chez des porteurs homozygotes et heterozygotes, Compt. rend. Soc. biol. 149: 641, 1955. G., and Borgomano, M.: Identification des hemoglobines et natholo~ioues Dar leurs courbes de relar~a~e et leur 13. Derrien, Y., Laurent, humaines normales <~ alcalino-resistance, XVme Congres des Pediatres de Langue Francaise, Mar- seille 23-25 May 1955, Communications p. 69. 14. Itano, lI. A.: The hemoglobines, Ann. Rev. Biochem. 25: 331, 1956. 15. Laurent, G., Bouscayrol, S., Dunan, J., and Borgomano, M.: Influence de la methemoglobinisation sur les courbes de relargage des hemoglobines humaines de type adulte et de type foetal, Compt. rend. Soc. biol. 150: 738 (no. 4), 1956. 16. Derrien, Y., Laurent, G., and Bouscayrol, S.: Sur une variation saisonniere de la teneur en fractions f (relargage) de l'hemoglobine de nouveau-ne, Compt. rend. Soc. biol. 150: 397 (no. 2), 1956. 17. Derrien, Y., Laurent, G., and Borgomano, M.: Isolement par denaturation frac- tionnee et etudes des courbes de relargage de la fraction alcalinoresistante des carboxyhemoglobines de nouveau-nes, Compt. rend. Soc. biol. 149: 137, 1955. 18. Derrien, Y., Laurent, G., and Roques, M.: Recherches sur la fraction alcalino- resistante de l'hemoglobine de l'homme adulte normal, Arch. sci. biol. 39: 650, 1955. 19. Derrien, Y., Laurent, G., and Borgomano, M.: Sur une proteine accompagnant l'hemoglobine de l'homme adulte et sa concentration dans la fraction alcal- inoresistante isolee de cette derriere, Compt. rend. Acad. Sci. 242: 1538, 1956. 20. Kunkel, H. G., and Wallenius, G.: New hemoglobin in normal adult blood, Sci- ence 122: 288, 1955. 21. lIelpern, M., and Strassman, G.: DifFerentiation of fetal and adult human hemo- globin, Arch. Path. 35: 776, 1943. 22. Betke, K., Richarz, H., Schubothe, H., and Vivell, O.: Beobachtungen zu Krank- heitsbild, Pathogenese und Aetiologie der akuten erworbenen hamolitischen Anamie (Lederer-Anamie), Klin. ~7ochnschr. 31: 373, 1953. 23. Singer, K., and Fischer, B.: Studies on abnormal hemoglobins. 7. The composition of the non-S fraction in sickle-cell anemia bloods. A comparative quantitative study by the methods of electrophoresis and alkali denaturation, [. Lab. Clin. Med. 42: 193, 1953. 24. Derrien, Y., Laurent, G., and Roche, J.: Sur la resistance a la denaturation alcaline des hemoglobines et de leurs derives, Compt. rend. Soc. biol. 147: 1934, 1953. 25. van der Schaaf, P. C., and Huisman, T. H. J.: The amino-acid composition of human adult and foetal carbonmonoxyhemoglobin estimated by ion exchange chromatography, Biochim. et biophys. acta, 17: 81, 1955. 26. Rossi-Fanelli, A., Cavallini, D., De Marco, C., and Trasatti, F.: Emogrlobina fetale. I. Analisi quantitative degli amino-acid) della emoglobina umana fetale

HETEROGENEITY OF HEMOGLOBINS A AND F DERRIEN 199 cristallizzata ed alcuni accorgimenti tecnici, Boll. Soc. it. Biol., sper. 31: 328 (fasc. 3-4), 1955. 27. Lissitzky, S., and Laurent, G.: Dosage de la leucine, de l'isoleucine et de la phenylalanine des proteines par chromatographie sur papier, Bull. Soc. Chim. biol. 37: 1177 (No. 11), 1955. 28. Roche, J. Derrien, Y., and Laurent, G.: Sur les fractions alcalinoresistantes des hemoglobines dans les enemies drepanocytaires, Compt. rend. Soc. biol. 147: 957, 1953. 29. Derrien, Y., and Reynaud, J.: Sur l'heterogeneite electrophoretique de l'hemo- globine humaine (sujets adultes normaux), Compt. rend. Soc. biol. 2~47: 660, 1953. 30. Derrien, Y., and Reynaud, J.: Etude comparee des hemoglobines humaines nor- males et pathologiques par electrophorese en veine liquide en tampon cacodylique dilue. Distinction des hemoglobines d'anemiques de Cooley et de nouveau-nest XVme Congres des Pediatres de Langue F`ran~aise, Marseille, 23-25 Mai 1955, Communications p. 178, and Compt. rend. Soc. biol. 149: 1595, 1955. 31. Berry, E. R., and Chanutin, A.: Electrophoretic studies of red cell extracts of stored blood, J. Clin. Invest. 36: 225, 1957. DISCUSSION Dr. H. J. Itano: Some time ago I suggested the possibility that these dis- continuities in the salting-out curve might arise from phase transitions.) ~ Dr. Perutz obtained different crystalline forms of human hemoglobin at different concentrations of phosphate.3 ~ think this possibility as well as heterogeneity should be considered in the interpretation of salting-out curves. Unfortunately, this possibility complicates the interpretation considerably. Perhaps both heter- ogeneity and phase transitions are contributing to these curves. The other point has to do with the electrophoresis. I showed that in two or three hours we w-ere able to separate components which differ by one charge. The diagrams of Dr. Derrien show separation after 20 hours. The amount or separation shown must correspond to a minute fraction of a charge. On a structural basis a fractional change in charge can result from a small change in pK of one of the groups. However, experimentally one must consider the fact that in these very prolor~ged runs convective disturbances may occur either from diffusion of salt from the electrodes, from the delta boundaries being pushed into the bottom section during reverse compensation, or from electrolysis at one of the electrodes. REF`EREN CES 1. Itano, H. A.: Solubilities of naturally occurring mixtures of human hemoglobin, Arch. Biochem. Biophys. 47: 148, 1953. (See also No. 2j 2. Oc:ston. A. G.. and Tombs, M. P.: An ambi~uity in the variable-solvent solubility ~ , , . test: tiomogene~ty of p,-lactoglobulin, Nature 178: 200, 1956. 3. Perutz, M. F~., Liquori, A. M., and Eirich, F.: X-ray and solubility studies of the haemoglobin of sickle-cell anaemia patients, Nature 167: 929, 1951. Dr. Derrien: The objectior~s formulated by Dr. Itano on the interpretation

200 PART III. ABNORMAL HEMOGLOBINS of the salting-out curves and electrophoresis diagrams I presented should not be accepted, in view of the following facts. In view of the electrophoresis diagrams of 1 per cent normal adult hemo- globin in very low ionic strength cacodylate buffer at pH 6.5, it is very dif- ficult to deny the individuality of the two major constituents 3 and 4 of Hb A. These begin separating within two hours and the process of their separation as a function of time does not show any convection disturbances. In accordance with Dr. Svensson, with whom I discussed this problem, the electrophoretic individuality of these two constituents has to be considered as certain under the experimental conditions. The difference in net charge seems to be only Q.3, which would exclude the possibility of the faster moving component (No. 4) being an intermediate form of oxidation. Furthermore, our experimental results are not in favor of the hypothesis that: discontinuities in salting-out curves represent changes in the solid phase of the same hemoglobin. As I said earlier the solubility curves of crystals show exactly the same discontinuities as the salting-out curves of solutions (precini- tation of solid amorphous phases>. Therefore, I think that if it were a matter of transition between different crystalline forms, it would be surprising that we should get exactly the same pictures in both types of experiments.

HETEROGENEITY OF HEMOGLOBIN AND METHOD OF ISOTOPIC BIOSYNTHESIS GEORGES SCHAPIRA, JEAN-CLAUDE DREYFUS AND JACQUES KRUHl The establishment of criteria of purity of proteins is a classical and a dis- appointing problem. No protein can satisfy all the different tests of purity and even the significance of the purity of a protein is now challenged. It seems that the reverse problem, the significance of the criteria for heterogeneity, must now be discussed, and eve shall propose a method of demonstrating protein heterogeneity which we have applied to hemoglobin. When a solution of a protein is studied by physicochemical methods one may observe associations with the solvent, associations with the salts and even association of the protein with itself. On the other hand, some proteins may show dissociation. In either case the solution of a homogeneous protein appears to contain more than orate component. This difficulty and its possible solution' were discussed in Professor Derrien's paper.3 When one prepares or stores a protein some "denaturation" may occur. Chemical and biochemical methods may then reveal heterogeneity which is not native but results from the manipulations or the time lapse involved. We are never able to claim the absence of some degree of alteration from the native state and it becomes difficult to claim that a heterogeneity is native. These difficulties also arise with hemoglobin. A part of the protein may be slightly modified during its fractionation the modification possibly affecting either globirl, demonstrated by partial denaturation, or the prosthetic group, as illustrated by a partial transformation into me/hemoglobin. Thus, a new compound differing f rom the native hemoglobin is artificialyv created and apparently two hemoglobins are found. D we thought that tile use of isotopes would overcome this difficulty by pro- viding results unmodified by manipulative artifacts. We have developed a method with very general scope which can be applied to the analysis and metabolic study of many proteins and other biological compounds.4 Our method is the following: a radioactive element is incorporated either in vitro or in vivo into the protein. Then, after the protein has been isolated, purified and submitted to fractionation, the isotopic composition i.e., the specific activity (S.A.) of each fraction is measured. 1. If the two fractions show different specific activities, the presence of at least two different proteins in the native state is certain. These proteins have different metabolic behavior. 2. If the two fractions obtained always display the same specific ac- ~ These studies were supported by grants from the Caisse Nationale de Securite Sociale (France) and the Institut National d'Hygiene (France). et this paper was presented by Dr. Schapira. 201

202 PART III. ABNORMAL HEMOGLOBINS tivities, we cannot conclude that actually more than one protein is present; one may be an artifact. A conclusion concerning heterogeneity cannot be made on this evidence alone. The validity of this method is based on two assumptions: 1.) It has been generally considered that our usual chemical fractionation methods should not separate isotopes. However, Piez and Eagles in i955 observed that incorpora- tior~ of C i-; ir~to amino acids slowed their movement on chromatographic columns with possible resultant errors. Fortunately there is an extremely close relationship between the ratio of C7'i to total carbon in the labelled molecule and the degree of resolution of the labelled from the unlabelled compounds. Perhaps the best answer to this possible criticism is the parallelism of results with radioactive hemin, globin, and iron in the biological and pathological variations which shall be presented below. 2.) We must also assume that, within certain limits, no impurities are present. This Still be discussed later. METHODS Rabbit hemoglobin was prepared according to Roche, et al.6 Human hemo- globin was crystallized according to DraLkin.7 The globin was separated according to Anson and Mirsky.8 It was col- lected ore the filter and washed until the acetone became completely colorless. The globin was lyophilized and hydrolyzed by refluxing for 48 hours in 3,000 volumes of 6 :N HC1. Hemin was precipitated from the acetone filtrate by addition of water and concentrating with heat. After precipitation it was washed by centrifugation with water and alcohol. In some experiments the heme was determined as pyridine hemochromogen, in others it was crystallized (lTischer9), weighed after evaporation of the solvent on the planchets, and counted. The DNP-glycine was isolated according to Perronei° after two or three passages on celite and was controlled by paper chromatography according t Biserte and Osteux,~ and A. Levy. In some experiments the DNP-glycine was oxidized by Fan Slyke's reagents and converted to barium carbonate. In another series the radioactivity was determined directly in a thin layer with the Geiger-Muller counter, following calorimetric measurement and cor- rection for thickness. Radioactive iron in the hemin solution was measured with a scintillation counter; hemin was converted to barium carbonate. In other experiments iron, after wet combustion of the hemin, was measured as -ferrous orthophenan- throline complex. Radioactive iron was measured with a Geiger-Muller counter following electrolytic plating.~3 In vitro synthesis was obtained according to Walsh, ef al.,~4 London, ei a1.,~5 and Borsook, et al.~6 Hemoglobin labelled in vivo was obtained by injection of the labelled compound and bleeding at different times, usually the fourth . r ~ ~ ~ c ay alter injection. The various hemoglobins were subjected to several fractionation procedures.

ISOTOPIC BIOSYNTHESIS SCHAPIRA, DREYF`US .AND KFLUH 203 H2lman cord blood hemoglobin was fractionated by: 1. ~ alkali denaturation, according to Singer, et al.,l; and 2.) ion exchange chromatography, according to lIuisman arid Prins.iS We separated both an "adult" and a "foetal" fraction. Adult rabbit hemoglobin was studied by: 1.) electrophoresis, according to Tiselius' method carried out at various pH,~9 ire which we analyzed anionic and cationic fractions; 2.) progressive alkali denaturation, according to Ramsey,20 which allowed the separation of one alkali-resistant and one alkali-sensitive fraction;~3 3.) paper chromatography, which gave us, using a pH 4.3 solution as mobile phase, elongated spots of hemoglobin displaying a narrowing from which we cut and eluted the two parts of the spot ;~ and 4.) alumina chroma- tography, which enabled us to demonstrate the adsorption of about 25 per cent of rabbit hemoglobin which was easily eluted by phosphate solutions. Adolf human hemoglobin was fractionated by: 1.) alumina chromatography, (Kruh2~), by which we demonstrated the adsorption of about 10 per cent of human hemoglobin; 2.) ion exchange chromatography, according to Huisman and Prins;~8 and 3.) starch electrophoresis, according to Kunkel and Wal- lenius.23 JESUITS 1. Haman cord bloody. Partial alkali denaturation permitted the sepa- ration of foetal and adult hemoglobin containing in vitro-incorporated Fed and alpha C1I' glycine (table I). A better separation can be obtained by ion exchange chromatography. The ratios of the S.A. of the fractions were 0.25 J for the hemin and 0.47 for the globin glycine.04 ~5 TABLE I DIFFERENTIAL INTCURPCRATI^N GE FEW AND CJJ' GLYCINE INTO HEMIN ARID GLOBIN OF HE~CGT CBINS FROM HUMAN CORD BLOOD UNDER in vitro CONDITIONS Experiment Number Peso Cats Hemin Hemin (c. min/mM iron) (c. min/mM hemin/8) r/s r/s CJJI Globin ( c. min / mM glycine ) r/s 9 10 11 12 13 a 13 b 13 c 14 15 Mean 0,34 0,42 0,43 0,45 0,58 0,66 0,69 0,60 0,55 0,61 0,40 0,65 0,62 0,75 0,59 0,64 0,67 0,82 0,66 0,54 + 0,04 0,626 + 0,037 0,86 0,78 0,75 0,85 0,74 0,51 0,87 0,77 0,70 0,53 0,73 6 + 0,04

204 PART III. ABNORMAL HEMOGLOBINS 2. Adult hemoglobin, rabbit and human. Adult rabbit and adult hu- man hemoglobins were studied after both in vivo and in vitro incorporation of radioactive iron or glycine. The hemoglobin which displayed the higher S.A. after the first stage of incorporation in vivo was called "Hemoglobin I," while that with the lower S.A. was called "Hemoglobin II." The fractions with the higher S.A. are resistant to alkali denaturation, non-adsorbed by alumina, anodic in electro- phoresis and slower moving on paper chromatography. Adult rabbit hemoglobin. More than fifty fractionations have been made of hemoglobin containing radioactive iron incorporated in vivo. A different isotopic composition in the two fractions was always observed, the ratio of specific activities always differing significantly from unity ~ table II ~ . The average ratios obtained with the various fractionation processes reported above have been: 1.11 with progressive alkali denaturation; 1.46 with electrophoresis in an alkaline buffer; 1.17 with electrophoresis in an acid buffer; 1.25 with alumina chromatography; and 1.27 with paper chromatography. The magni- tude of the ratio is an indication of the resolution obtained by the various methods. TABLE II DIFFERENTIAL INCORPORATION OF FE )0 INTO HEMCGI OBIN OF ADULT RABBITS in qJi¢V(J Normal Fractionation Anemic Experiment # ~ Ratio of S. A. ~ Experiment # ~ Ratio of S.A. Alkaline denaturation Paper chromatography Alumina chromatography Electrophoresis: pH 9.0-9.4 pH 5.0 10 4 11 3 1.11+0.041 6 1.27 + 0~07 1 4 1.25 + 0.06 7 1.46 + 0.19 1.17+0.05 1.22 + 0.0g 1.55 + 0.15 1.44 + 0.06 Similar results were obtained in preliminary assays using radioactive gly- cine (table IV) to be discussed later. Adult rabbit hemoglobins labelled in vitro with radioactive iron and subse- quently fractionated by alkali denaturation or by alumina adsorptions showed ratios of specific activities which are the reverse of those of fractions obtained from hemoglobin similarly labelled za vivo. Thus the in vitro kinetics of iron incorporation is the reverse of that found in viva. The experiments in which reticulocytes alone were used were exactly duplicated with a medium with blood rich in reticulocytes to which was added a suspension of bone marrow, liver extracts with hematopoletic action, vitamin Bee and folinic acid. Under these modified conditions, we found that the ratio of the specific activities of

