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

Chapter: BIOSYNTHESIS OF HEMOGLOBIN

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Suggested Citation:"BIOSYNTHESIS OF HEMOGLOBIN." 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:"BIOSYNTHESIS OF HEMOGLOBIN." 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:"BIOSYNTHESIS OF HEMOGLOBIN." 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:"BIOSYNTHESIS OF HEMOGLOBIN." 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:"BIOSYNTHESIS OF HEMOGLOBIN." 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:"BIOSYNTHESIS OF HEMOGLOBIN." 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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Page 71
Suggested Citation:"BIOSYNTHESIS OF HEMOGLOBIN." 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:"BIOSYNTHESIS OF HEMOGLOBIN." 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:"BIOSYNTHESIS OF HEMOGLOBIN." 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:"BIOSYNTHESIS OF HEMOGLOBIN." 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:"BIOSYNTHESIS OF HEMOGLOBIN." 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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Page 76
Suggested Citation:"BIOSYNTHESIS OF HEMOGLOBIN." 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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Page 77
Suggested Citation:"BIOSYNTHESIS OF HEMOGLOBIN." 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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Page 78
Suggested Citation:"BIOSYNTHESIS OF HEMOGLOBIN." 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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Page 79
Suggested Citation:"BIOSYNTHESIS OF HEMOGLOBIN." 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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Page 80
Suggested Citation:"BIOSYNTHESIS OF HEMOGLOBIN." 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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Page 81
Suggested Citation:"BIOSYNTHESIS OF HEMOGLOBIN." 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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Page 82
Suggested Citation:"BIOSYNTHESIS OF HEMOGLOBIN." 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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Page 83
Suggested Citation:"BIOSYNTHESIS OF HEMOGLOBIN." 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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Page 84
Suggested Citation:"BIOSYNTHESIS OF HEMOGLOBIN." 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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Page 85
Suggested Citation:"BIOSYNTHESIS OF HEMOGLOBIN." 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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Page 86
Suggested Citation:"BIOSYNTHESIS OF HEMOGLOBIN." 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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Page 87
Suggested Citation:"BIOSYNTHESIS OF HEMOGLOBIN." 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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Page 88
Suggested Citation:"BIOSYNTHESIS OF HEMOGLOBIN." 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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Page 89
Suggested Citation:"BIOSYNTHESIS OF HEMOGLOBIN." 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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Page 90
Suggested Citation:"BIOSYNTHESIS OF HEMOGLOBIN." 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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Page 91
Suggested Citation:"BIOSYNTHESIS OF HEMOGLOBIN." 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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Page 92
Suggested Citation:"BIOSYNTHESIS OF HEMOGLOBIN." 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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Page 93
Suggested Citation:"BIOSYNTHESIS OF HEMOGLOBIN." 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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Page 94
Suggested Citation:"BIOSYNTHESIS OF HEMOGLOBIN." 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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Page 95
Suggested Citation:"BIOSYNTHESIS OF HEMOGLOBIN." 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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Page 96
Suggested Citation:"BIOSYNTHESIS OF HEMOGLOBIN." 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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Page 97
Suggested Citation:"BIOSYNTHESIS OF HEMOGLOBIN." 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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Page 98
Suggested Citation:"BIOSYNTHESIS OF HEMOGLOBIN." 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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Page 99
Suggested Citation:"BIOSYNTHESIS OF HEMOGLOBIN." 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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Page 100
Suggested Citation:"BIOSYNTHESIS OF HEMOGLOBIN." 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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Page 101
Suggested Citation:"BIOSYNTHESIS OF HEMOGLOBIN." 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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Page 102
Suggested Citation:"BIOSYNTHESIS OF HEMOGLOBIN." 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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Page 103
Suggested Citation:"BIOSYNTHESIS OF HEMOGLOBIN." 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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Page 104
Suggested Citation:"BIOSYNTHESIS OF HEMOGLOBIN." 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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Page 105
Suggested Citation:"BIOSYNTHESIS OF HEMOGLOBIN." 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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Page 106
Suggested Citation:"BIOSYNTHESIS OF HEMOGLOBIN." 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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Page 107
Suggested Citation:"BIOSYNTHESIS OF HEMOGLOBIN." 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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Page 108
Suggested Citation:"BIOSYNTHESIS OF HEMOGLOBIN." 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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Page 109
Suggested Citation:"BIOSYNTHESIS OF HEMOGLOBIN." 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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Page 110
Suggested Citation:"BIOSYNTHESIS OF HEMOGLOBIN." 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:"BIOSYNTHESIS OF HEMOGLOBIN." 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:"BIOSYNTHESIS OF HEMOGLOBIN." 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:"BIOSYNTHESIS OF HEMOGLOBIN." 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:"BIOSYNTHESIS OF HEMOGLOBIN." 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:"BIOSYNTHESIS OF HEMOGLOBIN." 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:"BIOSYNTHESIS OF HEMOGLOBIN." 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:"BIOSYNTHESIS OF HEMOGLOBIN." 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:"BIOSYNTHESIS OF HEMOGLOBIN." 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:"BIOSYNTHESIS OF HEMOGLOBIN." 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:"BIOSYNTHESIS OF HEMOGLOBIN." 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:"BIOSYNTHESIS OF HEMOGLOBIN." 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:"BIOSYNTHESIS OF HEMOGLOBIN." 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:"BIOSYNTHESIS OF HEMOGLOBIN." 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:"BIOSYNTHESIS OF HEMOGLOBIN." 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:"BIOSYNTHESIS OF HEMOGLOBIN." 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:"BIOSYNTHESIS OF HEMOGLOBIN." 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:"BIOSYNTHESIS OF HEMOGLOBIN." 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:"BIOSYNTHESIS OF HEMOGLOBIN." 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:"BIOSYNTHESIS OF HEMOGLOBIN." 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:"BIOSYNTHESIS OF HEMOGLOBIN." 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:"BIOSYNTHESIS OF HEMOGLOBIN." 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:"BIOSYNTHESIS OF HEMOGLOBIN." 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:"BIOSYNTHESIS OF HEMOGLOBIN." 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:"BIOSYNTHESIS OF HEMOGLOBIN." 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:"BIOSYNTHESIS OF HEMOGLOBIN." 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:"BIOSYNTHESIS OF HEMOGLOBIN." 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:"BIOSYNTHESIS OF HEMOGLOBIN." 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:"BIOSYNTHESIS OF HEMOGLOBIN." 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:"BIOSYNTHESIS OF HEMOGLOBIN." 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:"BIOSYNTHESIS OF HEMOGLOBIN." 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:"BIOSYNTHESIS OF HEMOGLOBIN." 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:"BIOSYNTHESIS OF HEMOGLOBIN." 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 II. BIOSYNTHESIS OF HEMOGLOBIN THE BIOSYNTHESIS OF PORPHYRINS DAVID SHEMIN The over-all pathway of porphyrin synthesis in the cell is now known. This paper will first summarize this pathway, with the pertinent evidence, and then consider some further data which may eventually elucidate the details of those reactions which are concerned with porphyrin synthesis. The elucidation of the pathway of porphyrin synthesis was greatly aided, after the initial observations,~~~~3 by the early finding of an in vitro system capable of synthesizing this complicated-looking molecule from its compara- tively simple precursors. It was found that both avian erythrocytes4 and mammalian reticulocytes~ can effect this synthesis in vitro. As these systems were investigated it was found later that hemolyzed preparatiorls6~ ~ and extracts of avian erythrocytesS under proper conditions could also synthesize the porphyrin molecule. Only two precursors, glyc~ne and succinate, are required for all the atoms . of the porphyrin molecule. this was demonstrated by incubating duck ery- throcytes with labeled substrates and then degrading the porphyrin molecule ill a manner by which each carbon atom from a specific position could be isolated. It was found that the carbon atoms of the substrates occupy particu- lar positions in the porphyrin molecule.9~~0 Heme synthesized from glycine-2-C24 was shown to contain eight radio- active 4~~~ carbon atoms in specific positions.9 i~ Samples of heme, synthesized from Calf methyl and C7^J' carboxyl-labeled acetate, which were degraded, re- vealed a labeling pattern from which it was concluded that the acetate was converted to a four-carbon atom unsymmetrical compound via the citric acid cycled Further, it was concluded that this "active" succinate condensed with glycine, in some unknown manner, to form a precursor pyrrole. The relation- ship of porphyrin formation to the citric acid cycle is shown in figure 1. This relationship was documented by studies in which succinate- 1,4-C', succinate-2,3-C2'', a-ketoglutarate-l,2-Cl;, a-ketoglutarate-5-C7'i arid pri- mary carboxyl-labeled citrate were the substrates.6 13 In each of the experi- ments the predicted carbon atoms in the porphyrin contained the Call. The condensation of glycine and the active succinate (Reaction D, fin. 1) , . . . ~ . , . ~ , O divas then Investigated. An consideration of the possible methods of condensa- tion of succinate and glycine which would give rise to a product from which a pyrrole could reasonably be synthesized, a mechanism for detaching the ~ This work was supported by grants from the National Institutes of Health, IJnited States Public Health Service (A-1101, C-8), from the American Cancer Society, from the Rockefeller Foundation and from the Williams-Waterman Fund. 66