ISOTOPIC BIOSYNTHESIS—SCHAPIRA, DREYFUS AND KRUH 205 TABLE III )IFFEREN-TI.~L INCCKPCR.\TION- CF FE ~ ) IN-TO HE.~-GECEIN OF ANEMIC ADULT RABBITS 272 Intro Experiment Number Ratio of S.A. Without effectors + bone marrow + liver extract + vitamin Bee folinic acid 22 7 4 s 3 TABLE INS 0.71 + 0.31 1.33 +0.35 1.20 + 0.16 1.19 ~ 0.11 1.34 ~ 0.02 INCORPORATION OF RADIOACTIVE GLYCINE ALTO RABBIT HEMOGLOBIN to in. by - :~< A_ Ratio I / II Globin Hemin Powder Glycine . I II I 11 I 1 1 1 1 2,070 1,260 64 46 778 648 28 20.5 2,480 1,030 860 59 51 1 1 1 5,900 24,6()0 209 266 47,000 116.500 24,800 35,400 534 724 II 1,810 78,000 163,5G0 Hemin 1.64 1.20 1.20 0.65 0.70 Fractionations were carried out by alumina adsorption (unpublished data). Three days incorporation. - Globin Powder 1.40 1.36 1.16 0.78 0.74 Glycine 1.36 0.60 0.71 the two fractions is above unity again, close to that obtained by in viva iron incorporation and the reverse of that found with blood alone in vitro. Thus, these added factors modified the nature of the synthesis, hemoglobin I syn- thesis predominating (table III). Similar results severe obtained in our pre- liminary assays after labelling with radioactive glycine (table IV). Blood samples were taken for fractionation at various times after the in- . . . section of Fess. We found that the ratio of specific activities remained con- stant between the third and sixtieth day, but the reversed ratio was observed between the third and eighth hour. In fact, hemoglobin II was the first to become radioactive several hours after the iron injection, but this radioactivity subsequently decreased whereas that of hemoglobin I continued to rise until

206 00 75 ~0 PART III. ABNORMAL HEMOGLOBINS S.A ---fib Wh I ,\ 25 '' '`` /,, \4,- i,"' . , . lo 15 20 2S FIG. 1. Time curve of incorporation of Few into hemoglobins of a normal adult rabbit in ciao. Ordinates repre- sent specific activities of the two frac- tions in arbitrary units, the number 100 being the highest value obtained in the experiment. The numerals of the abscissa represent hours. (Repro- duced from Ciba Foundation Sympos- ium on Porphyrin Biosynthesis and Metabolism, J. & A. Churchill Ltd.) the two curves crossed and finally became parallel. Thereafter the ratio of specific activities remained constant.-7 (Fig. 1~. Adult human hemoglobin. We were able to study the synthesis of adult hemoglobin, isolated in crystalline state, in several patients, including five with acute leukemia.2S O9 In vitro experiments were also carried out with blood rich in reticulocytes.2S All of these studies were done with Few. In the synthesis of hemoglobins in persons without blood disorders we found the same pattern as was found in rabbits (table V). The pattern of incorporation of radioactive iron is different for the two fractions. The S.A. curves intersected in the same manner but the time of intersection was later in the human blood. Nevertheless, we were unable to observe any decrease of S.A. of the hemoglobin II. (:Fig. 2~. Hemoglobin synthesis in blood of patients with blood disorders was similarly studied in vitro. We found a predominance of hemoglobin II synthesis in the reticulocyte-rich blood of patients with various types of anemia. In starch electrophoresis according to Kunkel and Wallenius,~3 the main spot of adult human hemoglobin labelled in vitro with Cats glycine showed isotopic hetero- geneity of the hemin and globin. (See also the paper by Dr. Kunkel).30 I00 S.A ~ - Hb II Hb 75 SO 25 FIG. 2. Time curve of incorpora- tion of Flew into hemoglobins of a normal adult man ~n ~vi~vo. As in fig. 1, ordinate represents arbitrary units, the number 100 being the highest value obtained. Note, however, that in this figure the numerals of the abscissa represent days. (Reproduced from Ciba Foundation Symposium on Porphyrin Biosynthesis and Metabolism, J. & A. Chu rebill Ltd. ) i, 4:~ . , , ~,- O ~ 4 6 ~ DAYS

ISOTOPIC BIOSYNTHESIS—SCHAPIRA, DREYFUS AND KRUH 207 TABLE V DIFFERENTIAL INCORPORATION OF FESS INTO HEMOGLOBIN OF ADULT HUMAN BEINGS in Gino Ratio of S.~. II Controls 1.20 1.29 1.26 1.30 Acute Leukemia 0.83 0.77 0.50 0.44 0.43 ( in remission ) Ire vivo, the synthesis of the other hemoglobin is usually smaller in acute leukemia. In the five cases of acute leukemia iron incorporation took place according to a modality the reverse of that seen in normal subjects (table Nib. This disorder of hemoglobin formation was still present in ~ patient in com- . . . plete remission. DISCUSSION AND CONCLUSIONS 1. Human cord blood. There is general agreement that there are at least two hemoglobins, one foetal and one adult, in human cord blood. We have observed a different metabolic pattern for each of them. Iron and carbon are incorporated into hemin and glycine is incorporated into globin to a lesser degree in foetal than in adult hemoglobin. The differences are more apparent if the fractionation is performed by ion exchange chromatography according to Huisman, the ratio for hemins being 0.25 and that for the globins 0.47. Our results prove that the rate of synthesis of adult hemoglobin is higher than that of the foetal hemoglobin. Nevertheless the total amount of foetal hemoglobin synthesized during the experiments is higher, since it accounts for 75 per cent of the total. The synthesis of foetal hemoglobin is less active than that of adult hemo- globin, but this cannot explain the greater effectiveness of cord blood than adult blood in the incorporation of radioactive iron.3i 2. Adult hemoglobin. Impurities. The present method, like the meth- od of isotopic dilution, assumes the absence of impurities with different S.A. The following discussion is generalized to include incorporation of both glycir~e (incorporated into both hemin and globin) and iron in the hemo- globins of both man and the rabbit. Possible discrepancies due to non-heminic iron may be eliminated by the controls, the S.A. of hemin and of hemoglobin being the same.

208 PART III. ABNORMAL HEMOGLOBINS Porphyrin was not a contaminant, no fluorescence being detected in either the hemin or the hemoglobin solutions. The presence of proteins other than hemoglobin could not explain the differences of S.A. in the fractions studied. Five precipitations of the same hemoglobin with large losses did not change the S.A. The ratio of S.A. of the two fractions of rabbit hemoglobin remained constant for many days and the contamination of the hemoglobin with a protein of constant S.A. seems highly improbable. In several instances the S.A. of the hemin was measured, ex- cluding possible contaminating proteins as a source of error. Catalase and foetal hemoglobin are chromoproteins which could contami~ nate adult hemoglobin and should be discussed here. Our method of prepara- tion gave hemoglobin free of catalase activity, which is originally present at a concentration only 0.3 per cent of that of hemoglobin. Theorell, et al.32 ob- served the same order of magnitude of S. A. in catalase from erythrocytes and in hemoglobin. No evidence on the occurrence of foetal hemoglobin in adult rabbit blood is available. Less than ~ per cent of foetal hemoglobin may occur in normal adult human hemoglobin.33 On Amberlite XE-64 chromatography of adult human hemoglobin labelled in vivo with Few, less than 1 per cent emerged as a small preliminary frac- tion, which may represent the foetal hemoglobin. Its S.A. was less than that of the main fraction, which could be assumed to be the adult hemoglobin. If we submitted the major fraction to alumina adsorption the two parts had the usual ratio of S.A. Significance of heterogeneity of adult hemoglobin. Biochemical as- pects. We must discuss the significance of the heterogeneity of adult hemo- globin both from a hematological and a biochemical point of view. The same results are obtained whether the hemoglobin is labelled with carbon or with iron or if the glycine is isolated from the labelled globin. We can therefore claim that adult hemoglobin of both rabbit and man is a mixture of at least two hemoglobins which are metabolically different and whose physicochemical properties allow a true separation. The present methods of fractionation do not give complete separation of the chemical individuals, only an enrichment of each f faction being obtained. The chemical differences between the fractions are unknown as is the number of components. :For instance, as observed by Prins and Huisman,34 the apparent heterogeneity of hemoglobin as shown by chromatography on alumina depends on the experimental conditions, but the effectiveness of the separation, as we have demonstrated, may be tested by the differences of S.A. of the fractions obtained. Our method of isotopic biosynthesis reveals metabolic heterogeneity of foetal and adult hemoglobin from cord blood, and the metabolic heterogeneity of normal adult hemoglobin. It is a tool which allows us to follow the progress of fractionation and to recognize resolution. It may be applied to other pro-

ISOTOPIC BIOSYNTHESIS—SCHAPIRA, DREYFUS AND KRUH 209 teins and biological compounds, having been applied by others in the study of nucleic acids.35 Its primary advantage is that it eliminates the difficulties in interpretation arising from artifact heterogeneity. Hematological aspects. l hese hemoglobins have different metabolic be- haviors. Examples of different metabolism of protein according to its location are known, as, for example, the differences between hepatic and erythrocyte catalase observed by Theorell. We have observed, with Coursaget and F. Schapira,:3~; differences in the rate of incorporation of N45-labelled glycine into cardiac and gastrocnemius myosin of the rabbit. In these cases the pro- teins were perhaps the same but present in different organs. In the case of hemoglobin eve can assert that these hemoglobins are different and we can assume that these different hemoglobins are contained in different red cells. However, a distinction is to be made between red blood cells with a brief or aberrant span of life and those with a normal life span. The finding of a temporary decrease in the radioactive iron content of rabbit hemoglobin II leads to a major conclusion—the possible existence of erythrocytes levity a very short life span. This is evidenced by the very rapid rise in the specific activity of hemoglobin II, followed by a rapid fall. (Fig. 1~. The existence of erythrocytes with a short life span would account for the findings of London West Shemin and Rittenber~` and of Neuber~er.3S An. . ~ . . . a . ~ . . , O ~ When N45-labelled glycine is given, an early peak of excretion of heavy ni- trogen is found in the stercobilin isolated from the feces. The differences which remain after the disappearance of the short-lived red cells may be explained by new, as yet unproven, hypotheses. Different red cells in a pathological state may be synthesized in loci other than the bone marrow and their presence may explain the higher ratio of S.A. in the phenyl- hydrazine anemia of rabbits* and the reversed ratio in acute human leukemia. This explanation cannot apply to either the normal state or to in vitro syn- thesis, and we have to assume the heterogeneity of the population of red blood cells,40 different red cells containing the different hemoglobins which are syn- thesized at different rates. Alternatively, the heterogeneity may be intra-cellular, the same cell syn- thesizing two or more hemoglobins at different rates from different pools of precursors. We have indirect evidence for such a mechanism; in sickle hemo- ~lobin trait both Hb A and Hb S seem to be contained in the same red cell. To summarize: We considered whether the heterogeneity of a protein like hemoglobin is or is not native. A possible answer to this question can be ob- tained by the method of isotopic biosynthesis which establishes the hetero- geneity of adult hemoglobin while it avoids the difficulties of interpretation posed by the numerous possible artifacts introduced by the physical and chem- ical techniques of fractionation. -a These facts were indirectly confirmed by Benard, Dantchev, and GaJdos.39

210 PART III. ABNORMAL HEMOGLOBINS REFERENCES 1. Colvin, J. R., Smith, D. B., and Cook, W. H.: The microheterogeneity of proteins, Chem. Rev. 34: 687, 1954. 2. Roche, J., and Derrien, Y.: Les hemoglobines humaines et les modifications physi- ologiques et pathologiques de leurs caracteres, Le Sang 24: 97, 1953. 3. Derrien, Y.: These Proceedings. 4. Schapira, G., Dreyfus, J. C., and Kruh, J.: Metabolisme different de deux hemo- globines chez un meme animal adulte etudie a ['aide du fer radioactif, Compt. rend. Acad. sci. 230: 1618, 1950. 5. Piez, K. A., and Eagle, H.: Systematic edect of Cti-labeling on ion-exchange. Chromatography of amino acids, Science 122: 968, 1955. 6. Roche, J., Derrien, Y., and Moutte, M.: Solubilite dans les solutions salines con- centrees et caracteres specifiques des hemoglobines sanguines, Bull. Soc. chim. biol. 23: 1114, 1941. 7. Drabkin, D. L.: A simplified technique for a large scale crystallization of human hemoglobin. Isomorphous transformation of hemoglobin and myoglobin in the crystalline state, Arch. Biochem. 21: 224, 1949. 8. Anson, M. L., and Mirsky, A. E.: Protein coagulation and its reversal, J. Gen. Physiol. 13: 469, 1930. 9. Fischer, fI.: Hemin, Org. Synth. 21: 53, 1941. 10. Perrone, J. C.: Separation of amino-acids as dinitrophenyl derivatives, Nature 161: 513, 1951. 11. Biserte, G., and Osteux, R.: La chromato',raphie de partage sur papier des dini- trophenyl-amino-acides, Bull. Soc. chim. biol. 33: 50, 1951. 12. Levy, A. L.: A paper chromatographic method for the quantitative estimation of amino acids, Nature 174: 126, 1954. 13. Schapira, G., Dreyfus, J. C., and Kruh, J.: Recherches sur la biochimie de l'hemo- globine a ['aide du fer radioactif. I-Fractionnement des hemoglobines de lapin adulte par denaturation alcaline, Rull. Soc. chim. biol. 33: 812, 1951. Walsh, R. J., Thomas, E. D., Ghow, S. K., F~luharty, R. G., and Finch, C. A.: Iron metabolism. Heme synthesis in ~vitro by immature erythrocytes, Science 110: 396, 1949. 15. London, I. M., Shemin, D., and Rittenberg, D.: Synthesis of heme i~z vitro by the immature non-nucleated mammalian erythrocytes, J. Biol. Chem. 183: 749, 1950. 16. Borsook, H., Deasy, C. L., Haagen-Smit, A. J., Keighley, G., and Lowy, P. H.: Incorporation in q~itro of labeled amino acids into proteins of rabbit reticu- locytes, J. Biol. Chem. 196: 669, 1952. 17. Singer, K., Chernoff, A. I., and Singer, L.: Studies on abnormal hemoglobins. I— Their demonstration in sickle cell anemia and other hematologic disorders by means of alkali denaturation, Blood 6: 413, 1951. 18. Huisman, T. H. J., and Prins, H. K.: Chromatographic estimation of four dif- ferent hemoglobins, J. Lab. and Clin. Med. 46: 252, 1955. 19. Schapira, G., Kruh, J., Bussard, A., and Dreyfus, J. C.: Recherches sur la bio- chimie de l'hemoglobine a ['aide du fer radioactif. II-Fractionnement des hemo- globines de lapin adulte par electrophorese, Bull. Soc. chim. biol. 33: 822, 1951. 20. Ramsey, M.: A comparative study of hemoglobin denaturation, J. Cell. & Comp. Physiol. 18: 369, 1941. Kruh, J., Dreyfus, J. C., and Schapira, G.: Recherches sur la biochimie de l'hemo- globine a ['aide de fer radioactif. III-Fractionnement des hemoglobines de lapin adulte par chromatographie sur papier, Bull. Soc. chim. biol 31: 773, 1952.