BIOSYNTHESIS OF PORPHYRINS SHEMIN < . . | (F) ~7 TRICARBOXYLIC ACID CYCLE (F) (A) > oc-Ketoglutarate > Succinyl derivative (D ) 1 + Glycine (E) Pyrroles ~ Protoporphyrin (F) Succinate 1 FIG. 1. The relationship of the tricarboxylic acid cycle and porphyrin formation. carboxyl group of glycine from its a-carbon atom must also be taken into account. This must be considered since the carboxyl group of glycine is not utilized for porphyrin formation and in the initial condensation of glycine with succinate, the whole molecule of glycine is involved. The condensation of succi- {T ~ I C A R BOX Y L I C\ ACID GYCLE ~SU CC I NY L) UREIDO group of purines, SlJCGlN4~E I formate, etc. ~ `~' c(- 1< ETO G LU T A R A T E ~ f G/ycine -\ /SUCCI NATE- H th e 1-ca rbon ato m toy G L YC I N E HOOC-CH2- CH2-C - C - COOH ~ I _ gOOC-CH2-CH2-C- CHO o k e t o - 9 ~ u t o r a I d e h y d e / ~ A ~ ~ ~ 1501 HOOC- CH2_CH2_C_C0OH o Ct - keto-glutaric acid ~-amino-~-keto ad ~ pic acid ( I J-co2 HOOC-CH2-CH2- ~C, -CH2NH2 o c; - amino - levulinic aci d ( I: ) P O R P H Y R I I\J FIG. 2. The Succinate-G!ycine cycle: a pathway for the metabolism of glycine.

68 PART II. BIOS YNTHESIS OF HEMOGLOBIN nate on the a-carbon atom of glycine to form a-amino-~-keto adipic acid would appear to be in agreement with the experimental Endings and conclusion (fig. 29. The compound formed, a p-keto acid, could then undergo decarboxylation readily and thus provide a mechanism by which the carboxyl group is detached from its a-carbon atom. :Further, the product of decarboxylation would be an amino ketone, b-2minolevulinic acid. Condensation of two moles of this latter compound by a Kr~orr type of condensation would give a reasonable mechanism for the formation of a pyrrole in which the carbon atoms of glycine and succinate would be in the previously found positions (fig. 3~. In order to test this postulate, hemolyzates of duck erythrocytes were in- c~bated with b-aminolevulinic acid-5-C 5 and with b-aminolevulinic acid- 1,4-C~. i~ Not only were the heme samples much more radioactive than com- parable samples synthesized from radioactive glycine and succinate, but the labeling pattern in the heme was the same for both b-aminolevulinic acid- 5 C25 and glycine-2-C~-;, and for both b-aminolevulinic acid-1,4-C' and succinic acid-1,4-C-5. i;~~~0 These experiments demonstrated that 6-amino- levulinic acid is an intermediate in porphyrin synthesis. This conclusion was supported by the experiments of Neuberger and Scotti' and by Dresel and Falk.~S Furthermore, it was subsequently demonstrated that fractions ob- tained from liveri9~~° and avian red blood cells"'' catalyze the formation of the mono-pyrrole, porphobilinogen':3~''4 (fig. 3), which divas previously shown try be an intermediate in the formation of porphyrin." The above is a summary of the synthesis of porphyrin from its precursors, glycine and succinate. We may now consider some experiments which were carried out in order to shed some light on the intimate details of some of the steps. The Formation of b-Aminolev~linic Acid. The synthesis of b-aminole- vulinic acid from glycine and succinate appears to be a rather complicated reaction in regard to the nature of the activated derivatives and to the bio- logical system. Whereas hemolyzates of avian erythrocytes can synthesize COOH H2 l H2 COO H COGH CH2 1 COOH ~ H2 c-2 + ;~° -2~0 ~ ~ PROTO- -C 0 ~ H2 H2N 5-A MING LEVULINIC aCID (II) + (mu) ~ N NH2 H P R E C U RSOR P Y PRO LE FIG. 3.—A mechanism for the formation of the monopyrrole, porphobilinigen, by condensation of two moles of b-aminolevulinic acid. The carbon atoms bearing the closed circles were originally the of-carbon atom of glycine.

BIOSYNTHESIS OF PORPHYRINS—SHEMIN 69 porphyrins from al; cine and succinate, preparations obtained by homogeni- zation, freezing and thawing, acetone powders, and extracts can only utilize b-aminolevulinic acid as a substrate for protoporphyrin synthesists i;' Ap- parently, the system responsible for the synthesis of 6-aminolevulinic acid is quite labile and complex. We found several years ago that certain compounds would inhibit the formation of b-aminolevulinic acid. It was found that cysteine, pyruvate, and acetate-'0 would inhibit b-aminolevulinic acid formation. This was ascertained from experiments which demonstrated that porphyrin synthesis was inhibited by addition of these compounds when glycine and succinate were the sub- strates and not when b-aminolevulinic acid Bras the substrate. We have found recently that not only can pyridoxal phosphate increase the synthesis of porphyrins as demonstrated by Schulman and Richert,-` but that the cysteine inhibition can be overcome by this coenzyme.'S These experiments point to a necessary activation of the glycine. We have more recently found that forma- tion of b-aminolevulinic acid can be markedly inhibited by aza-L-serine.'0 This latter inhibition was not overcome by the addition of glutamine.~° It may be worth noting that azaserine has no inhibitory effect on the conversion of b-aminolevulinic acid to heme. Surprisingly, the addition of 6-diazo-5- oxo-L-norleucine, which is a more effective inhibitor than azaserine in purine ring synthesis,:~° has no inhibitory influence on the formation of b-amino- levulinic acid. At this moment it is difficult to describe definitely the details concerned with glycine activation, especially in consideration of the above experiments. The activation of succinate is as yet to be elucidated. The ex- periments which were done with labeled acetate definitely established the formation of an unsymmetrical succinate and it was suggested, at that time, that this may be a succinyl coenzyme derivative.~° As yet the nature of this derivative has not been established. We haste carried out model organic experiments in which glycine and succinate were activated in order to see if b-aminolevulinic acid can be formed under relatively mild condition. Glycine was converted into an oxazolone derivative and succinate was in the form of its anhydride. Base-catalyzed condensation of these molecules occurred and b-aminolevulinic acid was demonstrated after hydrolysis of the condensed product.3i The Formation of Porphobilinogen from b-Aminole~linic Acid. The enzymatic formation of porphobilinogen from two moles of b-aminolevulinic acid requires that two different types of reactions should occur; an aldol type condensation and a Schiff base type linkage. Gibson, Neuberger and Scott'° have obtained no evidence that these enzyme preparations consisted of two enzymes. The enzyme concerned with porphobilinogen may, however, only catalyze one of these reactions, e.g., the aldol condensation. This reaction only may need the catalysis, for once this occurs the Schiff base reaction may occur spontaneously. In order to shed some light on the mechanism of

70 PART II. BIOSYNTHESIS OF HEMOGLOBIN porphobilinogen formation we have investigated model organic reactions. Scott3> and wee have found that b-aminolevulinic acid in alkali under anaerobic conditions would to a small extent be converted to porphobilinogen. In order to increase the yield and if possible to isolate intermediates which could sub- sequently be converted to porphobilinogen and to attempt to understand the formation of porphobilinogen, we have acylated the amino group of b-amino- levulinic acid and then subjected these derivatives to alkaline and anaerobic ~ . . conditions. An acid Group on the amino croup which is not readily hvdrolvzed bv ~ =~ ~ — ~ > ~ 1~ — ~ — —~ — — —~ — — — ~ ~ . .. .., . . . . ~ . ~ . . . . alkali at room temperature will hinder the formation of pyraz~ne derivatives, while permitting an aldol condensation to occur between two molecules. Sub- sequent hydrolysis of the ac`,;1 Groups would permit a Schiff base reaction and ~ J J J D ~ - ~ the product WOU1d be a pyrrole. 1 he structure of the pyrrole will depend on the initial carbon atoms involved in the aldol condensation. N-Acetyl b-amino- levulinic acid subjected to the conditions mentioned above yielded, after several days, products which on exposure to air were converted to an intense red pigment and which gave an intense color with Ehrlich's reagent. The color intensity obtained with Ehrlich's reagent indicated a very high yield of these compounds. The structure or structures of the product await elucidation. N-Phthalimido derivatives which were subjected to the same conditions may yield open chain condensation products because of the resistance of the phthalimido grouping to complete alkaline hydrolysis.3i The Formation of Porphyrins frown the mono-pyrrole, porphobilinogen. The mechanism of the conversion of the mono-pyrrole, porphobilinogen, to the biological functioning porphyrin (III isomer) has not been elucidated. We have suggested a mechanism which is based on the organic experiment of Corwin and collaborators.33~34 Condensation of three moles of the porpho- bilinogen could lead to a tripyrrylmethane compound as represented in figure 4. The tripyrrylmethane then breaks down to a dipyrrylmethane and a mono- pyrrol. The structure of the dipyrrylmethane is dependent on the place of Ac P Ac P ~ Ac P Be P ~ A ~ f, .~ . -2~H2 CH2—NH2 Ac P P Ac FIG. 4.—A mechanism of porphyrin formation from the monopyrrole. Ac—Acetic acid side chain; P Propionic acid side chain; ~ a-carbon atom of glycine and 6-carbon atom of 6-aminolevulinic acid.

BIOSYNTHESIS OF POF(PlIVRINS—SHEMIN 71 splitting. Art A split would give rise to dipyrrylmethane A, and a B split should give rise to dipyrrylmethane B. Cor~densation of a mole of A and a mole of B would give rise to a porphyrin of the III series. In the formation of the porphyrins of the III series it can be seen from figure 4 that it is necessary to lose a one-carbon atom compound since there are three amino- methyl side chains and only two are required to condense the two dipyrroles to the porphyrin structure. Consistent with this hypothesis is our finding that on the conversion of porphobilinogen to porphyrins, either by heating under acid conditions or by enzymatic conversion in cell-free extracts, formaldehyde i, formed.~5 The formation of protoporphyrin and heme can occur in a cell-free extract of duck erythrocytes. After incubation of cell-free extracts, obtained by centrifugation at 100,000 g, with 6-aminolevulinic acid-5-C ;, the isolated bemire was radioactive. The radioactivity was constant after several re- crystallizations. The hemin was then subjected to a chemical degradation in order to isolate methylethvlmaleimide and hematir~ic acid. The methyl- ethylmaleimide can only arise from pyrrole rings A and B of protoporphyrir~. It was found that the sample of methylethylmaleimide was radioactive and equal to that of the hematinic acid. Furthermore, the sum of the radioactivity of the methylethylmaleimide and hematinic acid was equal to the value cal- culated from the radioactivity of the hemin.35 The picture or porphyrin synthesis which has been summarized emphasizes the general concepts which have emerged from the biochemical studies carried out during the past two decades: the relative simplicity of the reactions; the relative simplicity arid availability of the substrates utilized for the synthesis of complicated structures; and the biochemical unity in living matter. Pro- toporphyrin is synthesized from two simple and readily available compounds, glycine and succir~ate, by rather simple reactions and the synthesis is very closely linked to the main energy-yieldir~g reactions of most cells. Further, it appears that all porphyrins in nature, including chlorophyll, in all different types of cells are synthesized by the same basic pathway. The different por- phyrins merely arise by modifications occurring in the side chains ire the Q- positions of the pyrrole units. In further support of this conclusion it is worth mentioning our recent studies ore the biosynthesis of vitamin Bee. The struc- ture of the vitamin has recently been formulated to contain a porphyrin-like component.36~3' We have found that b-aminolevulinic acid is readily utilized for the synthesis of vitamin Bed :3s and that the predicted carbon atoms of the vitamin synthesized from b-aminolevulinic acid-l,4-C7; contained the radioactivities.~0 REFEFtEN CES 1. Shemin, D., and Rittenberg, D.: The utilization of glycine for the synthesis of a porphyrin, J. Biol. Chem., 159: 567, 1945.