ISOTOPIC BIOSYNTHESIS SCHAPIRA, DREYFUS AND KRUH 211 22. Krnh, l.: Recherches sur la biochimie de l'hemoglobine a ['aide de fer radioactif. IV—F`ractionnement des hemoglobines de lapin adulte par chromatographie sur alumine. Comparaison avec les resultats obtenus par d'autres methodes, Bull. Soc. chim. biol. 34: 778, 1952. 23. Kunkel, H. G., and Wallenius, G.: New hemoglobin in normal adult blood, Sci- ence 122: 288, 1955. 24. Dreyfus, J. C., Schapira, G., and Harari, M.: Incorporation du fer radioactif in ~itro dans les p;lobules roue:es du nouveau-ne (hemop:lobine foetale et hemo- globine adulte), Compt. rend. Soc. biol. 148: 1798, 1954. 25. Schapira, G., Dreyfus, J. C., Kruh, J., Paoletti, C., and Boiron, M.: Heterogeneite metabolique de l'hemoglobine de sang de cordon, etudiee a ['aide du fer et du glycocolle radioactifs, Compt. rend. Soc. biol. 149: 1178, 1955. 26. Kruh, J., Dreyfus, J. C., and Schapira, G.: Recherches sur la biochimie de l'hemo- globine a ['aide de fer radioactif. NT—Biosynthese des hemoglobines z~z ~vitro Bull. Soc. chim. biol. ]5: 1181, 1953. 27. Dreyfus, J. C., Schapira, G., and Kruh, J.: Phases initiales de l'hemoglobinopoiese etudiees a ['aide du fer radioactif, Compt. rend. Soc. biol. 147: 782, 1953. 28. Schapira, G., Dreyfus, T.- C., and Kroh, I.: Synthese des hemoglobines humaines marquees ~n ~z~o tnommes normaux et atteints de leucemie aigue) et i~z ~ztro, Compt. rend. Soc. biol. 147: 780, 1953. 29. Schapira, G., Tubiana, M., Dreyfus, J. C., Kruh, l., Boiron, M., and Bernard, ~.: Recherches sur l'anemie des leucoses aigues. I—Metabolisme du fer dans la leucose aigue etudie a ['aide du Fer 59, Revue Hematol. 9: 3, 1954. 30. Kunkel, H. G.: These Proceedings. 31. Jensen, W. N., Ashenbrucker, H., Cartwright, G. S., and Wintrobe, M. M.: The uptake in qJitro of radioactive iron by avian erythrocytes, J. Lab. Clin. Med. 42: 833, 1953. 32. Theorell, H., Beznak, M, Bonnichsen, R., Paul, K. G., and Akeson, A.: On the distribution of injected radioactive iron in guinea pigs and its rate of appear- ance in some hemoproteins and ferritin, Acta Chem. Scand. 5: 445, 1951. 33. Chernoff, A. I.: The human hemoglobins in health and disease, New Engl. J. Med. 253: 322, 1955. 34. Prins, H. K., and Huisman, T. H. T.: Some observations about the heterogeneity of hemoglobin in aluminum oxide chromatography, Biochim. et bioph. acta 20: 570, 1956. 3 5. Bendich, A., Russell, P. J., Jr., and Brovvn, G. B.: On the heterogeneity of the desoxyribonucleic acids, J. Biol. Chem. 201: 305, 1953. 36. Schapira, G., Coursaget, J., Dreyfus, J. C., and Schapira, F.: Incorporation dans la myosine du glycocolle marque a l' azote. Roles de l' atrophie et de la topo- graphie musculaire, Bull. Soc. chim. biol. 35: 1309, 1953. 37. London, I. M., West, R., Shemin, D., and Rittenberg, D.: On origin of bile pig- ments in normal man, ;. Biol. Chem. 184: 351, 1950. 38. Neuberger, A.: Studies on mammalian red cells, Ciba Foundation Conference on Isotopes in Biochemistry, p. 68. j. & A. Churchill, Ltd., London, 1955. 39. Benard, H., Dantchev, D., and Gajdos, A.: Augmentation de la proportion d'hemoglobine alcalino-resistante au cours de la reparation de certaines enemies experimentales chez le lapin, Le Sang 25: 78, 1954. 40. Ponder, E.: L'idee d'heterogeneite appliquee aux globules rouges, a leurs stromas et aux cellules en general, Revue Hematol. 11: 123, 1956. . . .

STARCH ELECTROPHORESIS OF HEMOGLOBIN: FINDINGS IN THALASSEMIA SYNDROMES PARK S. GERALD * For the past year we have employed starch electrophoresis in the investiga- tion of hemoglobin from patients with various thalassemia syndromes and from their families. The electrophoretic technique which has been used is a modification of that described by Kunkel.i We have utilized this tool primarily ire the study of families in which the propositi were found to have thalassemia major. The diagnosis was based upon the presence of a severe hemolytic anemia faith marked anisocytosis and poi- kilocytosis, nucleated red cells in the peripheral circulation, and hepato- splenomegaly. When not rendered impossible by frequent transfusion, the presence of any other abnormal hemoglobin was excluded by electrophoresis. We have examined twenty-three parents from twelve such families, all of Mediterranean or European ancestry. All but one of these parents showed microcytosis of the red cells. The single normocytic parent was subsequently eliminated from the study when paternity was excluded on the basis of blood groups. Of the twentv-twn Parents with microcvtosis. all hilt one .~howed an . . . 1 A ~ · ~1 - elevation in the Ao fraction. l his single exception was further distinguished bv the presence of an abnormal hemoglobin and consequently will be described as a separate entity. The remaining parents, twenty-one in all, comprise a homogeneous group with the following three features in common micro- cytosis, elevated As content and parentage of a thalassemia major child. The simultaneous presence of microcytosis and elevated As content appears at present to be diagnostic of thalassemia trait. As I shall subsequently demon- strate, microcytosis alone, even though hereditarily transmitted, is not a suffi- . . . client criterion. , ~ . o an, . . The degree of elevation of the A, content in the thalassemic parents is of considerable interest. In figure 1 are depicted our most recent data. The values for the normal adults are the results of single analyses, while those for the thalassemic parents are the means of paired analyses. The use of duplicate analyses in the thalassemic population was necessitated by the increase of standard deviation with increase in As content. With two exceptions the thalassemic adults comprise a homogeneous croup. O O ~ Excluding these two ex- ceptions, the average As content is 4.8~. This is surprisingly close to twice 2.5% the median value for the normal group. The two thalassemic parents with As content between 3 and 4~70 would seem to be different from the homogeneous majority. We have studied in detail one of these individuals. Over a two months period the As level has Public Health Service Research Fellow of the National Heart Institute. 212

ELECTROPHORETIC FIN1)INGS IN THALASSEMIA GERALD 213 O Normal Adults an LLI c' tar Lie to to o 00 00 000 0 000 000000 2.0 3.0 40 5.0 A2 CONTENT goof total Hqb) 53 Parents of Thalassemia Major Children · ooO FIG. 1.- Distribution of the Hb AS content among normal adults and among parents of thalassemia major children. remained constant within the limits of experimental error. Oxide relative has been found with microcytic red cells. His electrophoretic fractionation has similarly given a value for As in this intermediate range. The possibility thus exists that hermetic factors control the degree of elevation of the A2 level in thalassemia trait. I should now like to discuss briefly the parent with microcytosis and an abnormal hemoglobin which I previously mentioned. The propositus for this family clinically appeared to have typical thalassemia major. Examination of the hemoglobin revealed 80: to be alkali-resistar~t and the A2 component comprised an increased amount of the non-alkali-resistant fraction. No ab- normal hemoglobin could be demonstrated. The child's mother exhibited the microcytosis, anisocytosis and poikilocytosis typical of thalassemia trait. Upon electrophoresis of this woman's hemoglobin at pEI 8.6, an abnormal component faith S-like mobility could be detected Dig. 2~. This abnormal hemoglobin was unusual in that it constituted only 11~ of the total pigment present. We have examined further members of this patient's family and have ob- served four additional instances of the same abnormality. All haste had normal or slightly decreased total hemoglobin levels and identical morphologic changes ir, their erythrocytes. All have had a normal As content and 10-12~ of an S-like hemoglobin. Sickling tests were negative in the three individuals FIG. 2. Starch electrophoretic pat- tern of the hemoglobins in the "Le- pore abnormality" and in sickle cell trait. Electrophoresis of the cyanmet- hemoglobin derivatives at pH 8.6 (ve- ronal buyer, ionic strength 0.05 ) duration of run, 40 hours; tempera- ture, 8 ° C.; distance from origin to front of Hb A', 11.0 cm.

214 PART III. ABNORMAL HEMOGLOBINS tested. The consistent Ending of microcytosis and an abnormal hemoglobin in the previously published hemoglobinopathies. Since we have not uniquely characterized the abnormal component by physico-chemical means, we have refrained from designating it as a new hemoglobin. Instead we are calling the syndrome the "lLepore abnormality" after the family in which it was found. From the f~miIV data available, Aid. 3.), it would appear reasonable to ~ , low concentration distinguish this abnormality from any of conclude that the "Lepore abnormality" is transmitted as a single gene defect. Nonetheless, it is still conceivable that this syndrome, like hemoglobin H disease, may be the result of the interaction of two genes. In particular, the failure to demonstrate the S-like hemoglobin in the propositus could be evi- dence of segregation of such genes. FIG. 3. Family pedi- ~ree depicting the here- ~ ` - 1 1< E; ~ ~ data ry tr ansm~ salon of the "Lepore abnormality." O Normal (~)Thalassem~a trait Lepore abnormality Thalassemia major During the course of our studies on thalassemia, we encountered two patients (one of Filipino and one of Italian ancestry) who appeared to have very mild forms of thalassemia major. Electrophoretic analysis proved them to be instances of hemoglobin H disease. These cases were unusual in that the Hb lI comprised only 6~ of the total pigment. Except for this, the find- ir~gs were identical with those of previously published reports; namely, a mild hemolytic anemia, anisocytosis and poikilocytosis of the red cells, formation of inclusions in the erythrocytes after vital staining with cresyl blue, and spleno- megaly. The abnormal hemoglobin moved faster than Al at pH 8.6, had an isoelectric point below pH 6.5, and was denatured by freezing. In addition to the presence of the abnormal hemoglobin, the electrophoretic patterns were unusual in exhibiting a reduction in the amount of the As frac- tion. Quantitation revealed that in both cases the slow component constituted only 1.0% of the total pigment; i.e., less than half the normal amount. Such low levels have otherwise been observed only in very severe iron-deficiency anemia or with high concentration of alkali-resistant hemoglobin. Neither of these conditions was present in our patients. In view of the current hypothesis that hemoglobin H is found only in com- bination with thalassemia trait,2 we had expected an increase in the As con- tent as evidence of the presence of the thalassemia gene. Investigation of the family of the Italian patients partially explained this discrepancy. In figure 4 we have diagrammed the relationship of the family members contacted to date. On the mother's side are have been unable to detect any abnormalities. On

ELECTROPHORETIC FINDINGS IN THALASSEMIA GERALD 215 l l | O Normal (by ( I I 1- [A ~ I I ~ Hgb H ~ b b ~ ~ ~ Microcytosis FIG. 4. Family pedigree of an hereditary microcytosis associated with Hb H disease. the father's side we have found three members with microcytosis and a mini- mal degree of anemia. Excepting for the propositus, the electrophoretic pat- terns are qualitatively and quantitatively normal. The hereditary microcytosis of: this family accordingly differs from thalassemia trait in lacking an elevation of the A2 content. We have thus encountered three electrophoretically distinguishable varieties of hereditary microcytosis. That usually present in thalassemia major families of Mediterranean extraction and characterized by an increased A2 content we have called thalassemia trait. That form without increased A, content and so far observed in a single fIb H family perhaps warrants the designation "pseudo-thalassemia." Finally, that form associated with an S-like hemo- globin will be named after the abnormal hemoglobin if it proves distinct from hemoglobins S and D. REFERENCES 1. Kunl~el, H. G., and Wallenius, G.: New hemoglobin in normal adult blood, Sci- ence 122: 288, 1955. 2. Motulsky, A. G.: Genetic and haematological significance of haemoglobin H. Na- ture 173: 1055, 1956. 3. We are indebted to Dr. F. H. Gardner for permission to report the hematologic findings in this family. DISCUSSION J9r. A. Josephson: I just want to make a point about the A2. We have studied approximately 35 people with thalassemia, proven, and although many fall into a range comparable to the severity of the disease, there are a number of exceptions. Some of our thalassemia minimas have had elevation of the A2 level. One complicating feature is that we have studied the A2 in other hema- tologic disorders, primarily red cell disease, and found that in three out of six patients with pernicious anemia the A2 has been elevated. Dr. /1. G.~ll/lo~zllsky: I would first like to confirm Dr. Gerald's results. We have studied six patients with Hb H-thalassemia disease from three fami- lies. A diminished amount of the As component was found. I would like to turn now to another component we found in normal hemo- lysates. These studies were fully presented at the meeting of the American Federation for Clinical Research in Atlantic City.

216 PART III. ABNORMAL HEMOGLOBINS Using paper electrophoresis at acid pH (pH 5.8-6.0) and staining with bromphenol blue, we found a distinct minor component with diminished mo- bility in the hemolysates of normal subjects and in patients with a variety of disorders (fig. 1~. This component was markedly diminished in cord blood. Artifacts as source of the component could be ruled out. We first thought we were dealing with a heterogeneous hemoglobin. FIG. I. Paper electropho- resis at acid pH of hemoly- sates of normal subjects and patients. Location of minor component discussed in text is indicated by arrows on the normal column. ( Note that line of origin and relative size are not same for left column as for other two.) Initially this hypothesis appeared substantiated when the component was found to have identical electrophoretic mobility to Hb H at acid pH (fig 19. However, when electrophoretic runs were done at multiple pH's, it was found that the component under study had a different electrophoretic behavior from Hb H near the neutral and alkaline range. The component could be concen- trated by salting out at 72.5~ ammonium sulfate. No precipitate was ob- tained from cord broad at this ammonium sulfate concentration (fig. 2~. PIG. 2.—Paper electrophoresis of ( NfI4) .,SO4 precipitates of hemo- lysates.