72 PART II. BIOSYNTHESIS OF HEMOGLOBIN 2. 7. a. Shemin, D., and Rittenberg, D.: The biological utilization of glycine for the syn- thesis of the protoporphyrin of hemoglobin. J. Biol. Chem., 166: 621, 1946. Shemin, D., and Rittenberg, D.: The life span of the human red blood cell, J. Biol. Chem., 166: 627, 1946. 4. Shemin, D., London, I. M., and Rittenberg, D.: The synthesis of protoporphyrin in vitro by red blood cells of the duck, J. Biol. Chem., 173: 799, 1948; 183: 757, 1950. SO London, I. M., Shemin, D., and Rittenberg, D.: Synthesis of heme in Vitro by the immature non-nucleated mammalian erythrocyte, .~. Biol. Chem., 173: 797, 1948; 183: 749, 1950. 6. Shemin, D., and Kumin, S.: The mechanism of porphyrin synthesis. The formation of a succinyl intermediate from succinate, J. Biol. Chem., 198: 827, 1952. London, I. M., and Yamasaki, M.: Heme synthesis in non-intact mammalian and avian erythrocytes, Federation Proc., 11: 250, 1952. Shemin, D., Abramsky, T., and Russell, C. S.: The synthesis of protoporphyrin from 6-aminolevulinic acid in a cell-free extract, J. Am. Chem. Soc., 76: 1204, 1954. 9 Wittenberg, J., and Shemin, D.: The location in protoporphyrin of the carbon atoms derived from the or-carbon atom of glycine, J. Biol. Chem., 185: 745, 1950. 10. Shemin, D., and ~rittenberg, J.: The mechanism of porphyrin formation. The role of the tricarboxylic acid cycle, J. Biol. Chem., 192: 315, 1951. 11. Radin, N., Rittenberg, D., and Shemin, D.: The role of glycine in the biosynthesis of heme, J. Biol. Chem.. 184: 745, 1950. 12. Muir, H. M., and Neuberger, A.: The biogenesis of porphyrins. 2. The origin of the methyne carbon atoms, Biochem. J., 47: 97, 1950. 13. Wriston, J. C., Lack, L., and Shemin, D.: The mechanism of porphyrin formation. Further evidence on the relationship of the citric acid cycle and porphyrin formation, J. Biol. Chem., 215: 603, 1955. 14~ Shemin, D., and Russell, C. S.: 6-Amino-levulinic acid; its role in the biosynthesis of porphyrins and purines, J. Am. Chem. Soc., 75: 4873, 1953. 15. Shemin, D., Russell, C. S., and Abramsky, T.: The succinate-glycine cycle. I. The mechanism of pyrrole synthesis, l. Biol. Chem., 215: 613, 1955. 1 6. SchiRmann, E., and Shemin, D.: Further studies on the utilization of b-amino- levulinic acid for porphyrin synthesis, J. Biol. Chem., 225: 623, 1957. 17. Neuberger, A., and Scott, J. J.: Aminolevulinic acid and porphyrin synthesis, Nature (London) 172: 1093, 1953. 18. Dresel, E. I. B., and Falk, J. E.: Conversion of 6-aminolevulinic acid to por- phobilinogen in a tissue system, Nature (London) 172: 1185, 1953. 19. Gibson, K. D., and Neuberger, A., and Scott, J. l.: The enzymic conversion of b-aminolevulinic acid to porphobilinogen, Biochem. J., 58: xii, 1954. 20. Gibson, K. D., Neuberger, A., and Scott, I. J.: The purification and properties of 6-aminolevulinic acid dehydrate, Biochem. J., 61: 618, 1955. 21. Schmid, R., and Shemin, D.: The enzymatic formation of porphobilinogen from b-aminolevulinic acid and its conversion to protoporphyrin, I. Am. Chem. Soc., 77: 506, 1955. 22. Granick, S.: Enzymatic conversion of 6-aminolevulinic acid to porphobilinogen, Science, 120: 1105, 1954. 23. Westall, R. G.: Isolation of porphobilinogen from urine of a patient with acute porphyria, Nature (London) 170: 614, 1952. 24. Cookson, G. H., and Rimington, C.: Porphobilinogen. Chemical constitution. Nature (London) 171: 875, 1953.

BIOSYNTHESIS OF PORPHYRINS—SHEMIN 73 25. Falk, J. E., Dresel, E. I. B., and Rimington, C.: Porphobilinogen as a porphyrin precursor, and interconversion of porphyrin:; in a tissue system, Nature (London) 172: 292, 1953. 26. Labbe, R., and Shemin, D.: Unpublished observation. 27. Schulman, M. P., and Rickert, D. A.: Heme synthesis in vitamin B.`; and panto- thenic acid deficiencies, J. Biol. Chem., 226: 181, 1957. 2S. Dain, J., and Shemin, D.: Unpublished observation. 29. Weliky, I., and Shemin, D.: Unpublished observation. 30. Levenberg, B., Melnick, I., and Buchanan, J. M.: Biosynthesis of purines. XV. The effect of aza-L-serine and 6-diazo-5-oxo-L-norleucine on inosinic acid biosynthesis de Volvo, J. Biol. Chem. 225: 163, 1957. 31. Winestock, C., and Shemin, D.: Unpublished observation. 32. Scott, J. J.: Synthesis of crystallizable porphobilinogen, Biochem. J., 62: 6P, 1956. 33. Corwin, A. H., and Andrews, J. S.: Studies in the pyrrole series. III. The rela- tion of tripyrrylmethane cleavage to methene synthesis, J. Am. Chem. Soc. 59: 1973, 1937. 34. Andrews, J. S., Corwin, A. H., and Sharp, A. G.: 1, 4, 5, 8-Tetramethyl-2, 3, 6, 7- tetracarbethoxy-porphyrin and some derivatives, J. Am. Chem. Soc. 72: 491, 1950. 35. Abramsky, T., and Shemin, D.: Unpublished observations. 36. Hodgkin, D. C., Pickworth, S., Robertson, J. H., Trueblood, K., Piozen, R. J., and White, J. G.: Structure of vitamin B..,, Nature 176: 325, 1955. 37. Bonnett, R., Cannon, J. R., iohnson, A. W., Sutherland, I., Todd, A. R., and Smith, E. L.: The structure of vitamin B.., and its hexacarboxylic acid degradation product, Nature 176: 328, 1955. 38. Shemin, D., Corcoran, J. W., Rosenblum, C., and Miller, I. M.: On the biosyn- thesis of the porphyrin-like moiety of vitamin B~.,, Science 124: 272, 1956. 39. Corcoran, J. W., and Shemin, D.: The biosynthesis of vitamin B~.,, Biochim. et biophys. acta (in press). .\

THE ENZYMATIC SYNTHESIS OF UROPORPHYRINOGENS FROM PORPHOBILINOGEN* LAWREN CE B O GORAD You have heard ~ report on the elegant work of Dr. Shemin and his colleagues relating b-aminolevulinic acid to the Krebs cycle on the one hand and to porphyrin biosynthesis on the other. Also, several laboratories have reportedi~'~3 the isolation of an enzyme which catalyzes the condensation of two molecules of b-aminolevulinic acid to form one of porphobilinogen COOH COOH ,. I CH2 CH, COOH 1 CHEF C O CON He - DAL Cow Ha\ CHEF 1 a' ,—CHINO \ N ~ PORPHOBILINOGEN FrG. 1. Structural formulae for b-aminolevulinic acid and porpho- bilinogen. (PBG) (fig. 1~. The utilization of PBG in the biosynthesis of porphyrins, including protoporphyrin IX, has been demonstrated using a number of different sources of enzymes;4~5~6 table I shows the nature of the porphyrins recovered after the incubation of PBG with a frozen and thawed preparation of Chlorella cells.4 This work supported a great deal of earlier evidence (e.~., reference 7) which suggested that protoporphyrin IX is derived from ~ - ~ 7 - ~ 0= ~ ~ ~ ~ an octacarboxyl~c tetrapyrrole and furthermore Indicated that all the naturally- occurring porphyrins originate by the condensation of four molecules of the same pyrrole, i.e. porphobilinogen. The present report deals primarily with investigations of enzymes which catalyze steps in the synthesis of uroporphyrins, and their immediate pre- cursors, from PBG. There are four possible uroporphyrin isomers but only two, uroporphyrin I and uroporphyrin III (fig. 2), are known to occur in nature. Uroporphyrin I can be visualized as being formed by the linear con- densation of four PBG molecules followed by ring closure and oxidation, ¢' this work was supported by grants frorr the National Institute of Arthritis and Metabolic Diseases, United States Public Health Service, and from the National Science Foundation. It was also supported in part by the Wallace C. and Clara A. Abbott Memorial Fund of the University of Chicago. 74

BIOSYNTHESIS OF UROPORPHYRINOGENS—BOGORAD 75 TABLE I PCRPHYRINS PRODUCED DURING THE INCUBATION OF PBG AND FROZEN AND THAWED CHLORELLA PREPARATIONS4 Porphyrins Fraction Present of Total Porphyrin Synthesized Aqueous Acetone-HC1 (2N HC1) Uroporphyrin 2, 3, 4, 5, 6, 7* 2,4, 5* = Protoporphyrin 285fo 52% 6% 1470 * Numbers refer to number of carboxyl groups per molecule. Underlined porphyrins present in greatest abundance. P H AC AC Am// up HI NH H; AC ~N /~AC P H P Uroporphyrin 111 AC = -CH2-COOH P =—CH2- CH2- COOH AC W P N N HC~\ BACH IN N=: P in\ ILIAC Ac H p U~oporphyrin I FIG. 2. Structural formu- lae for uroporphyrin I and III. but a tetrapyrrole with this arrangement of propionic and acetic acid side chains is unsuitable as an intermediate in the synthesis of protoporphyrin IX, unless a mechanism for shifting these substituents exists. On the other hand! the arrangement of side chains as on uroporphyrin III would fit the re- quirements of a precursor of protoporphyrin IX. Thus, the problem with respect to the biosynthesis of protoporphyrin IX is not merely to make a tetrapyrrole from PBG but to make tile proper one. In the initial studies of individual enzymes in this phase of porphyrin bio- synthesis,S~9 aqueous extracts of acetone powders of spinach leaf tissue were subjected to ammonium sulfate fractionation. The course of PBG con- sumption and the appearance of uroporphyrin in the presence of one fraction are shown in figure 3. Melting point determinations and paper chrom- atographyi° have shown that the porphyrin produced in such a reaction is about 99/ uroporphyrTr~ I. Further purification of this enzyme, porphobilin- oger1 deaminase, in this fraction was accomplished by mild heat treatment and zone electrophoresis. Purified preparations differ from the cruder ones in that, when they are used to catalyze the reaction, the appearance of porphy- rin lags far behind the consumption of PBG. As is shown in figure 4, using one such preparation, at the time of near exhaustion of the substrate less than

76 PART II. BIOSYNTHESIS OF HEMOGLOBIN 0.5 0.4 0.3 0.2 O. 1 \ PBG / ~ IBM Porphyrin x 4 30C 200 - <, a' 100 O 60 120 180 240 3< )0 minutes Ml NUTES 1 :~ PI - 60 0 :r At, , _ _. FIG. 3. (left) The course of the consumption of PBG and the appearance of uro- porphyrin I. The reaction shown here was catalyzed by PBG deaminase in a 40-50C/c ammonium sulfate fraction of an aqueous extract of spinach leaf tissue. FIG. 4. (right) Lag in production of porphyrin when using purified preparation of PBG deaminase. Compare with faster reaction shown in figure 3. 15% of the PBG consumed can be accounted for as porphyrin, while the maximum final yields of porphyrin have, in some experiments, approached l OO 'Jo . It was found ' ' later that, using either the crude or more purified preparations, anaerobic conditions have no effect on the rate of PBG con- sumption but the appearance of porphyrin is completely suppressed. During the consumption of PBG, either aerobically or anaerobically, one mole of ammonia is released for each mole of PBG which disappears. These data suggest that no oxidative step, e.g. the formation of a pyrrole aldehyde, need occur in the course of the condensation of PBG molecules to form the color- less intermediate. The following observations make it clear that this colorless material is uroporphyrinogen I, an interesting and important intermediate in porphyrin biosynthesis. It can be oxidized to uroporphyrin I rapidly by iodine, or, more slowly, by aerobic incubation, or an enzymatic oxidation can be accomplished by the addition of a small amount of crude deaminase preparation. (Thus, the crude deaminase preparations from spinach leaf tissue appear to contain two enzymes which are active in porphyrin biosynthesis, porphobilinogen deaminase and this oxidase, the specificity of which has not yet been determined). From the point of view of subsequent steps in porphyrin biosynthesis it is especially interesting that the colorless product of the deaminase reaction can serve as a substrate for enzymes present in frozen and thawed preparations of Chlorella which catalyze the synthesis of porphyrins with fewer than eight

BIOSYNTHESIS OF UROPORPHYRINOGENS BOGORAD 77 carboxyl groups per molecule. Since these enzymes cannot use uroporphyrin I as a substrate, it is obvious that this colorless material is not oxidized to uro- porphyrin I prior to its being acted upon by these enzymes. The data in TABLE II PORPHYRIN CC~VERSIONS BY FROZEN AND THAWED CHLORELLA PREPARATIONS*; Porphyrin Substrate Recovered ,uM x 4 Nature of porphyrins recovered* % % Uroporphyrin other soluble Porphyrins 5.76 AM PBG-equiv- alents from PBG + PBG-D incubation 4.21 18.1 81.9 5.52 AM PBG ~ 4.02 ~ 22.0 1 78.01 3.49 AM PBG equiv- alents as uroporphy- rinogen I 2.29 1 24.5 75.5 ~ 2.55 EM PBG equiv- alents as uroporphy- . ran I 2.14 1 100.0 0.0 * Frozen and thawed Chlorella preparation incubated with substrate anaerobically, then aerobically to oxidize porphyrinogens. -I Mostly coproporphyrin but includes traces of porphyrins with 2, 3, and .o - COOH groups/ molecule. Table II show that this colorless material and reduced uroporphyrin I, i.e., uroporphyrinogen I, serve equally well as substrates for these enzymes. These data, and others, show that the colorless product of the deaminase reaction is uroporphyrinogen I ~ fig. 5 ~ . Thus, judging from the fact that the product is the completely symmetrical uroporphyrinogen I, PBG deaminase appears to catalyze the linear con- densation of PBG molecules and ring closure of the tetrapyrrole but, as pointed out above, the arrangement of side chains on this isomer probably renders it valueless as an intermediate in the biosynthesis of protoporphyrin IX. Another enzyme, uroporphyrinogen isomerase, which participates in the synthesis of uroporphyrinogen III from PBG, has been partially purified from aqueous extracts of wheat germ. Crude aqueous extracts catalyze the consumption of PBG and the appearance of uroporphyrin III, sometimes mixed with uroporphyrin I, but two ammonium sulfate fractions are of par- ticular interest. One of these, Fraction B. catalyzes the consumption of PBG and the appearance of uroporphyrin III, sometimes mixed with uroporphyrin

78 PART II. BIOSYNTHESIS OF HEMOGLOBIN COOH COOH COO H COOH 1 1 1 1 COOH CH2 COOH CH2 COOH CH2 COOH CH2 CH2 CH2 CH2 CH2 CH2 C ~ 2 CH2 CH 2 OWL cili ~~ H: N N N H CH H H HCH HCH ~ ~ HCH \:H ~ ~ HI CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 COOH CH2 CH2 COOH CH2 COOH CH2 COOH COOH COO H COOH COOH lJROPoRPHyRlNoGEN m UROPORPHYRINOGEN I FIG. 5.—Structural formulae for uroporphyrin I and III. I. After aging for two days at 4°C. or heating for 15 minutes at 55°C. the capacity of this fraction for catalyzing PBG consumption is unaltered, but only uroporphyrin I is produced. This suggested that at least two enzymes might be involved in the biosynthesis of uroporphyrin III and that one enzyme is more heat labile than the other. Fraction C, from wheat germ, is of greater interest. Preparations of this fraction vary in their capacity to catalyze the consumption of PBG when incubated alone w-ith this pyrrole; some preparations are slightly active, while others possess no measurable activity. If, however, a preparation which is inactive by these standards is incubated with PBG deaminase prepared from spinach leaf tissue, as well as w-ith PBG, the substrate is consumed at a rate commensurate with the concentration of the deaminase but the III, rather than the I isomer of uroporphyrin, is produced. Up to 100/ of the PBG consumed has been accounted for as porphyrin in some of these experiments. The product has been characterized as uroporphyrin III by paper chromatography10 and melting point determinations of the octamethyl ester and by similar analyses of the methyl ester of coproporphyrin produced by partial decarboxylation of the uroporphyrin. ~ Pooled material from 5 experiments was estimated to contain 85-90 t70 uroporphyrin III and 10- 15~/o of the I isomer). As in the case of the enzymatic production of uro- porphyrin I, the activity of the deaminase-isomerase system is not impaired by anaerobiosis but, a~ain, the uroporphyrino~en, rather than uroporphyrin, accumulates. The uroporphyrinogen III so produced can be utilized by frozen and thawed Chlorella preparations as a substrate for the synthesis of a number of porphyrins, including protoporphyrin. (Also see reference no. 12~. Thus we are clearly on the right track for protoporphyrin biosynthesis.