DISCUSSION 217 Searching for a non-hemoglobin red cell constituent to explain the fraction, we were fortunate in that Dr. Huex~nekens (Department of Biochemistry, University of Washington J recently had isolated a yellow protein from human red cells which had TPNH-activated methemoglobin reductase activity. At all pH's studied, this yellow protein had electrophoretic mobility identical to that of the fraction under study (fig. 3~. We concluded therefore that the component represented methemoglobin reductase. —2 —1 my 1 o +2 ` \ HbA Hb H\ \ b.U b.3 t.U f . ~ b.u b.0 Y.u pH - ~ _ O- ~ + - — ~ O- ~ + .~ Component I I I I I I ~ 1 1 ~ 6.0 7.0 8.0 9.0 pH Methemog robin __- Reductase I ~ 1 ~ 1 1 1 · 1 1 1 1 6.0 7.0 8.0 9.0 pH FIG. 3. Relative electrophoretic mobilities of the subject component, hemoglobins A arid H and methemoglobin reductase. Dr. J. L. Cook: In his remarks made earlier concerning our column chrom- atographic procedure, Or. Morrison mentioned our results with regard to thalassemia. I would like now to show two typical ion-exchange chromato- grams of patients from the Hematology Clinic at the University of Rochester. Figure 1 shows thalassemia minor. The forerunnir~g component has its peak in the position characteristic of the feta1 peak from cord hemoglobin. The second peak is in the region of the principal component of normal adult hemoglobin. The last peak is in the area of the final peak of normal adult hemoglobirl, but differs in being significarltly larger than in the normal. Attention is invited to the peak at tube 40, which we find characteristic of thalassemia minor, though it is absent in thalassemia major and in normal hemoglobin. Figure 2 shows typical thalassemia maj or. Note the sharp peak which moves in the fetal region. This component represents about 30 per cent of

218 800 700 - o 600 - ~S 500 he z 400 o Z 300 o ~ 200 o LL I 100 PART III. ABNORMAL HEMOGLOBINS FIG. 1 _ _ THALASSE M IA M I NOR ALKALINE RESISTANT HO 3 % 0: by my G V I 1 - 1 1 ~ _ = REGION OF NORMAL / l ADULT PEAK - o J I I, I , I , \ 1 0 20 30 40 50 60 TUBE NUMBER 900 800 700 600 500 400 300 200 THALASSEMIA MAJOR ALKALINE RESISTANT HS 24 % AREA I 30 % —- REGION OF NORMAL ADULT PEAK 1 1 1 11 FIG. 2 ol J ~ 10 20 30 40 50 60 TUBE NUM 3ER (Figure 1 appears in Federation Proceedings 16: 765, 1957, and is reproduced by permission of the publishers.) the hemoglobin and, when isolated, is alkaline-resistant. In thalassemia major, as well as thalassemia minor, our technique demonstrates an increase in the final component in the tube 54 region. Dr. 1. M. London: One of the questions that has arisen, and has in fact been asked by some of the people here in the audience, is concerned with the question of heterogeneity, particularly in normal human hemoglobin. Do the various groups that are using different techniques—whether these be zone electrophoresis, column chromatography, or paper electrophoresis feel that the minor components that they are observing are the same? Would any of you care to comment on the possible identity of the minor components that you note by these various techniques? Dr. H. G. Kinked: This is not a question that can readily be answered. The minor components that we have studied are observed with a wide variety of electrophoretic procedures. Free solution electrophoresis demonstrates the A2 component particularly in patients with thalassemia, as Drs. Josephson and Singer have shown. Paper electrophoresis also shows the minor components when special conditions are employed. There also is a correspondence between these and the subfractions obtained by Drs. Morrison and Cook. We are at present exchanging samples to resolve some quantitative differences, particu- larly in one of the components. The heterogeneity of hemoglobin noted in low ionic strength cacodylic acid does not correspond well with these other studies. Here the quantities involved are far greater than the minor subfractions re- ported. Dr. Martin Morrison: I think it is clear that the rapidly-moving com-

DISCUSSION 219 ponent on our column and Dr. Kunkel's rapidly-moving electrophoretic com- ponent at pH 8.6 are very close to being the same, both in percentages and in electrophoretic movement. I do not think there is any question about this. With regard to the slow-moving component, we did get Dr. Kunkel's sample and it does move very much like ours. It is exactly like ours in position. The percentages may be off by virtue of the partial oxidation of Hb A, let us say one iron atom per molecule of normal A, which might give us a small in- crease. That is, if the major component A is partially oxidized, it would move in the same region as the third component. As much as 10 per cent of our third component could be composed of such molecules and we may not have detected that. Dr. Alfred Charting: One obtains three distinct components when a red cell extract of a freshly-drawn sample of blood is analyzed by free electro- phoresis in a cacodylic acid buffer at pH 6.5 and 0.06 M concentration. The fastest-moving component is colorless and has been designated as F. The two remaining components designated as A and B comprise about 60 and 30 per cent, respectively, of the total area of the pattern. These two components appear to be hemoglobins. It has been observed that the slow-moving com- ponent (B) gradually disappears when ACD blood is stored at 4°. It can be demonstrated that this component may be restored to its original concentra- tion by the addition of inosine. We believe that the evidence obtained in our laboratory supports the concept of heterogeneity of hemoglobin in the red cell of a normal individual. REFERENCE 1. Berry, E. R., and Chanutin, A.: Electrophoretic studies of red cell extracts of stored blood, J. Clin. Invest. 36 225, 1957.

OBSERVATIONS OF THE AMINO ACID COMPOSITION OF HUMAN HEMOGLOBINS WILLIAM H. STEIN With the availability within the last ten years of more or less routine micro J methods for the amino acid analysis of proteins, it has become feasible to use these methods to compare proteins from different sources. A recent instance of such an application can be found in the work of Brown, Sanger, and Kitai1 and of Harfenist and Craig on the insulins of different animal species. It was clearly shown by amino acid analysis that the insulins of the cow, the pig, and the sheep were distinct chemical compounds, after which Sanger and his colleagues) were able to pin-point the exact nature of the structural dif- ferences. Amino acid analysis began to be applied in a similar way to the problem of the several hemoglobins very soon after the existence of the problem was appreciated, that is, soon after Pauling, Itano, Singer, and Wells3 showed that sickle cell and normal human hemoglobin could be differentiated electro- phoretically. Schroeder, Kay and Wells4 analyzed these two types of hemo- globin in 1950, but they were unable to correlate the electrophoretic proper- ties with any clear-cut differences in amino acid composition. More recently, hemoglobin preparations have also been analyzed by Rossi-Fanelli and his colleagues, by Huisman and his coworkers ~ and by Dustin, Schapira, Dreyfus, and Hestermans-~[edard.S As a result of these investigations it has become clear that fetal hemoglobin (Hb F) has an amino acid compo- sition markedly different from that of the adult hemoglobins. It has also be- come clear that normal adult hemoglobin and the various abnormal varieties are very similar in composition, although Huisman has claimed that hemo- globin C contains slightly more lysine than do the others. The work to be described in this brief communication has been done jointly with Dr. Darrel H. Spackman, Dr. R. David Cole, and Dr. Stan- ford Moore.9 All the hemoglobin samples studied were prepared by Dr. Henry Kunkel at the Rockefeller Institute by the use of zone electrophoresis on starch.~° In figure 1 is shown an effluent curve obtained from an acid hydrolysate of sample of hemoglobin ~ obtained f tom a normal adult white male. The amino acid analysis was performed by chromatography on columns of sulfonated polystyrene resin using the automatic recording equipment developed in our laboratory by Dr. Sparkman. Single analyses were performed on 22-hour and 70-hour hydrolysates of each sample of hemoglobin. It is of particular interest to note (fig. 1) that the base line is perfectly flat between methionine and leucine, the area in which isoleucine would show 220

AMINO ACID COMPOSITION STEIN 221 ._ .g ~ ' '' 'C " "" ' 'a Cat..,,, -..0 ~3 ~C:':,,'' C''~. 'in ~ c:::: ~ d a: ·_ C:: . ...... - ~-4 C:>— ~ it ., o ~ tD C; I:: be, "_~ no _ -, - . . {:: , 8 ~ =;~ =; 3 ~ M~suap IvoTldo Id = 53 ~ , ~ d O L ~ ~ ~ - - ~ ~ ' in. . . . .,., ,, ,.~ , ,; O lo.< ~ is s c;, -c . ~2 · 4 ~ ,~ q' ._ cc: a' A: ._ "c <-. ~ lo: .. ............. . . -. ·. -.-.-. ~ t It i I I t t 1 1 1 8 A, A ~ ~ ~ _ ~2 A; .. .... .. c .. oT ~¢ ;~ ~ - o ~ ~ :c - ~ - oL ct ~ ~ ~ £ ~ _ —. oo o _ Ct o cn C~ . _ ~ V s:; — V — ~ ~ V X ~ ~ o o.= ~ o r s =( 0 z 0 ~ O ;- \8 . 0 , c~ ~ 0 `> 2 b~ ~ . _ s" o ~ ° o ~ ~ V o Z; C C ° =- O Z o ~; ~ o ') ~ a, 0 - 0 := ~ ~-= ~ V - ~ X ~ == ~ =° ¢ >~ O ·- s °o ~ ._ ~ C C) ~ ~ ~ :- · _ ~ s:: - ~ w~ 0 0 ~ C) ~ S ~ o C~ ~: o

222 PART III. ABNORMAL HEMOGLOBINS up if it were present. Apparently, this sample of electrophoretically purified hemoglobin A is devoid of isoleucine, because the constancy of the base line, which is characteristic of the recorder procedure, coupled with the log scale of the plot, renders the method very sensitive to small quantities of amino acids. In fact, an amount of isoleucine equivalent to one residue per molecule of hemoglobin, less than 0.2 per cent, would appear as a peak about 173 of the size of the methionine peak. The absence of isoleucine was somewhat surprising, inasmuch as all the hemoglobin samples analyzed by Schroeder and by Huisman were reported to contain from 0.2 to 0.4 per cent of this amino acid. The single sample ana- lyzed by Rossi-Fanelli, however, like the one referred to in figure 1, did not appear to contain any isoleucine. Although a difference of a few tenths of a per cent in an analytical value is not normally considered to be very significant, when this difference is between zero and one or two residues per molecule, it is no longer a question of the accuracy of the analytical figure but rather of the presence or the absence of an impurity. In view of the interest in comparing different hemoglobins, the question of purity is obviously of some moment. Accordingly, other hemoglobins were investigated. Samples of hemoglobin A from a normal Negro and from an individual exhibiting the thalassemic trait were analyzed, as were samples of hemoglobin C and hemoglobin E. All were prepared electrophoretically by Dr. Kunkel. All were devoid of isoleucine. To make sure that isoleucine was not being destroyed in some unexpected way, a sample of fetal hemoglobin was analyzed, and found to contain the anticipated 1.45 per cent of isoleucine. Finally, Dr. Schroeder was kind enough to send us a sample of a hydrolysate he had analyzed, and found to contain 0.2 per cent isoleucine. We were able to confirm his figure exactly. Thus, there can be no doubt; when isoleucine is present, we find it; but hemoglobin free of isoleucine can be obtained. Cysteine was another amino acid the content of which was low in the elec- trophoretically-prepared hemoglobin ~ table I ~ . According to Ingrami2 and to Benesch, Lardy, and Benesch,43 urea-denatured hemoglobin A contains 8 sulfhydryl groups per molecule by amperometric titration. Hommes, Drink- waard, and Huisman:4 found 8 sulihydryl groups in hemoglobins A, B. and C, and 8 half-cystine residues as well, determined as cysteic acid by the chroma- tographic method of Schram, Moore, and Bigwood.~5 As can be seen from table I, however, the electrophoretically-purified hemoglobins contain only 4 to 5 -SH groups per molecule by amperometric titration in 8 M urea. The total quantity of half-cystine, determined as cysteic acid,~5 is, in most cases, in acceptable agreement with the number of sulfhydryl groups determined amperometrically, indicating the absence of disulfide bonds in all of the samples. This was further confirmed with one sample of hemoglobin A which, when titrated after treatment with sodium sulfite, showed no increase

AMINO ACID COMPOSITION STEIN 225 4. Schroeder, W. A., Kay, L. M., and Wells, I. C.: Amino acid composition of hemoglobins of normal negroes and sickle-cell anemics, J. Biol. Chem., 187: 221, 1950. 5. Rossi-Fanelli, A., Cavallini, D., and De Marco, C.: Amino-acid composition of human crystallized myoglobin and haemoglobin, Biochim. Biophys. Acta, 17: 377, 1955. 6. van der Schaaf, P. C., and [Iuisman, T. H. J.: The amino acid composition of human adult and foetal carbonmonoxyhaemoglobin estimated by ion exchange chromatography, Biochim. Biophys. Acta, 17: 81, 1955. 7. Huisman, T. H. J., van der Schaaf, P. C., and van der Saar, A.: Some charac- teristic properties of hemoglobin C, Blood, 10: 1079, 1955. 8. Dustin, J. P., Schapira, G., Dreyfus, J. C., and Hestermans-Medard, O.: La com- position en acides amines de l'hemoglobine foetale humaine, Compt. rend. sac. biol., 148: 1207, 1954. 9. Stein, W. H., Kunkel, H. G., Cole, R. D., Spackman, D. H., and Moore, S.: Ob- servations on the amino acid composition of human hemoglobins, Biochim. Bio- phys. Acta, 24: 640, 1957. 10. Kunkel, H. G., and Wallenius, G.: New hemoglobin in normal adult blood, Sci- ence, 122: 288, 1955. 11. Spackman, D. H., Stein, W. H., and Moore, S.: Automatic recording apparatus for use in chromatography of amino acids, Federation Proc., 15: 358, 1956. 12. Ingram, V. M.: Sulphydryl groups in haemoglobins, Biochem. J., 59: 653, 1955. 13. Benesch, R. E., Lardy, H. A., and Benesch, R.: The sulihydryl groups of crys- talline proteins, J. Biol. Chem., 216: 663, 1955. 14. Hommes, F`. A., Drinkwaard, J. S., and Huisman, T. H. J.: The sulfhydryl groups of four different human hemoglobins, Biochim. Biophys. Acta, 20: 564, 1956. 1 5. Schram, E., Moore, S., and Bigwood, E. J.: Chromatographic determination of cystine as cysteic acid, Biochem. J., 57: 33, 1954. 16. Hirs, C. H. W., Stein, W. H., and Moore, S.: The amino acid composition of ribonuclease, J. Biol. Chem., 211: 941, 1954. DISCUSSION Dr. [Y. J. Schroeder: I wish to add to Dr. Stein's remarks about the iso- le~cine problem. ()f the many samoles we have analyzed. only one have we found to be free of isoleucine and that was onta~ne~ oy co~umn e~ectro- phoresis. Yesterday, Dr. Edsall remarked that the work which Dr. Rhinesmith, Prof. Pauling and I reported some time ago gave 3.6 N-terminal residues to the hemoglobin molecule. The non-integral number is an absurdity. F`urther work leads us to believe that there are 4.0 N-terminal residues as Dr. Edsall had surmised. We come to this conclusion by a study of partial hy- drolysis of DNP-globin. It we partially hydrolyze DNP-globin for various periods of time, we find that very rapidly, and indeed, within 15 minutes, 90 per cent of two residues per molecule has been released in the form of DNP- valyl-leucine. On continued hydrolysis, the DNP-valyl-leucine simply de- creases in the way in which you would expect DNP-valyl-leucine itself to 1 - J ~ J 1 · ~ 1 1 1

224 PART III. ABNORMAL HEMOGLOBINS in the number of sulfhydryl groups. Apparently, electrophoresis not only removes art impurity that contains isoleucine, but also one that is rich in sulfhydryl groups. The value for the sulfLydryl groups seems to vary some- what from one hemoglobin sample to the next, and in some cases an integral molar quantity is not found. This may be a result of experimental error, or it may mean that the impurity has not been completely removed even from the electrophoretically-prepared samples. In agreement with Hommes, Drink- waard and Huisman, less half-cystine was toured in fetal hemoglobin than in hemoglobin A, but the value is three to four rather than six residues per molecule. Unlike these authors, however, we have not been able to secure any evidence for the presence of a disulEde bond in fetal hemoglobin. Except for isoleucine and cysteine, the analytical results obtained in the present studies (table I) tend to be slightly lower than, but are in general quite similar to, other values to be found in the literature. It should be emphasized, however, that the single analyses performed on each 22- and 70- hour hydrolysate are insufficient to provide definitive information relative to the amino acid composition of hemoglobin A. Nor have sufficient analyses been carried out to decide with assurance whether or not the various hemoglobins (table I) have the same amino acid composition, particularly in view of the fact that there is reason to suspect from the suliLydryl analyses that these preparations may not be completely pure. The nature of the impurity or impurities present in many of the hemo- globins analyzed heretofore cannot be decided at this time. Minor hemoglobin components are removed by electrophoresis, but it is doubtful whether they contain sufficient isoleucine or cysteine to account for the results. For exam- ple, there would have to be a 10 to 25 per cent contamination by fetal hemo- globin to contribute 0.2 to 0.4 per cent isoleucine, whereas only 1 per cent or so of fetal hemoglobin has been reported to be present in adult hemoglobin. Fetal hemoglobin, of course, could not account for the high cysteine values, since it contains fewer -SH groups than does adult hemoglobin. Unfortunately, this brief discussion has provided few final answers. The results have been presented simply to call attention to the fact that the absence of isoleucine is one criterion for evaluating the purity of hemoglobin, and that determination of sulThydryl groups and of half-cystine as cysteic acid may be useful in following the purification of this protein. REFEREN CES 1. Brown, H., Sanger, Flu., and Kitai, R.: The structure of pig and sheep insulins, Biochem. J., 60: 556, 1955. 2. Harfenist, E. J., and Craig, L. C.: Differences in the quantitative amino acid composition of insulins isolated from beef, pork and sheep glands, l. Am. Chem. Soc., 74: 4216, 1952. 3. Pauling, L., Itano, H. A., Singer, S. J., and Wells, I. C.: Sickle cell anemia, a molecular disease, Science, 110: 543, 1949.