BIOSYNTHESIS OF UROPORPHYRINOGENS BOGORAD 79 In order to determine whether both the deaminase and the isomerase need be present simultaneously for uroporphyrinogen III biosynthesis, or whether these enzymes can act serially on PBG, the following experiments were performed. In one group of experiments PBG was incubated with the deaminase anaerobically, and then, at the time of exhaustion of the substrate, uroporphyrinogen isomerase was added. This had no effect ore the nature of the product, i.e. uroporphyrin I was finally recovered. So the possibility of the shifting of acetic and propionic acid side chains as a mechanism of iso- merization appears to be as unlikely as would have been predicted. In another series of e~cperimer~ts it was found that the exposure of PBG to the isomerase first and then to PBG deaminase is an equally ineffective means of producing uroporphyrin III. Experiments were performed in which PBG was incubated with uroporphyrinogen isomerase for three hours at 37 °C. When the isomerase was inactivated by heating the solution at 5 5 ° C. for 30 minutes, PBG deaminase was added, and the solution was incubated, uroporphyrin I was recovered (fig. 6, no. 14~. Uroporphyrin I was also recovered where uroporphyrinoger~ isomerase was never included in the incubation mixture, (fig. 6, no. 18) or when the isomerase was inactivated by- heating before PBG was added to the solution (fig. 6, no. 18~. OR the other hand, as already described, when urops~rphyrinogen isomerase, and PBG are ir~cubated all together (fig. 6, no. 17~' uroporphyrin III is produced. Cooksor~ and Rimingtoni4 have suggested that the switching of the amino- methyl group from one a-position to the other of PBG might be involved ire, at least, the non-enzymatic synthesis of uroporphyrinogen III from PBG. Then one molecule of isoPBG could condense with three molecules of PBG, or a linear tripyrrole produced from three molecules of PBG, to make uro- porphyrinogen III. The results of the experiment described above clearly exclude the possibility that the isomerase might act to catalyze such a shift when PBG is incubated alone with it. (Such a shift would not be reflected . . O W IOB O . O ~ O O O O ~ ~ ~ ,°2. ~ ~ ,'. " ~ 13 14 ~16 17 18 Ui U ~ FIG. 6. Paper chromato- gram (Falk and Benson methodic ) of methyl esters from preincubation experi- ment. ( See text for treat- ments. ) Dotted lines show position of pigments at the beginning of the second de- velopment; solid lines show final positions. UI = uro- porphyrin I marker; IT III uroporphyrin III marker.

80 PART II. BIOSYNTHESIS OF HEMOGLOBIN in a change in the concentration of PBG using the Ehrlich p-dimethylamino- benzaldehyde assay). This is particularly obvious from the observation that uroporphyrin I is produced when the isomerase is inactivated after the first incubation with PBG but prior to the introduction of PBG deaminase (fig. 6, no. 14), while uroporphyrin III is recovered if the inactivation step is omitted ~ fig. 6, no. 13 ~ . Thus, uroporphyrinogen isomerase appears to be incapable of contributing to the synthesis of uroporphyrinogen I I I in the absence of the deaminase. The requirement for the presence, simultaneously, of PBG deaminase, uroporphyrinogen isomerase, and PBG for the enzymatic synthesis of uroporphyrinogen III is apparent. The next question which arises is: Is there any direct interaction between PBG and uroporphyrinogen isomerase ? At present only indirect evidence from kinetic studies is available. As I have indicated, the rate of consumption of PBG in the presence of the deaminase-isomerase system is not measurably different from the rate in the absence of the isomerase. This is true at relatively high substrate levels (100-400 ~g./ml.~; however, kinetic data reveal marked differences in the two reactions. Table III shows that there is a difference in the apparent TABLE III vm PBG-Deaminase + PBG 0.076 ~M/ml; PBG-Deaminase + PBG ~ Uroporphyrinogen Isomerase Ks 7.2 x 10 - 5 M/L. 0.125 ~M/ml. 10.4 x 10 - ~ M/L. Michealis-~:enten constant and that maximum reaction velocity is attained at a higher substrate concentration in the presence of both enzymes than when only the deaminase is present. (The uroporphyrinogen isomerase prep- aration used in these experiments failed to catalyze any measurable change in PBG concentration under these conditions when incubated alone with this pyrrole at any of the substrate concentrations studied). These data are compatible with the conclusion that there is a direct interaction between the isomerase and PBG at some point in the course of the synthesis of uropor- phyrinogen III and that this enzyme requires two substrates: PBG and some product of the action of PBG deaminase of PBG short of a cyclized tetra- pyrrole, presumably a linear di- or tripyrrole. Finally, what is the mechanism by which uroporphyrinogen III is assembled from PBG enzymatically? Dr. Shemin has introduced a scheme involving a tripyrrylmethane intermediate. This proposal13 and another one involving a "T" tetrapyrrylmethane intermediates appear to be in conflict with the observation that uroporphyrinogen III can be synthesized from PBG enzy- matically under anaerobic conditions, since the production of the "T" struc- ture from a di- or tripyrrylmethane and PBG would require an oxidation.; ~ See further qualifications and comment in Addendum at the end of this paper.

BIOSYNTHESIS OF UROPORPHYRIi\OGENS :~3OGOR.ND 8 The proposal of Cookson and Kimingtoni~t has already been mentioned. The present observations on the deaminase-isomerase system neither confirm nor contradict the principles of their group transfer hypothesis but do sug- gest that, if such a mechanism is involved in the enzymatic synthesis of uro- 1 o~-phyrinogcn III, the transfer of the a-substituent must, most likely, occur when both substrates of uroporphyrinogen isomerase ~ PBG and a linear polypyrrole formed from PBG by the action of PBG deaminase ~ are on the surface of the enzyme. This conclusion is supported by studies on the effects of certain PBG analogues on this system. The final cyclization might then be catalyzed by the isomerase, PBG deaminase, or by another enzyme which may be present in the wheat germ preparations, for it should be pointed out that, while the purified deaminase preparations most probably certain only one enzyme which is active in nornhvrin biosynthesis. i.e. PBG ~ . 1 1 ~ ~ deam~nase, wheat germ ~ Action A; may contain, in addition to uroporp~y- rinogen isomerase, other enzymes which are active in uronorobvrin III svn- J ~ 1 , thesis. It is also possible that "uroporphyrinogen isomerase" is, in fact, group of enzymes. The lability of the isomerase and technical problems in the ready determination of the relative proportions of uroporphyrins I and III in mixtures have thus far discouraged attendants to purify this material further. Summary: This report describes two enzymes involved in the synthesis of porphyrinogens f rom porphobilinogen. One of these, porphobilinogen deaminase, catalyzes the consumption of PBG and the production, from it, of uroporphyrinogen I. The second, uroporphyrinogen isomerase, appears to be unable to catalyze any modification of PBG when incubated with it alone, but, when this enzyme is incubated with PBG and PBG deaminase, uro- porphyrinogen III is produced. These enzymatic products can serve as sub- strates for enzymes present in frozen and thawed preparations of Chlorella which catalyze the synthesis of porphyrins with fewer than eight carboxyl groups per molecule. ADDENDUMS Two hvdrogens, one from the "free a-position" of PBG and one from the methane bridge of the di- or tripyrrylmethane, must be removed in order to form any of the "T" structures which have been suggested4~~3 as intermediates in the enzymatic synthesis of uroporphyrinogen III. The next step in such schemes, howsoever, involves a cleavage of the "T" intermediate by the ad- dition of one hydrogen to each of the products. Thus, there is no net oxi- dation, and an enzyme which could accept two hydrogens and then give them up to the final products of the reaction could act anaerobically; there- fore, the observation that uroporphyrinogen III can be formed enzymatically * This Addendum and its accompanying figure were not presented at the Con- ference but were later submitted as relevant "afterthoughts," particularly with re- gard to the "T" structures discussed in the paper.

89 PART II. BIOSYNTHESIS OF HEMOGLOBIN UROPORPHYRINOGEN I ~ 2NH~ P BG - Dl+ PBG H zN H 2C ~ C ~ C 13 H ~ 2 N H 3 -If+ Enz (UG-Ist) /~2NH2C9lC|Enz Hl:;3~C:H If- PBG H H C H \Enz+H2NH2C~CiNjlCN 2 ~Hl3 \ ~ ~ P = CH2 CH2 COOH PBG-D = PORPHOBILINOGEN DEAM I NASE = CH2 COO H -~H2NH2C(N\:C ; ~CH2NH2 ,l(PBG- D:) US - Is = UROPORPHYRINOGEN I SOMERASE URO POR PHYR I NOGEN m ~ 2 NH3 FIG. 7. Formula diagram for possible mechanism of biosynthesis of uroporphyrin ogen III f rom PBG. from PBG under strictly anaerobic conditions does not necessarily exclude the possibility of "T" pyrrylmethanes as intermediates. Another possible mechanism of uroporphyrinogen III biosynthesis from PBG could involve an enzyme with transpyrrylase activity (fig. 7 ). According to this hypothesis, first a linear tripyrrylmethane would be formed through the action of PBG deaminase on PBG. Then a transpyrrylizing enzyme would act to catalyze the exchange of a molecule of PBG for a dipyrrolic segment of the tripyrrole. This would then leave, in the reaction mixture, two dis- similar dipyrrylmethanes; the condensation of one molecule of each of these two dipyrrylmethanes would lead to the formation of uroporphyrinogen III. One of the condensations required is of the sort which PBG deaminase ap- pears to catalyze; the other condensation might possibly also be catalyzable by PBG deaminase, but another enzyme might be required. If this is the mechanism of uroporphyrinogen III biosynthesis, uroporphyrinogen isomerase acts as a transpyrrylase. Results obtained in experiments w ith PBG ana- logues argue against a dipyrrylmethane as the substrate for the transpyrrylase postulated here.

BIOSYNTHESIS OF UROPORPHYRINOGENS BOGORAD 83 REFERENCES 1. Granick, S.: Enzymatic conversion of 6-aminolevulinic acid to porphobilinogen, Science 120: 1105, 1954. Schmid, R., and Shemin, D.: The enzymatic formation of Porphobilinogen from 6-aminolevulinic acid and its conversion to protoporphyrin. J. Amer. Chem. Soc. 77: 506, 1956. 3. Gibson, K. D., Neuberger, A., and Scott, J. J.: The enzymic conversion of 6-amino- levulinic acid to porphobilinogen, Biochem. J. 58: xii, 1954. 4. Bogorad, L., and Granick, S.: The enzymatic synthesis of porphyrins from porphobilinogen, Proc. Natl. Acad. Sci. (U.S.) 39: 1176, 1953. 5. Foals, J. E., Dresel, E. J. B., and Rimington, C.: Porphobilinogen as a porphyrin precursor, and interconversion of porphyrins, in a tissue system, Nature 17~?: 292, 1953. 6. Schulman, M. P.: Enzymatic synthesis of Porphobilinogen from b-aminolevulinic acid and its conversion to porphyrins, Fed. Proc. 14: 277, 1955. 7. Granick, S.: The metabolism of heme and chlorophyll. In "Chem. Pathways of Metabolism" Vol. I, D. M. Greenberg, ea., Acad. Press, N. Y., 1954. 8. Bogorad, L.: Enzymatic synthesis of uroporphyrin, Fed. Proc. 14: 184, 1955. 9. Bogorad, L.: Intermediates in the biosynthesis of porphyrins from porphobilinogen, Science 121: 878, 1955. 10. Falk, J. E., and Benson, A.: Separation of uroporphyrin esters I and III by paper chromatography, Biochem. J. s5 101, 1953. 11. Bogorad, L.: The enzymatic synthesis of uroporphyrin III, Pi. Physiol. 30: xiv. 1955. 12. Neve, R. A., Labbe, R. F., and Aldrich, R. A.: Reduced uroporphyrin III in the biosynthesis of heme, J. Amer. Chem. Soc. 78: 691, 1956. 13. Shemin, D., Russel, C. S., and Abramsky, T.: The succinate-glycine cycle. I. The mechanism of pyrrole synthesis, J. Biol. Chem. ~?15: 613, 1955. 14. Cookson, G. fI., and Rimington, C.: Porphobilinogen, Biochem. J. 57: 476, 1954.

ENZYMATIC STUDIES OF PROTOPORPHYRIN SYNTHESIS* S. GRANICK Introduction. The enzymes of protoporphyrin biosynthesis may be di- vided conveniently into three groups for purposes of study: "Active' glycine ' "Active" succinate ~ ~ Air > PBG—URO-gen ~ COPRO-gen ~ PROTO Group I Group II Group III The first group is that which may be considered to convert "active glycine" and "active succinate" to lo-amino levulinate (bAL).i The second group con- verts bAL to coproporphyrinogen (COPRO-gen). The third group converts COPRO-gen to proto porphyrin. Groups I and III enzymes are connected with Articulates; group II comprises soluble enzymes. \N7e shall consider in turn the properties of the three groups of enzymes.) I. ENZYMES OF THE FIRST GROUP The first group of enzymes which cor~verts "active glycine" + "active succinate" to bAL is a complex of enzymes and enzyme systems.' We have been interested in this group because controls of porphyrin biosynthesis un- doubtedly reside here. For example, one would like to know what steps govern the differentiation of the proerythroblast into a cell in which the predominant syntheses are those of porphyrins and globin. Marked enhance- ment of porphyrin synthesis is also observed in certain Chlorella mutants and in a strain of Tetrabymena toward the end of the rapid phase of growth; here also some governing reactions toward porphyrin synthesis must be in- volved. To study this first group of enzymes we have followed protoporphyrin synthesis in chicken red cells with inhibitors in order to map out what possible enzyme systems might be involved. We were able to assign the action of these inhibitors to the enzymes that acted between glycine and SAL, or to the enzymes that acted between bAL and PROTO, by making tests with glycine or with bAL as substrates. From experiments with inhibitors one may obtain suggestive leads as to the enzymes involved. Because of the ~ These investigations were supported in part by a research grant from the Division of Research Grants and Fellowships of the National Institutes of Health, U. S. Public Health Service R. G. 4922. -t SAL— 6-amino levulinate, bAL-ase enzyme which converts bAL to PBG PBG porphobilinogen; PBG-ase enzyme which converts PBG to URO-gen, URO — uroporphyrin; IJRO-gen — uroporphyrinogen; U D-ase enzyme which decarboxylates URO-gen to COPRO-gen; COPRO coproporphyrin; COPRO- gen — coproporphyrinogen; PROTO — protoporphyrin; CoASH coenzyme A; GSH glutathione; EDTA ethylenediamine tetraacetate; Tris — tris hydroxy- methyl aminomethane. 84