'e He · - me - of c-) c) 4- ~ oh c' ~ ~ - o o o ~ o c) x ~ c) ¢ (7 as ¢ He 1 a) ~ ~ o c) ~ Go ¢ o to o ~ ~ a, ~ Cal l o ~ ~ He c) ~ o ~ [~ # o .= an ~ o ~ ~ of co o ~ ~ an o ~ x ~ - ~ ¢ (v o ~ an ~ · ¢ . - o a, Ad · - en - cd ~ l-~mo-` =~ =~ ~ "c ~ ~ ~ ~ o ~ . . . . . ~ o om-mo~ ~ ~ =`—~ o o ~ N O ~ ~ ~ O ~ _ V~ O ~ =\ O O ~ C u2 V 5: d" ~ C~ J 0 c ~ ~ -0 ~ ~ ~ ~ ~ ~ O ~ .~ 0 ~ \0 ~ ~ O oe >' ~ N ~ ~ ~ ~ C) ~ 00 ¢* ~ .$~ =~ o . ~ ~ ~ N O ¢- =0 O =~ C~ s~ == ~=3 Sol~ ¢° . ss == ~ ._ Ct - ~o .s ~ oo C~ o o ~ ~ oo ~ ~ O ~ ~ oo cr~ - . _I _ e~ ~ ~o ~ ~ ~ o~ oo o _ . . · . ~ ~ ~ o ~ ~ V oo C~ 00 ~ et . . . . . . o ~o ~ ~ ~ ~ _' oo oo ~o ~ ~ ~ ~ . . . . . . ~o ~ C~ o ~ _ _ C~ oo . . . . . . o~ ~ ~ ~ o _' C~ ~ ~ ~ ~ ~ o C . . . . crx ~o d" ~ ° ~ o ~ ~ ~ V C ~C . _ V~ ~ _~ ~ 0\ ~ O O ~ ~ O O ~ ~ _ ~ 00 ~ ~ _ O ~ ~ O 0 O ~ ~ _ V ~ \0 ~ ~ ~C O C~ ~ C~ ~ . . . . . O ~ ~ _ ~ V O 00 ~ _ O C~ ~ _( V ~ 00 0 Ch~ O0 00 ~ O \0 ~ . . . . . ~ ~ 00 U~ ~ ~ ~ ~ ~ O ~ ~ O . . . . ~ ~ ~ ~ ~1 crx cr~ crx ~ °° ~ 0 . . · . . O0 C~ _t ~ C~ 00 ~ \0 0 . . . . . ~ ~ 00 ~ ~ ~ ~ O C~ 00 0 0 . . . ~ ~ ~ - , ~ ~ O0 ~ _ ~ O ~ O ~ ~ 00 V, _ ~ ~ ~ 00 oo _ ~ cr~ 00 . . . . . 00 _ ~ ~ ~ C~ ~ O . . . . . ~ ~ 00 C~ ~ 'u U : ·~, = a ~`,, ,, a ° C .= :- ~ =- a 00~0 . ~o~ -- ~\ -' == 0 ._ _ ~ ._ ._ C~ Ct . .C) ~ C) U, ~ 5 37, 0 .~: U: 5- ~ ;^ t—, -5 tt _ I¢ o o ~ b4— oO ~ ~ ~ . ., ~, CO X o . b4 ~ s~ ~ ~ g s-= ~o¢0 s.~= o o pC' o~ ~o C~ (l,~ C,: ~ · ~ .= sq q~ o¢ ~ . = ~o o~o * s~ * ~ .. CO .. CO ~V . ~o ~a O ~ ~ .- O C~ ' _ t4 p, .= ;- O O ~ O · 0= ~ =0 0 o2 9 ~ g¢ ~ C} ~ =~.~ - b4 U: `~, 9 t~ .~ - Ce g CO ~ ~ C) ~ ~ -~ O o. - O C) P' ~ :' ¢ E~ . a * ~ ~,= ~S C) — CO

226 PART III. ABNORMAL HEMOGLOBINS decrease under the same condition of hydrolysis. No increase is observed after ]5 minutes. There is very little DNP-valine after 15 minutes of hydrolysis, but thereafter it increases with continued hydrolysis more rapidly than DNP- valine is released from DNP-valyl-leucine. Because we know the rate at which DNP-valine is released from DNP-valyl-leucine, we can calculate the amount that must be coming from the other two chains that do not release DNP-valyl-leucine. When we do this, we find that the first order reaction rate constant is something like 0.70 hr.-i, whereas the constant for DNP- valyl-leucine itself is 0.143 hr.-~. The DNP-valine from the two chains other than those that release DNP-valyl-leucine is coming off more slowly. By cal- culation we find that in 22 hours of hydrolysis there still should remain ap- proximately 20 per cent of the two resistant chains unhydrolyzed. Since there are two chains, this is equivalent to 0.4 of an N-terminal group. Our previous value was 3.6 and the addition of 0.4 comes out so perfectly to 4.0 as to be almost unrealistic. There are a good many implications in these results that I don't have time to discuss. They certainly show, however, that there are two kinds of chains in the molecule. This work is described in an article scheduled for publication.) REFEREN CE 1. Rhinesmith, H. S., Schroeder, W. A., and Pauling, I.: N-terminal amino acid residues of normal adult human hemoglobin: A quantitative study of certain aspects of Sanger's dinitrophenyl (DNP) method, J. Am. Chem. Soc. 79: 609— 615, 1957.

THE STRUCTURAL BASIS OF DIFFERENCES IN ELECTROPHORETIC BEHAVIOR OF HUMAN HEMOGLOBINS I. HERBERT SCHEINBERG Sickle-cell hemoglobin, hemoglobin C, and several other variants of human hemoglobin differ in electrophoretic mobility from normal human adult hemo- globin (hemoglobin A).1 2 Since all of these hemoglobins probably have the same size and shape,3 their differences in electrophoretic mobility are pro- portional to differences in the net electrical charge of the molecules. The present paper is an attempt to relate these differences in charge to differences in chemical structure. HEMOGLOBIN A ( Total Charge 186 Net Charge —14 Total Charge— 189 Net Charge —1 1 HEMOGLOBIN S - - - - ...... )~\ ~+~+++~ )~> K+++++++ <~ ------)> I- · )_41 w---- HEMOGLOBIN S - Total Charge— 183 Net Charge —11 FIG. 1. Total and net charges of hemoglobins A and S. including two different total charges of S with the same net charge (pH 8.6). Figure 1 shows that two hemoglobins can have the same difference in net charge with more than one total charge. In both cases illustrated, sickle-cell hemoglobin, or hemoglobin S. is shown at pH 8.6 with a net charge which is three units more positive than normal hemoglobin. In one case, however, this is the result of three more positive charges on hemoglobin S than on normal hemoglobin. In the other case it is the result of three less negative charges on hemoglobin S than on normal hemoglobin. Electrophoretically, there should be no difference between both of these hypothetical hemoglobin S molecules but, as will be clear below, both represent different chemical structures. Near the physiological pH probably all of the charges on hemoglobins come from their constituent amino acids. Neutral amino acids contribute one terminal carboxyl and one terminal amino group to each polypeptide chain which is not a ring. Additional positive charges are contributed by the nitro- genous groups of the three basic amino acids, Rb (fig. 2~. Additional negative charges are contributed by the second carboxyl group of aspartic and glutamic acids, Ra. Within the pH range of about 6.5 to 9.0 all of these groups (except the imidazole of histidine above pH 8.0) generally exist in their charged forms4 (fig. 31. Thus, in this pH range a greater net positive charge on sickle- cell hemoglobin may be the consequence of a greater number of positively 227

228 H3N~ - Rb - COO- basic amino acid PART III. ABNORMAL HEMOGLOBINS lysine, if -Rb- is-CH- (CH-~) 4 arginine, if-RI,- NH;3+ is -CH- (CH .), AH NH3+ histidine, if -Ret,- is -CH- \C NTH (AH.,- C - CH 1 1 HN NH+ C H FIG. 2. Structure of the positively charged forms of the three basic amino acids. Rb-NHm = Rb-N pH 9.5 = pH 11.5 R., is lysine or arginine ;Rb-NH~ = Rb-N pH 6.0 = pH 8.0 Rb is histidine Ra—COO— = Ra—COOH pH 5.0 = pH 3.0 Ra is asp.artic or glutamic acid FIG. 3.—The approximate ranges of pH in which various charged groups of amino acids ionize. charged nitrogenous groups, or a smaller number of carboxyl groups on this molecule in comparison with the normal. Figure 4 shows the result of a paper electrophoresis experiment performed in a buffer of pH 8.6 with hemoglobins A, S and C. The vertical line is the locus of the points of application of the hemoglobins, and the anode, as indi- cated at the top of the figure, is at the right. All three hemoglobins moved toward the anode indicating that, at this pH, all three have a net negative charge. This charge is obviously least negative, or most positive, on hemoglobin C and most positive, or least negative, on hemoglobin A. Since all of the groups mentioned above, except the imidazole of histidine, are charged at this pH, the observed differences in net charge may be due to differences in the numbers of free carboxyl, amino, or guanidinium groups on these hemoglobins (fig. 3~. This experiment suggests that the differences in charge between the three hemoglobins are not due to differences in their content of histidine since

STRUCTURE AND ELECTROPHORETIC BEHAVIOR—SCHEINBERG 229 ::~::~ ::: :~ .~.~ ~ I. .A , ~ s ~ ~-~ FIG. 4. Paper electrophoresis experiment in a veronal buffer of pH 8.6. In this and succeeding figures the letters to the left of the starting points denote the type of hemo- globin applied, and the + and — signs in- dicate the sides on which the anode and cathode, respectively, were placed. charge differences persist at pH 8.6 where imidazole groups are largely un- charged.4 If electrophoretic mobilities are determined at other than near neutral pHs it is possible to observe the effect of selectively making some groups electrically neutral. If we raise the pH of the buffer used for electrophoresis to about 12.0, almost all of the lysine and some of the arginine residues of hemoglobin will have lost their electrical charger (fig. 3~. An experiment performed at pH 11.7 is shown in figure 5 and it is apparent that differences in mobility, and therefore in charge, persist at this pH. It is true that the difference are less A. .~ ,^ FIG. 5.- Paper electrophoresis experi- :3: : ment in a phosphate buffer of pH 11.7. :::

230 PART ITI. ABNORMAL HEMOGLOBINS marked than those seen at pH 8.6 but this is due to the fact that the net charge of hemoglobin A rises from about -14 at pH 8.6 to about -67 at pH 12 so that a difference of about three in the net charge is relatively less at the higher pH. This result suggests that the differences in charge between these hemo- giobins are not due to differences in lysine or, probably, arginine since the differences in charge remain even when most lysine and arginine residues have become electrically neutral. If we now lower the pH at which the electrophoresis is carried out we first notice that the relative Nobilities change in sign. Thus, at HI 5.25 hemoglobin C now moves fastest, and hemoglobin A, slowest (fig. 6 ). This is, calf course, a consequence of being below the isolectric point of all three forms of hemoglobin so that each now has a net positive charge, with hemoglobin still possessing a greater net positivity than hemoglobin S. and hemoglobin S possessing greater net positivity than the normal form. ........ a . ~. ,,., , >,.,.,< ~ .. ... . , ~ FIG. 6. Paper electrophoresis experiment in an acetate buffer of pH 5.25. FIG. 7. Paper electrophoresis experiment in an acetate buffer of plI 4.01. F`IG. 8.- Paper electrophoresis experiment in a glycine buffer of pH 3.35. Figure 3 shows that carboxyl groups lose their charges between about pH 5 and 3. If we perform electrophoresis experiments at about pH 4 and below, the carboxyl groups of the hemoglobins should be uncharged. Figure 7 shows that at pH 4.01 there is essentially no difference in mobility between the three hemoglobins. This is also true at pH 3.3i in a glycine buffer (fig. 8~. These results indicate that the differences in net charge between the three hemoglobins are due to differences in their content of free carboxyl groups because the differences in charge are abolished at a pH at which only the carboxyl groups of the proteins' amino acid residues are uncharged. That this disappearance of the differences in Nobilities between the hemo- globins is not due to irreversible denaturation at acid pH is shown by the following experiment. One set of the three hemoglobins was dialyzed against a buffer of pH 4.01, and another set was dialyzed against a buffer of pH 3.35

STRUCTURE AND ELECTROPHORETIC BEHAVIOR SCHEINBERG 231 for the length of time that the electrophoretic runs were carried out at these pHs. Some hemoglobin precipitated, but the supernatant solutions were re- turned to pH 8.6 and subjected to electrophoresis. Figures 9 and 10 show that the differences in mobility characteristically observed at pH 8.6 are still present in these supernatant hemoglobin solutions. Even the presumably de- natured and precipitated hemoglobins retained these differences when dissolved in sodium hydroxide and subjected to electrophoresis in a buffer of pH 10.8 (fig. 11~. FIG. 9. Paper electrophoresis experiment in a veronal buffer of pH 8.6 with hemo- globins which had been dialyzed against an acetate buffer of pH 4.01 for 88 minutes, and then against veronal for 16 hours. (Supernatants) FIG. 10. - Paper electrophoresis experiment in a veronal buffer of pH 8.7 with hemo- g;obins which had been dialyzed against a glycine buffer of pH 3.35 for 74 minutes, and then against veronal for 16 hours. (Supernatants) FIG. 11.- Same as Fig. 9, but using precipitated hemoglobins dissolved in NaOH. The observed differences in net charge between these hemoglobins do not depend on differences in small-ion binding since complete removal of small- ions by ion-exchange resins results in different isotonic points for the three proteins.5 Differences in tyrosine or sulfhydryl content should not contribute to differences in charge except at pH values of about 10 and higher.4 The phosphorus content of hemoglobins A and S is not large enough, according to Havinga, to account for the difference in charge.6 Pauling and his co-workers calculated, on the basis of the difference in isoelectric points between hemoglobins A and S. that hemoglobin S possessed two to four more net positive charges per molecule than hemoglobin A.i On the basis of that calculation, and the results reported above, it appears that hemoglobin S possesses about two to four less free carboxyl groups, and hemo- globin C possesses perhaps five or six less free carboxyl groups than normal adult human hemoglobin.7 Several possible structures could account for these differences as shown below. The abnormal hemoglobins may have fewer

232 PART` III. ABNORMAL HEMOGLOBINS aspartic or glutamic acid residues or a greater number of carboxyl groups existing as amides or esters than is true of the normal protein. Possible Bases for Differences in Content of Free Carboxyl Groups in Hemoglobins A, S and C. Per molecule of hemoglobins S and C in comparison with hemoglobin A, one or more of the following possibilities should account for the differences: 1) Smaller number of aspartic and/or glutamic acid residues; 2) Larger number of terminal and/or aspartic and/or glutamic car- boxyl groups existing as amides; 3 ~ Larger number of esterified terminal and/or aspartic and/or glutam- ~c carboxyl groups. REFEREN CES 1. Pauling, L., Itano, H. A., Singer, S. J., and Wells, I. C.: Sickle cell anemia a molecular disease, Science, 110: 543-548, 1949. 2. Itano, H. A., and Neel, J. V.: A new inherited abnormality of human hemoglobin, Proc. Nat. Acad. Sci., 36: 613-617, 19 50. 3. Perutz, M. F`., Liquori, A. M., and Eirich, F.: X-ray and solubility studies of the hemoglobin of sickle cell anemia patients, Nature, 167: 929-931, 1951. 4. Cohn, E. J., and Edsall, l. T.: Proteins, amino acids and peptides, Chapter 20: 440505, Monograph Series No. 90, Reinhold Publishing Corp., New York, 1943. 5. Scatchard, G., and Black, E. S.: The effect of salts on the isotonic and isoelectric points of proteins, J. Phys. & Colloid Chem., 53: 88-99, 1949. 6. Havinga, E.: Comparison of the phosphorus content, optical rotation, separation of hemes and globin, and terminal amino-acid residues of normal adult human hemoglobin and sickle cell anemia hemoglobin, Proc. Nat. Acad. Sci., 39: 59-64, 1953. 7. Scheinberg, I. H., Harris, R. S., and Spitzer, J. L.: Diderential titration by means of paper electrophoresis and the structure of human hemoglobins, Proc. Nat. Acad. Sci., 40: 773-783, 1954. DISCUSSION Dr. R. Benesch: I would like to make one comment on Dr. Scheinberg's paper. He said that charge contributions from RS- in determining the elec- trophoretic mobility of proteins could only be expected at pH 11 or above. N/Yhile this would be true for tyrosine, it would not be true for -STI because, while we do not know the exact pK in hemoglobin, it is certainly a lot lower, semewhere around 9.

THE CHEMICAL DIFFERENCE BETWEEN NORMAL HUMAN AND SICKLE CELL ANAEMIA HAEMOGLOBINS V. M. INGRAM Previous articles have told the history of sickle cell anaemia, the first- and best-studied of the "molecular diseases.") They have also detailed the chemical evidence on the difference between the haemoglobins A and S. One should add the important finding of Perutz and his colleagues' that the solubility of deoxygenated haemoglobin S is very low and that this causes tactoids to appear which distort the red cell into the characteristic sickle shape. They also noted that x-ray diffraction patterns from crystals of the two haemo- globins were indistinguishable. This indicates that the difference between them is a small one and is not likely to be a difference in folding of the polypeptide chains since this involves shifting many atoms and would probably have been detectable. To summarize, by 1956 the known chemical difference between the taco proteins was that haemoglobin S contains about two carboxyl groups3 fewer per molecule than does haemoglobin A. I can now report that these carboxyl groups belong to glutamic acid and that they are replaced by two valine resi dues in haemoglobin S. This appears to be the only chemical difference be- t~veen the two proteins. The determination of the particular amino acids involved is made very difficult by the large size of the haemoglobin molecule. Experiments4 were therefore begun in 1956 to degrade these protein molecules into a number of small peptide fragments. It was hoped that if a rapid method could be found for characterizing the chemical properties of these peptides, then perhaps a replacement of even a single residue for another might be easily detectable. Accordingly, trypsin was allowed to digest samples of heat-denatured haemo- g;lobin A and S. since it splits specifically those bonds in the polypeptide chains which are formed by the carboxyl groups of the amino acids lysine and arginine. It is known that the haemoglobin molecule of 66,700 is composed of two iden- tical half molecules.' 5 In each of these there are about 25 lysines and ar- ginines6 and hence approximately 25 peptides are expected to be formed. In- deed, under the conditions used mixtures of about 25 peptides, on the average less than 10 amino acids long, resulted from each of the two haemoglobins. This is additional proof that haemoglobin is composed of equal halves, for otherwise some 50 different peptides would result. The two mixtures were compared by a two-dimensional combination of paper electrophoresis and paper chromatography.4 As a result the peptide spots were spread out in a characteristic map or "fingerprint" (figs. 1, 2 ~ . By working under very rigorously standardized conditions it was easily possible to obtain fingerprints 233

234 PART III. ABNORMAL HEMOGLOBINS o Y80 Hb A o ~0 o oU to Hb S o FIG. 1. -"Fingerprints" of tryptic digests of hemoglobins A and S. Of haemoglobin A and S in which all peptides occupied identical positions except for one, called peptide no. 4, which appeared in a new position in the haemoglobin S fingerprint. It must therefore have a different structure and will represent the portion of the polypeptide chains where the chemical dif- ference between the two proteins lies. The structures of these two peptides, the Hb A and Hb S no. 4 peptides, are shown in fig. 3. The structure of these two peptides has now been established, mainly by partial acid hydrolysis; the fragments are shown in fig. 3, separated on a finger- print. In addition, use was made of end group analyses, qualitative amino acid analyses, and Edman stepwise degradation of some of the fragments of

CHEMICAL DIFFERENCE BETWEEN HE A AND HB S INGRAM 235 ~0~ tic OC o c: o - c' o H) I lib (a) (a) fib d lIb S (b) (b) E o :;.v L ~ ' ',l-PV TV L-L) ,' .~-V~ P~ vC, , ~ P vat L, Gt~6 g7+c{;~ fib A ~ S F`IG. 2. Further examination of tryptic digests of hemoglobins A and S. (a) Slowest moving positively charged fractions; (b) neutral fractions. (From Nature 178: 792, 1956.) ~ ~ ~ _ _ H's - Val -Leu-Leu-Thr-Pro-Glu -Glu -Lys 4, 4. . _ ~ _ H_ Hi s - Va I - Leu - Leu- Thr - Pr o- Val - Glu - Ly s FIG. 3.—Acid degradation and structure of the no. 4 peptides from the hemoglobins A and S. partial acid hydrolysis. The indicated charge distribution was inferred from the electrophoretic behaviour of the peptides. Both peptides contain the same nine amino acids except for one; the first glutamic acid of the Hb A no. 4 carboxyl peptide, changes to another, valine, in the lob ~ peptide. l hus a group is lost. Since there are two identical half molecules, this change occurs twice in the whole haemoglobin molecule and the fact that haemoglobin S has about 2 carboxyl groups fewer is now explained. The two haemoglobins differ very little; only one out of nearly 300 amino acids in the half molecule changes. However, the present experimental results do not help to explain the abnormally low solubility of deoxygenated sickle cell haemoglobin,2 which is the cause of the anaemia. In particular, there is as yet no evidence to indi- cate the position of the no. 4 peptide along the haemoglobin peptide chains nor where this peptide is located when the chains are folded in the globular molecule of haemoglobin. In order to show that the two no. 4 peptides really do carry the only change

236 PART III. ABNORMAL HEMOGLOBINS in the molecule, the other peptide spots in the fingerprints were compared for amino acid composition. No differences were found. Furthermore, since haemoglob~n has a tryps~n-res~stant core about 30 ~ of the molecule this large piece was in each case isolated and digested with chymotrypsin. This treatment readily yielded again two mixtures of peptides, one from the haemo- globin A core and the other from haemoglobin S. They were compared by fingerprinting and chromatographic ex~minntinn of the neutral oentides (fig. 4~. Again no differences could be detected. One is therefore led to con- clude that the only difference lies in the two no. 4 peptides. 1 1 O ~ O ~ I' ~ o ~0 :6 Q 0 o f:.~° Lo + (a) Hb A _ + Hb S FIG. 4. (a) "Fingerprints" and (b) chromatography of neu- tral peptides of the chymotryptic digests of the hemoglobins A and S "trypsin resistant cores." Hb A Hb S (b) It is widely believed that haemoglobin is the first protein product made by the gene; it follows that changes in the gene should be faithfully reflected by changes in the protein. Neel has shown that a single mutation of a haemo- globin gene produces the abnormal "sickle cell" gene.7 It appears now from the results briefly presented here that what is presumably an alteration of a portion of the gene results in an alteration of a portion of the polypeptide chain of the corresponding protein,- in this case haemoglobin. In the "sickle cell" mutation the change in the protein is very small indeed, indicating that this mutation is extremely localized in the gene. Perhaps this affects only a single base pair in the very long chain of the DNA of the gene. These ideas fit in very well with the demonstration by BenzerS and Streisinger,9 working with intact bacteriophage, that genes can be divided into hundreds of sub- units. Similar divisibility had also been shown for the genes of Aspergillusl° and Ne2crospora.l1

CHEMICAL DIFFERENCE BETWEEN HE A AND HB S INGRAM 237 It is also possible to report progress in similar investigations on haemoglobin C, carried out in collaboration with Mr. l. A. Hunt. This was the second abnormal human haemoglobin to be discovered)' and it results from another single mutation of the haemoglobin gene. As earlier papers indicated, it has even fewer net negative charges per molecule than does haemoglobin S and is therefore easily distinguishable electrophoretically. The solubility of reduced haemoglobin C is very near the normal. Haemoglobin C has been submitted to the detailed comparison with haemo- globin A outlined above for sickle cell haemoglobin. Trypsin digests of the whole protein and chymotrypsin digests of the resistant core were prepared and were examined by fingerprinting (fig. 5) and by chromatography. Again G t: ~ . + HbA - 1 1° j:~: MU HbC FIG. 5. Portion of the "finger- prints" of tryptic digests from the hemoglobins A and C. the only peptide affected by the mutation is the no. 4 peptide of the tryptic haemoglobin A digest, the same one that showed the "sickle cell" change. Its place in the corresponding haemoglobin C digest is taken by two new peptides, one neutral, the other positively charged. It is too early yet to speculate on the chemical changes underlying these observations; the structural analyses of the peptides are not far enough advanced. It is however, strikingly evident that the same very small region of the protein is affected by this second muta- tion. Genetic evidence showsi2 that the haemoglobin S and C mutations are allelic, i.e., on the same place in the gene, or at any rate closely linked. The chemical evidence to date on the primary protein products of these genes in- dicates that the same very small portion of the peptide chains is affected in troth cases. This is the exact chemical counterpart of the concept of allelic mutations. REFEREN CES 1. Pauling, L., Itano, H. A., Singer, S. J., and Wells, I. C.: Sickle cell anemia, a molecular disease, Science 110: 543, 1949. 2. a ) Perutz, M. F., and Mitchison, J. M.: State of haemoglobin in sickle-cell anaemia, Nature 166: 677, 1950. b) Perutz, M. F`., Liquori, A. M., and Eirich, i?.: X-ray and solubility studies of haemoglobin of sickle-cell anaemia patients, Nature 167: 929, 1951. 3. Scheinberg, I. H., Harris, R. S., and Spitzer, J. L.: Differential titration by means of paper electrophoresis and the structure of human hemoglobins, Proc. Nat. Acad. Sci. ¢0: 777, 1954.

238 PART III. ABNORMAL HEMOGLOBINS 4. Ingram, V. M.: A specific chemical difference between the globins of normal human and sickle-cell anaemia haemoglobin, Nature 178: 792, 1956. 5. Schroeder, W. A., Rhinesmith, H. S., and Pauling, L.: In press. 6. Stein, W. H.: These Proceedings and in press. 7. Neel, J. V.: Inheritance of sickle cell anemia, Science 110: 64, 1949. 8. Benzer, S.: The elementary units of heredity, in The Chemical Basis of Heredity, McElroy, W. D., and Glass, B., eds., Johns Hopkins Press, Baltimore, 1957. 9. Streisinger, G., and Franklin, N. C.: Mutation and recombination at the host range genetic region of phage T2, Cold Spring Harbor Symp. Quant. Biol. At: 103, 1956. 10. Pritchard, R. H.: The linear arrangement of a series of alleles of ~spergillas Nidula7~s, Heredity 9: 343, 1955. 1 1. Giles, N. H., Partridge, C. W. H., and Nelson, N. J.: The genetic control of adenylosuccinase in Neurospora (grassy, Proc. U. S. Nat. Acad. Sci. 43: 305, 1957. 12. Itano. H. A.: The hemoglobins, Ann. Rev. Biochem. 25: 331, 1956. 13. Zuelzer, W. W., Neel, J. V., and Robinson, A. R.: Abnormal hemoglobins, Prog- ress in Hematology 1: 91, 1956. DISCUSSION Dr. falter L. Hughes: After Dr. Ingram's very beautiful sleuthing job, I hate to get down to a very practical problem, but I do avant to initiate a discussion on sulfhydryl groups in hemoglobins. All my analyses have been done by methyl mercury determinations. We have used methyl mercury as the most specific reagent known to me for -SH interaction. Certainly everyone agrees that mercury forms the most specific bonds with sulfur, and methyl mercury, the smallest mercurial, should react most easily. To initiate the discussion, I will briefly present the data I have accumulated sporadically over the years. I have studied three species of hemoglobin—bovine, horse, and human and found -SlI contents for bovine, 2; horse, 4; and~human, 6. More par- ticularly, I have found two such groups in bovine hemoglobin, whether native or denatured, and also in globin isolated from this hemoglobin. In the case of equine hemoglobin, whether native or denatured, I have found four groups. I might say that these values are not exact integers. However, I believe the sulfhydryls must be an integral part of the molecule. I do not think Dr. Stein's finding of impurities can explain all of these species differences and so I have rounded off the values. The precise values obtained were: for bovine, 2 to 2.5; for equine, 4 to 4.5; and for human, 5.5 to 6. The bovine hemoglobin was not crystallized and I suspect some impurity may be raising the value obtained. The equine and human have been crystal- lized. In the case of the human hemoglobin, there appear to be two types of sulfhydryl groups; two are very active and four more are less active and can hardly be recognized completely except on denaturation. I think I can draw from published data arguments in support of my own.

DISCUSSION 239 Dr. Ingram is here and so I do not mind presenting his data, even though I have taken some liberties with them. These are his data of two years ago.i (Table I). He has prepared a more recent paper which I have not seen and upon which he may wish to comment. TABLE I -SH CONTENT OF HEMOGLOBINS Reagent AgN O3 HgCl~ HgCl., PCMB Moles Bound Bovine Human 4.0 8.3 6 2 Moles of Mercurial Abolishing Silver Binding (2) 6 3 -a} * Adapted from: Ingram, V. M., Sulphydryl groups in haemoglobins, Bioch. J. so: 6D3, 1955. If you compare the bovine and human hemoglobins which Dr. Ingram studied, you will notice that he indicates that bovine has four sulfhydryl groups by silver titration, but he finds that two mercuries will block all four. Simi- larly, he finds eight in human hemoglobin by silver titration which can be blocked by six mercuries. He obtains a similar picture with mercurials. Since the more specific mercurial can completely block the excess silvers, I believe this rules out the possibility that the extra groups can be sulfhydryl. They must instead be some other group which is titrating with an amenity for silver close enough to that of suliLydryl so that they are included in the curvature at the end point of the amperometric titration. I think the final word on the -SH content will come via some good sulfur and methionine de- terminations which will establish an upper limit to the sulihydryl values. I can cite what I believe are the best data on equine hemoglobin. Trickery re- ports eight sulfurs and Brand3 reports four methionines. Then by difference we obtain a maximum of four -SH groups in perfect agreement with our sulfhydryl analysis. REFERENCES 1. Ingram, V. M.: Sulphydryl groups in haemoglobins, Biochem. J. 59: 653, 1955. 2. Trickery, H. B., and White, A.: Proportion of cystine yielded by hemoglobins of the horse, dog and sheep, Proc. Soc. Exper. Biol. and Med. 31: 6, 1933. 3. Brand, E., and Grantham, J.: The methionine and isoleucine content of hemo- globins, J. Am. Chem. Soc. 63: 724, 1946. Dr. R. Benesch: I would like to mention an experiment which confirms the very interesting suggestion first made by Dr. Ingram that title -SH groups of hemoglobin occur in closely-spaced pairs or ever triplets. It is possible to dis- place the proton of a sulfhydryl group quantitatively with ~ specific reagent (Benesch, R., and Benesch, R. E.: Biochim. Biophys. Acta 23: 643, 1957~.

240 PART III. ABNORMAL HEMOGLOBINS Since there are four reactive -SH groups in native human hemoglobin, the liberation of four protons would be expected upon treatment with a protorl- displacing reagent, such as salyrgan in the RlIgCl form (4 RHgC1 + Pr (SH ) 4 Pr ( SHgR) 4 + 4H+ + 4 Cl- ) . The value which we found, however, was two, thus confirming the contention that reaction of one -SH group of a pair with this large mecurial blocks the other one to access by the same reagent. A final point which has general applicability in protein chemistry. Many proteins, including hemoglobin, have been treated with urea in order to de- nature them and to break hydrogen bonds. What is not often realized is that urea is capable of forming quite stable complexes with metals, iron being one of them. Barbieri (Barbieri, G. A.: Atti accad. naz. Lincei, Vol. 22 No. I 867, 1913) was the first to crystallize hexa-(urea)-iron (III) chloride, o4 0-3 O D o—o t - O x—x t ~ 30 mans —~ t = 20 hrs 02 01 _ /! / i/~\ ~ ~ o ' ' ' ~ ~ _ 520 540 560 580 600 ~ (mix) FIG. 1. Denaturation of hemoglobin in the presence of urea and silver ions. TABLE I -SH GROUPS AND STABILITY OF HEMCGLOBIN Tris buffer pH 7.4 To decrease time in ~ 576 Tris buffer pH 7.4 Urea 4 M To decrease time in ~ 576 Human Hb control 20 furs. 2 20 furs. 5.5 8 moles Ag+ per mole protein — 30 mins. 41 same 75 mins. 3.6 75 " 49 same — — 270 " j 1 same 20 furs. 14 20 furs. 59 Sheep Hb control 20 furs. 14 20 furs. 14 8 moles Agm per mole protein 20 " 21 20 " 29

DISCUSSION 241 (Fe(OC(NH~6)Cl3. This is one fact which should be taken into account when one treats a protein like hemoglobin with urea. This is illustrated in the figure and the table. The findings may be sum- marized as follows: When human oxyhemoglobin is treated with eight atoms of silver to block what we believe are all the sulfhydryl groups, nothing much happens to the hemoglobin. Likewise, nothing happens to the hemoglobin when it is treated with urea. When the protein is treated with eight atoms of silver in 4 ~ urea, however, a very rapid breakdown takes place (fig. 1 and table I). As illustrated here, the breakdown is essentially to me/hemoglobin, although it then proceeds further. In summary, what it really comes to is that when the -SH groups are blocked, the urea is capable of pulling the iron out of the molecule. Dr. Howard Dintzis: In support of Dr. Hughes' observation and in th matter of chemical semantics, I should like to point out that the people who are titrating with silver and mercury are not necessarily titrating -SH groups. This is a matter of great confusion at present. They are titrating silver and mer- cury binding sites. Evidence from x-ray crystallography on hemoglobins cer- tainly is that, under some conditions, there are groups other than the -SH groups which combine mercury. We showed this by blocking the -SH groups and found that mercury was still bound under some conditions. In myoglobin the situation is very similar. There are groups that bind both mercury and silver even though there is no -SH group under some conditions. It was found that there are specific binding sites for silver and specific binding sites for some mercurials. This point should be made clearer than it has been so far. Dr. Max Perutz: There is some x-ray evidence regarding the location of the -SH groups in hemoglobin. You have seen on the diagrams which I showed yesterday that there are two -SH sites in hemoglobin which can be blocked by mercury. The same two sites are found if hemoglobin is blocked with four silver atoms making fIbAg4. The second pair of silver atoms goes close to the first pair, the four silver atoms giving rise to only two oblong peaks on a Fourier projection, each peak apparently representing two silver atoms. This kind of picture has been obtained both in horse and bovine hemo- globin, which are the only two species so far examined. I do not know what the picture would look like in human and in sheep hemoglobin, but I should like to express the hope that in due course we shall all agree on all these hemoglobins having just four -SH groups and no more. Dr. V. M. Ingram: In discussions with various people who have used these methods it has become quite clear that, under truly identical conditions and with samples prepared in the same way, there is very good agreement on the numbers of silver or mercury atoms which are bound by a protein. Disagree- ments have arisen because we were not aware of the profound effect of altera- tions in the experimental conditions. I was very glad to hear Dr. Dintzis' remarks. I, too, think that we should

242 PART III. ABNORMAL HEMOGLOBINS talk of silver and mercury binding sites and not assume that those will neces- sarily be -SH groups. No doubt most of them will be sulihydryl groups, but, as the work of Dr. Stein and his colleagues has shown, independent con- ~ . . Creation Is necessary. In the light of his experiments it would appear that there are no more than six -SH groups in normal human hemoglobin. My earlier findings that eight silver atoms and six mercury atoms are bound by the denatured protein seem to need a new explanation. One could assume that the six -SH groups bind si:; silver or mercury atoms and that, in addition, two of them are capable of binding an additional silver atom each. Such a compound has been postulated by Cecil. Whereas the original interpretation called for two pairs of Ag atoms held by two pairs of very closely spaced -SH groups, we now have two very close pairs of Ag atoms held by only two individual sulfhydryl groups. The mercury binding on sites which cannot be -SH groups, which Dr. ~~ntz~s mentioned, is very much less firm and of quite a different order of magnitude. It is not detectable under the conditions of amperometric titration. As far as Dr. Hughes' contribution is concerned, I am glad that we seem to be in complete agreement on mercury binding in denatured hemoglobins. Two, four and six mercuries are the numbers which I, too, found in denatured ~ . . . . . Ox, horse and human hemoglobin. In addition, we also agree that in human hemoglobin two mercuries are bound very readily and the other four not so firmly; in fact, I found that one has to denature the molecule before they will combine. Dr. [MY. H. Stein: In a protein such as hemoglobin where the absolute values for -SH groups or half-cystines are low, it is difficult to be certain of the results to better than +1 residue per molecule. Hence, the preserve of four, five, or six -SH groups per molecule would be compatible with our data. Eight residues seems unlikely. We would certainly agree with the previous speakers that amperometric titrations can, under various conditions, yield differing results. It is for this reason that the determinations of half-cystine as cysteic acid were performed. The cysteic-acid method has been checked in a number of different ways in the past, and though not necessarily foolproof, does not possess the same kind of uncertainties found in the titration pro- cedures. Moreover, it is difficult to see how hemoglobin can contain more -SH groups by amperometric titration than there are half-cystine residues. A higher value by amperometric titration than by the cysteic-acid method would appear to favor the presence of binding sites for silver or mercury other than -SH groups, as has been mentioned by others. Such a discrepancy was not encountered in our studies, however. I should like to return to Dr. Ingram's very beautiful work for a moment, because it seems to me to carry an important message relative to the homo- geneity of hemoglobin. If hemoglobin were a complex mixture of proteins, treatment with trypsin would be expected to yield a far greater number of

DISCUSSION 243 peptides than would be predicted from the arginine plus lysine content, assuming the protein to be pure, and the yields of these peptides would be low. I gather, however, that Dr. Ingram is getting about the expected number of peptides, and that the yields are high. A similar kind of result was obtained by Sanger with insulin, and in our own studies on ribonuclease, and these two proteins are of quite well-established homogeneity. Dr. Ingram's work would thus impose quite definite limits on the heterogeneity-of the hemoglobin he employed, and would indicate that the source of the gross heterogeneity found by other procedures must reside in structural differences which are not re- flected in alterations in the sequences of the amino acid residues. Would you agree to that, Dr. Ingram? Dr. Ingram: Yes, I would. Dr. John F. Taylor: I think it is important to point out that we may some- times be interested in the total -SH and sometimes in the number of -SH groups that are detectable in native hemoglobin under mild conditions. The former is evidently related to the total hemoglobin sulfur and its partition, about which there is still some uncertainty. The latter appears to be the more significant in relation to hypotheses about the mechanism of heme-heme in- teractions or other aspects of hemoglobin structure and function. We have measured the apparent -SH of several native mammalian hemo- globins,~ with the different reagents that have beer discussed, including ·1 .1 1 1 I /1 ~ ~X7-,L ~ _ , _ ~ , ~ silver, methyl mercury and t~iV1~. W 1tn any one hemoglobin preparation the extent of reaction depends upon the reagent and, especially with silverer, upon the reaction conditions. Inasmuch as PC~B treatment affects the heme- heme interactions, the number of -SH sites revealed with this reagent seems especially significant. With any one reagent the number of apparent -SH groups depends upon the source of the hemoglobin and its history. Careful handling and the use of EDTA in the preparation of a sample favor the preservation of its titratable -SlI groups. Inasmuch as heme-heme interactions are rather similar among the mammalian hemoglobins, while the number and the nature of the PC~B-reactive sites differ, the more significant number of sites would seem to be the smallest that has thus far been observed. This number is two in bovine hemoglobin and also, with PLUMB, in human hemo- ~l~hin Fach site might include more than one -SH group, of course. There are several indications that not all the reactive -SH groups are identical, when more than two are detectable. For example, in canine hemo- globin, which reacts with four moles of PCNIB (per Hb of 68,000) taco sites appear to differ from the other two, both in their reaction with ferricyanide (described on page 155) and in their reaction with cystine. Two of the four PCMB-reactive sites of canine oxyhemoglobin, and both of the two sites of bovine and human oxyhemoglobin, are abolished upon treatment with excess cystine at pH 9. From the resulting increase in electrophoretic mobility, com- pared with untreated HbO~, on either side of the isoelectric pH, it may be a, _ e,

244 PART III. ABNORMAL HEMOGLOBINS suggested that cystine reacts with the hemoglobin -SH to form a mixed disulfide: HEMO-~-SlI + 2 C S SC ~ HE)/IO-~-S-SCy Eldjarn and Pihl' have described and studied quantitatively the analogous re- action of cystamine with several proteins, including hemoglobin. The reaction j ust described, applied to several mammalian hemoglobins which contain differing numbers of reactive -SH groups in the native state, leas enabled us to prepare a series of hemoglobins that have been modified with respect to their titratable -SH groups without detectable change in absorption spectrum or in their ability to form oxy- or me/hemoglobin. Some samples of modified canine hemoglobin have been crystallized. The properties and re- actions of these substances are under investigation. Depending upon the sub- stance used to react with the hemoglobin, it will also be possible to vary the number and kind of the polar or non-polar side chains introduced into the protein in place of -SH. Finally, by extending the methods developed by Eldjarn and Pihl it will be possible to use the above reaction as another inde- pendent measure of the number of reactive -S. H sites in the molecule of hemoglobin or other protein. + 2CySH . REFEREN CES 1. Taylor, J. Fit.: Sulfhydryl groups of hemoglobin, Third International Congress of Biochemistry, Resume of communications, p. 17, 1955. 2. Eldjarn, L., and Pihl, A.: On the mode of action of x-ray protective agents. I. The fixation in ciao of cystamine and cysteamine to proteins, J. Biol. Chem. 223: 341-352, 1956. Dr. R. Benesch: I agree, of course, that silver and mercury are bound by many groups besides -SH groups. However, the essential difference is that the affinity of these metals for -SH groups exceeds (usually by several orders of magnitude) that for other sites on the protein. Therefore, when proteins are treated with these reagents the -SH groups react first and only after these sites are saturated does further nonspecific interaction with other groups take place. This is illustrated by the equilibrium dialysis experiment with hemo- globin taken from the paper by Benesch, et. al., (Benesch, R. E., Lardy, H. A., and Benesch, R.: J. Biol. Chem. 216: 663, 1955.) (Fig. 1.) In the case of the amperometric silver titration, the specificity is further enhanced by the following factors: Owing to the high sensitivity of the rotating platinum electrode, the absolute concentration of protein, and therefore of silver, is extremely low (about 2 x 10-;'M>. This may be contrasted, for example, with the equilibrium dialysis against p-mercuribenzoate, shown below, where con- centration ten times higher had to be employed to obtain significant results .

DI SCUSSION C 12 - o Q O 8 c o D US ¢ o E 245 fix x / O x ~ 4 x~ l/ / -/x~o x ~ 0 ~0 x O ok/ 0/ 0 / 0 - x-x = s he e p H b 02 0-0= human HbO2 , ~ I 10 20 30 p-Mercuri benzocte c x ~ 0 4 FIG. 1.—Reaction of hemoglobins with p-mercuribenzoate. (From the Journal of Biological Chemistry 216: 672, 1955; by permission. ) 2. Only a comparatively small excess of silver is necessary for an accurate determination of the end point. 3. The silver is used in the form of a relatively stable complex arid will therefore have little tendency per se to react with groups other than -SH groups. 4. A long list of biologically occurring compounds, such as amino acids, nucleic acids, etc., has been found not to react with silver at all under the conditions employed in this method Dr. Makio M~crayama': I would like to carry this discussion further by re- porting, in some detail, our studies of titratable sulfhydryl groups of normal, sickle cell, "C" and fetal hemoglobins. It was observed in our laboratory* that a deoxygenated sickle cell hemo- lysate has a negative temperature coefficient of "elation, i. e., a deoxygenated , ~ sickle cell hemoglobin solution of sufficient concentration gels at 38° C.,~ but the gel liquifes upon being cooled to 0° C. The reaction is reversible. This observation suggested that some alteration ir1 the molecular architecture of hemoglobin occurs during the process of deaggregation, i. e., the complemerl- tary combining sites on adjacent hemoglobin molecules must be altered at 0° C. One of the objects of the present investigation was to learn whether there would be a change in the number of titratable -SH Groups between the ice point and body temperature. ~ 1 Riggs reported that a sickle cell hemolysate which gels at 38° C. loses its gelling property where dialyzed against deionized water. Oxygenation also prevents gel formation of a sickle cell hemolysate.7 s, ii We investigated the relative accessibility of -SH groups at 0° and 38° C. for the following prep- arations: a) normal alla sickle cell deoxygenated hemolysates; b) normal and sickle cell oxygenated hemolysates; c) normal and sickle cell hemoglobins ~ While Dr. A. C. Allison was visiting the Gates and Crellin Laboratories of Chemistry, California Institute of Technology, in the summer of 1954.

246 PART III. ABNORMAL HEMOGLOBINS crystallized and then dialyzed against distilled water, deoxygenated; d ) normal and sickle cell hemoglobin solution crystallized and then dialyzed against distilled water, oxygenated; and e) hemoglobin C and normal fetal hemoglobin crystallized and then dialyzed against water, deoxygenated. I will summarize the data obtained by argentometric-amperometric titra- tior~ methods, as well as the mercurimetric-amperometric titration technique on Hb A and Hb So ~ and Hb C and Hb F.4 ~ It was found that the maxi- mal number of titratable -SH groups is the same for Hb A, lIb S and Hb C; however, the mercurimetric-amperometric titration data suggest that their spatial arrangement appears to be different. The fetal hemoglobin molecule has fewer titratable -SH groups than Hb A and the mercapto group spatial arrangement is also different from that of the normal adult hemoglobin mole- cule. The hemolysates were prepared as has been described elsewhere.5 The apparatus t and technique used for amperometric titrations are essentially those described in the same reference.5 Resells and Conclusions. About four -SH groups per molecule are titra- table argentometrically for both normal arid sickle cell hemolysates at 0° C. There is one less titratable -SH group per molecule of normal hemolysate at 38° than at 0° C. There are two less titratable -SH groups per molecule of sickle cell hemolysate at 38° than at 0° C. In contrast, the number of mole- cules of PC~B bound by normal and sickle cell oxygenated hemolysates was essentially the same at 0° and 38° C. For undialyzed fresh hemolysates, about three moles of PCMB per oxyhemoglobin molecule are bound in both ir~- stances. A preliminary study of Hb C and Hb ~ indicates that there are about two to three PCMB-bindir~g sites in these molecules also. At O ° about four -SH groups per molecule are titratable for dialyzed ~ deoxygenated and oxygenated ~ hemoglobins A, S and C by the argento- metric method, whereas fetal (crystallized and dialyzed) hemoglobin binds about six silver atoms per molecule at both 0° and 38° C. The increment of titratable -SH groups equals plus four for hemoglobins A, S and C when the temperature of Tris buffer is raised from 0° to 38° C. The results of mercurimetric-amperometric titration were quite unexpected. About four mercury atoms are bound per molecule of normal and C hemo- g;lobins at 0°; the result is represented very schematically in figure 1A. Since there are also about four argentometrically-titratable -SH groups at 0° C. in Hb A and Hb C molecules, it is suggested that the titratable -SH groups at 0° C. are located far apart—the -SH groups are separated so that no pair- ing is possible; thus, they form -S-Hg-C1 instead of -S-Hg-S-. On the other ~ An automatic amperometric titration apparatus is available; however, it was not used for this investigation. It is described in Encyclopedia of Instrumentation for Industrial Hygiene, Gaffe, C. D., Byers, K. H., and Mosey, A. D., eds., University of Michigan, Ann Arbor, 1956. See pp. 430~32, Automatic amperometric titration assembly.

DISCUSSION 247 hand, dialyzed sickle cell hemoglobin binds about three mercury atoms per molecule at 0°; this is represented schematically in figure 1B. It is suggested that one mercury atom could be bound ~ in -S -Hg-S-, the centers of the sulfur atoms are separated by twice the covalent Hg S bond distance, giving -S-!Ig-S 5.60 A) and that the other two titratable -SH groups are sepa- rated too far. A B O O . . O O O 0~0 0 , o 5.60A FIG. IA. A schematic representation . . . Of the spatial arrangement of titratab~e -SH groups at 0° of dialyzed normal hu- mar~ hemoglobin molecule as well as for Hb-C molecule. FIG. 1B. A schematic representation of the spatial arrangement of titratable -SH groups at 0° of dialyzed sickle cell hemo- globin molecule. A O O B O O O O . O O ~ A,..... O 5.60A O O O O O ,... O O - .~ O >5.60A O O O ~ . O O -..._: O 5.60A O O O ~ ~ . ~ o 5.60A O O ~ --—~ O O C D FIG. 2A. -A conformation of sulfur atoms (of titratable -Sf1 groups at 38 ° ) of normal adult hemoglobin molecule. FIG. 2B. A conformation of sulfur atoms (of titratable -SKI groups at 3g ° ) of sickle cell hemoglobin molecule. FIG. 2C. A conformation of sulfur atoms (of titratable -SH groups at 38°) of the "C" hemoglobin molecule. FIG. 2D. A conformation of sulfur atoms (of titratable -SH groups at 0°-and 38°) of the normal fetal hemoglobin molecule. (Figure 2A—D appears in Federation Pro- ceedings 16: 758, 1957, and is reproduced by permission of the publishers.) At 38° C. about six mercury atoms are bound per molecule of dialyzed normal deoxygenated hemoglobin. There are about two mercury atoms more than at 0°. In the argentometric titration an increase of about four Ag atoms per molecule is obtained when going from 0° to 38° C. It is suggested that the four -SH groups which become titratable ~ accessible ~ at 38 ° are so arranged that two mercury atoms can be bound by them. This is schematical- ly shown in figure 2A. It can be seen that two -S-Hg-S- linkages are possible in this molecule. Dialyzed sickle cell hemoglobin binds about five mercury atoms per mole-

24S PART III. ABNORMAL HEMOGLOBINS cute at 38°. This is about two atoms of mercury more than at 0°. For silver the increase leas about four atoms per molecule. In accordance with the previous picture, this could mean that four new -SH groups are close enough (two and two) to be handled by just two mercury atoms. This is schemat- ically shown in figure 2B. It can be seen that sickle cell hemoglobin probably has three -S-Hg-S- linkages at the equivalence point in the mercurimetric- amperometric titration. Dialyzed hemoglobin C binds about eight silver or eight mercury atoms per molecule at 38°. Furthermore, the data suggest that eight titratable -SH groups are separated far apart these are all "lone" -SH groups; at the equivalence point of the mercurimetric-amperometric titration eight -S-Hg-Cl's are formed. These findings are schematically represented in figure 2C. As can be seen from the schematic diagram, there are no -S-Hg-S- bridges pos- sible (centers of the sulfur atoms are separated by a distance greater than S.60 A) in the Hb C molecule. The total number of titratable -SH groups is the same for Hb A, fIb S. and Hb C, but their spatial arrangement appears to be different. The amino acid compositions of these hemoglobins are similar.)' -! i~ Pauling et al.~, suggested that the difference in structure between a "normal" and an "ab- normal" hemoglobin molecule may be a difference in the way in which the polvpeptide chains are folded. The present findings suggest that there is one . . T ~ ~ . T ~ ~ A ~ 1 mercapto pair more in rib ~ than in lib ~ and, furthermore, that there are two mercapto pairs less in Hb C than in Hb A. There are six titratable -SH groups in fetal hemoglobin "purified" from the cord blood. Dialyzed Hb F binds about six silver and about three mer- cury atoms per molecule at 0° as well as at 38°. There are three -S-Hg-S- linkages possible in this molecule, which is schematically represented in fig- ure 2D. Discussion. Pauling's steric hindrance theory of heme-heme interaction in hemoglobin provides an obvious explanation of the action of oxygen in pre- venting the sickling of sickle cell anemia erythrocytes as well as the gelling of the sickle cell hemolysates.8 He has visualized the sickling process as one in which complementary sites on adj acent hemoglobin molecules combine. It was suggested that oxyhemoglobin and carbonmonoxyhemoglobin do not aggregate because of steric hindrance of the attached oxygen or carbon monoxide molecules. This steric hindrance effect might distort the comple- mentary sites through the forcing apart of layers of protein, as suggested by the isocyanide experiments. It was known for a long time that oxygen exerts a marked inhibitory effect ore the sickling produced by any means. Thus oxygen also exerts an inhibitory effect on the sickling produced by sulfhydryl compounds and is capable of reversing the phenomenon. Thomas and Stetsoni4 reported that sulihydryl compounds, notably FINS, 2,3-dimercaptopropanol (BAL), and cysteine,

DISCUSSION 249 Fuse sickling. They also found that sickling is completely inhibited by mall amounts of -STI blocking agents (o-chloromercuribenzoate, iodoaceta- .~ide, iodosobenzoate, sodium maleate ~ . Ions of various heavy metals also inhibit sickling. These -SH blocking agents presumably act to destroy the complementariness of configuration of the surface ot the molecule. vv nen deoxygenated sickle cell hemoglobin gel (at 38°) is cooled to 0°, the com- plementary sites must be likewise altered, resulting in liquefaction of the gel. An architectural alteration of the sickle cell hemoglobin molecule appears to be reflected by the change in the number of titratable -SH groups. Hb A and Hb C also undergo architectural alterations faith temperature, whereas Rib F does riot. ~ ~ 1 1 1 ~ to Not all of the -STI groups of normal and sickle cell hemoglobin molecules are titratable in the hemolysates. The -SH groups are more accessible to the titrants (Ag~ and Hgtt) after dialysis against water. There are four -SH groups per molecule not titratable at 0° in these hemoglobins. These four non- titratable groups might be imbedded in the protein moiety; they are not titra- table probably due to steric hindrance between -SH groups and a part of globin in the hemoglobin molecule. Similarly PC\1B binding is not equiva- lent to silver. About three PC~:B molecules are bound by oxygenated normal ar,d sickle cell hemolysates. The data suggest that there are about three -SH groups which are different: they are less sterically hindered by a part of the globin molecule. Dialysis of hemoglobin solution against water is known to produce con- , ~ ~ siderable alteration of the molecular architecture. For example, the oxygen ~ l 1 1-2 ~ - l _ - _ _ r L _ ~1 _ L 2 ~ affinity is increased many Iola.~> LJlalysls OI nemog;looln agalIl5t WE1~E 1b believed to cause loosening of the structure through the operation of electro- static repulsive forces between the similarly charged portions of the molecule. In the presence of salt, these repulsive forces are diminished by the ion at- ~nosphere that surrounds the charged groups. The loosening of the protein structure would make imbedded -SH groups of hemoglobin molecule easily accessible. Some of the -SH groups appear to be arranged in pairs, and they are able to bind mercury atoms forming -S-Hg-S- bridges. Evidence for the pairing of -SH groups comes from x-ray diffraction data. Studies of Perutz~ and Perutz, Liquori' and Eirichi° have shown that the molecules of horse and human hemoglobin possess dyed axis of symmetry. Furthermore, -ray dif- fraction measurements of horse hemoglobin with four equivalents of silver showed only one slightly elongated peak in the electron density map for each pair of silver atoms, indicating directly that two -SH groups must be close together. Acknowledgments: I wish to express my indebtedness to Prof. Linus Paul- ing for helpful suggestions and to Prof. Dan H. Campbell for his interest and encouragement during the investigation. I am also indebted to Prof. David

250 PART III. ABNORMAL HEMOGLOBINS L. DraLkin for his interest in this problem. I thank Drs. Phillip Sturgeon and W. R. Bergren of the Children's Hospital in Los Angeles for providing blood specimens from patients with homozygous sickle cell anemia. I am also in- debted to Drs. Neva Abelson, l. D. Alexander, Carl E. Bechman, P. I. Jensen, A. McClements, and E. ~I:ertens, all of Philadelphia, who generously provided blood specimens. REFEREN CES 1. Harris, J. W.: Studies on the destruction of red blood cells. VIII. Molecular orientation in sickle cell hemoglobin solutions, Proc. Soc. Exper. Biol. & Med. 75: 197, 1950. 2. Huisman, T. H. J., Jonxis, l. H. P. and van der Schaaf, P. C.: Amino acid com- position of four different kinds of human hemoglobins, Nature 175: 902, 1955. 3. Murayama, M.: Titratable sulfhydryl grroups of normal adult and sickle-cell hemoglobins, Fed. Proc. 15: 318, 1956. T. Murayama, M.: Titratable sulfhydryl groups of fetal and "C" hemoglobins, Fed. Proc. 16: 223, 1957. 5. Murayama, M.: Titratable sulihydryl groups of normal and sickle-cell hemo- globins at 0° and 38°, J. Biol. Chem. 228: 231-240, 1957. 6. Murayama, M.: (In preparation). 7. Pauling, L.: The hemoglobin molecule in health and disease, Proc. Am. Phil. Soc. 96: 556, 1952. 8. Pauling, L., Itano, H. A., Singer, S. J., and Wells, I. C.: Sickle cell anemia, a molecular disease, Science, 110: 543, 1949. 9. Perutz, M. F.: X-ray analysis of haemog;lobin, Nature 149: 491, 1942. 10. Perutz, M. F~., Liquori, A. M., and Eirich, F`.: X-ray and solubility studies of the haemoglobin of sickle-cell anaemia patients, Nature 167: 929, 1951. St. George, R. C. C., and Pauling, L.: The combining power of hemoglobin for alkyl isocyanides, and the nature of the heme-heme interactions in hemoglobin, Science 114: 629, 1951. 12. Schroeder, W. A., Kay, L. M., and Wells, I. C.: Amino acid composition of hemo- globins of normal negroes and sickle cell anemics, J. Biol. Chem. 187: 221, 1950. 13. Sidwell, A. E., Jr., Munch, R. II., Barron, E. S. G., and Hogness, T. R.: The salt effect on the hemoglobin-oxygen equilibrium, l. Biol. Chem. 121: 335, 1938. 14. Thomas, L., and Stetson, C. A., fir.: Sulihydryl compounds and the sickling phenomenon; a preliminary report, Bull. Johns Hopkins Hosp. 81: 176, 1948. 15. van der Schaaf, P. C., and Huisman, T. H. J.: The amino acid composition of human adult and foetal carbonmonoxyhaemoglobin estimated by ion exchange chromatography, Biochim. Biophys. Acta 17: 81, 1955. Dr. T. H. J. H2'isman (CommunicafionJ:* In recent years many studies have been carried out in order to increase our knowledge of the structure of the human hemoglobins. Especially in regard to the structure of fetal hemo- globin, the chemical analyses of the N-terminal residues, C-terminal residues arid sulfLydryl content seem of importance. It was found ~ ' 3 that Hb F contains two valyl residues in N-terminal position, while there are indica- ~ Dr. Huisman was unable to attend the Conference and this communication was presented by title but not read. It was announced at the time that it would be in- cluded in these Proceedings.

DISCUSSION 251 tions that only one histidine and one tyrosine molecule are in C-terminal position.4 Chemical analyses of the total half-cystine content revealed the presence of six hal f-cystine residues per mole hemoglobin.5 The data ob- tained with two amperometric titration methods ~ Ingram6 and Benesch of alp) made it appear likely that fetal human hemoglobin contains four -SH groups. (Table I). The normal adult hemoglobin, on the contrary, contains TABLE I THE NUMBER OF SULFHYORYL GROUPS IN FETAL AND ADULT HUMAN HEMOGLOBIN ( M. W.—68,000 ) Method Hb-A Hb F Assumed Assumed Chemical g.28 g 5.67 6 Amp. titrations;, ~ 7.6 8 4.0 4 Amp. titration ~ 9.6 4.4 Amp. titration + 4 M urea 7.6 8 4~1 4 fives 3 valyl residues in N-terminal position' one histidine and one tyrosine in C-terminal position) and eight half-cystine residues per mole hemoglobin, which are all present as free sulfhydryl groups.;; ~ (Table I>. The results obtained for fetal hemoglobin suggest that two half-cystine residues are present in ~ disulfide linkage in this protein. It therefore may be possible that either the Hb Fat is built up of two different polypeptide chains linked to each other by one disulEde bridge, or the disulfide bridge is present in one of the poly- peptide chains. In order to prove the first hypothesis some investigations were carried out by reducing this disulfide bridge with thioglycolic acid according to the method of Lindley.S With this technique it is possible, as shown for instance for insu- lin, to split a protein, which is built up by polypeptide chains linked by one or more -S-S- bridges, into the separate chains. The reduced protein was there- fore studied after coupling of the free -SH groups with iodoacetamide both by paper electrophoresis and by moving boundary electrophoresis. If after this reduction- two different polypeptide chains are formed, it may be possible to prove their existence by these electrophoretic techniques. The patterns for Hb F and Hb A are given in figure 1. The patterns for the fetal pigment ~ C, D and E ~ show two well-dis- tinguished components (marked 2 and 3 ~ while for the adult hemoglobin (A and B) one component was found (marked 1~. Electrophoretic studies of the two hemoglobins under the same conditions but without the reduction with thioglycolic acid (0.1 M glycine buffer solution pH S.1 with addition of 4 M urea) resulted in one component for both proteins. These results indicate that it is likely that fetal globin is divided into two different parts by the reduction with the thiol component. This supports the hypothesis that fetal hemoglobin i, built up by two different polypeptide chains connected by a disulfide bridge.

PART III. ABNORMAL HEMOGLOBINS . ~ . ~ ~ ~ , .., FIG. 1. The electrophoretic pattern of the Hb A and Hb ~ after reduction with thioglycolic acid and coupling with iodoacetamide. Buffer solution: 0.1 M lithium thioglycolate pH 5.1 + 4 M urea (300 volts at 10 mA). A and B—normal adult Hb; A after 2~/~ hours, B after 4~/~ hours. C, D and E fetal Hb; C after 3 hours, D after 44/~ hours, E after 5 hours. REFERENCES 1. Porter, R. R., and Sanger, F.: The free amino groups of haemoglobins, Biochem. J. 42: 287, 1948. 2. Schapira, G., and Dreyfus, J. C.: Groupes N-terminaux de l'hemoglobine de la maladie de Cooley, Compt. rend. Soc. de Biol. 148: 895, 1954. 3. Huisman, T. H. J., and Drinkwaard, J.: The N-terminal residues of five different human haemoglobins, Biochim. Biophys. Acta 18: 58S, 1955. 4. Huisman, T. H. J., and Dozy, A.: The action of carboxypeptidase on different human haemoglobins, Biochim. Biophys. Acta20: 400, 1956. 5. Hommes, F. A., Santema-Drinkwaard, J., and Huisman, T. H. J.: The sulihydryl groups of four different human haemoglobins, Biochim. Biophys. Acta 20: 564, 1956. 6. Ingram, V. M.: Sulphydryl groups in haemoglobins, Biochem. J. 59: 653, 1955. 7. Benesch, R. E., Lardy, H. A., and Benesch, R.: The sulfhydryl groups of crystal- line proteins. I. Some albumins, enzymes, and hemoglobins, J. Biol. Chem. 216: 663, 1955. S. Lindley, H.: The reduction of the disu]fide bonds of insulin, J. Am. Chem. Soc. 77: 4927, 1955.

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