BIOSYNTHESIS OF PROTOPORPHYRIN GRANICK 85 Glycine Active succinate ? fFyridoxal- P0~1 Glutamine >---^ c9AL l Liver factors, succinote O2 I~ CoASH ~ ~ ~ Cyt a3l FIG. 1. Some enzyme systems Y Lipoic ~~` c /~PO4 and substances involved In the con- TPP ~ " FADb | version of glycine to CAL. a:-] ~elogl utarate DP~H J Portion of ::lectron citric acid tron sfer c: cle system limited time available for presentation, only the summary of the results and the conclusions we have drawn from them are presented (fig. 1~. On the basis of centrifugation studies some of the enzymes involved in group I appear to reside in cell Articulates. Cyanide and CO inhibit bAL synthesis from glycine. Therefore an oxi- dative metabolism connected through cytochrome oxidase and the electron transport cytochromes is inferred. Dinitrophenol inhibits bAL synthesis. Therefore an oxidative phosphory- lation is assumed. Various members of the citric acid cycle when added as substrates to- gether with glycine increase the bAL synthesis. thus confirming the involve- ment of a citric acid cycle. an, . i, ~ ne electron transport system, oxidative phosphorylation, and the citric acid cycle are known components of mitochondria. Therefore one may con- clude that mitochondria play an active part in bAL synthesis. The action of malonate is not simply to block succinic dehydrogenase since SAL synthesis from glycine is inhibited equally whether succinate or a-keto- glutarate are added as substrates. If malonate inhibited only succinic deLy- drogenase one might have expected that SAL synthesis should not be inhibited when glycine and a-ketoglutarate were the substrates. Arsenite markedly inhibits bAL synthesis either when succinate or a-keto- glutarate are added as substrates. Arsenite combines with lipoic acid and relight block bAL synthesis in this manner. Shemin and Cumins concluded that the citric acid cycle at succinate can back up to form succinyl-CoA. Since bAL is not formed when either a-ketoglutarate, or succinate is added in the presence of arsenite, it may be that the "active succinate" is a lipoic derivative rather than a CoA derivative. Desoxypyridoxine inhibits bAL synthesis and the inhibition may be over- come by pyridoxal phosphate. This observation confirms the findings of Schulman and Richer that pyridoxal phosphate is necessary for bAL synthesis. We have also found that pyridoxal phosphate is required for SAL synthesis in homogenates of red cells. The early nutritional studies of Wintrobe

86 PART II. BIOSYNTHESIS OF HEMOGLOBIN and Cartwright5~6 demonstrated that the lack of vitamin Be led to the de- velopment of an anemia in which small pale erythrocytes were produced. V`7e may now interpret these findings as indicating that pyridoxal phosphate has two predominant effects, namely on porphyrin and on amino acid syn- thesis. The paleness of the red cells is due to the requirement of pyridoxal phosphate for SAL synthesis; the smallness of the cells is due to a decreased globin synthesis. The function of pyridoxal phosphate in bAL synthesis is unknown. Perhaps it serves at the step which links "active succinate" to "active glycine." Azaserine inhibits bAL synthesis. This inhibition is overcome by glutamine but not by pyridoxal phosphate or the aromatic amino acids. This result suggests that glutamine may be involved in bAL synthesis. Perhaps it might be involved in the formation of active glycine. Isonicotinic acid hydrazide also inhibits hAL synthesis but its inhibition is overcome by pyridoxal phosphate. Studies on hemolysates of chicken erythrocytes indicate that progressive enhancement of protoporphyrin synthesis may be obtained by the addition of the following substances: glycine, pyridoxal phosphate, some non-protein fractions from pig liver. In addition, slight increases were occasionally ob- served with inosine and with CoASH. None of the other coenzymes which were added was found to enhance the synthesis. Control mechanisms which may be involved in porphyrin synthesis are suggested by the inhibitory effects of certain amino acids and keto acids. When chicken red cells are incubated with glycine and arty one of the fol- lowing amino acids in equimolar concentration: serine, alanine, proline or arginine, the PROTO synthesis is decreased by 30 - 50 per cent; still greater inhibition is found in the presence of cysteine. It is probable that the inhibition is not by way of competition for pyridoxal phosphate since aspartate and glu- tamate, among others, did not inhibit. On the basis of these results one might hypothesize that when such amino acids as serine, cysteine, etc. become limiting, a partial block to porphyrin formation might be removed. Pyruvate and a-keto butyrate also inhibit SAL synthesis. It is suggested that these keto acids, and perhaps ot'~e's, might compete faith a-ketoglutarate for coenzymes. II. ENZYMES OF THE SECOND GROUP lathe enzymes of this group are all soluble, and serve to convert bAL to coproporphyrinogen.7 Three colorless enzyme fractions have been separated by zone electrophoresis on starchS at pH 7.65 in tris buffer, ionic strength 0.16 at 90mA, 5°C., and 3.1 NT/cm drop across the starch block. The enzymes of interest migrate toward the anode, and hemoglobin migrates toward the cathode. Figure 2 is an example of the enzyme fractions separated from a chicken red cell supernatant solution. Red cells from three species were

BIOSYNTHESIS OF' PROTOPORPHYR1N GRANICK Log n~9 3 prote~n De- ccrboxyl(!se P3G-ose bAL-a<~e 2 1 0 1 08 045 - Cm. 5 10 ~ ~ ~L ~ 1 ~L J , 20 25 30 35 ~ F`IG. 2. Zone electrophoresis on a starch blocl~ 2 cm. thick, 15 cm. wide, 45 cm. long. A supernatant solution of a chicken red cell hemolysate was applied as a streak across the width of the block at "Origin." The activities of eluates from 2 cm. wide seg- ments of the starch block and their protein contents are shown at the end of the run. Quantitative activities of bAL-ase and PBG-ase are presented (see text) and also the qualitative distribution of UD-ase (decarboxylase). examined: rabbit reticulocytes, chicken erytl~rocytes and cells from a case of erythroblastosis foetalis. hAL-ase. One fraction, bAL-ase, converts bAL to PBG. Another fractior~, PBG-ase, converts PBG to uroporphyrinoger1 (URO-gen). The third frac- tion, uroporphyrinogen decarboxylase (UD-ase), converts URO-gen to cop- roporphyrinogen ~ COPRO-gen ~ ~ fig. 3 ~ . The rabbit preparation yielded Group lI 2 `3A~ ~AI~-ase P~G deomin~x COOH COOH CH2 1 1 CH2 CH2 H2~- H2C N H porphobilinogen (P :ES G) A: P ] H2 N-H2C hl ~ pyPryl methone_ ~ C 1 + n NH3 Pr, tl Ac AC~/ ~P~ Intermedicte Ac4~// \^Pr deaminox N HN thene oxid tion N~ N C Ch2 ~ product HC~ ~C ~C~ (500 my' band) ~¢ h'~Ac Ac H2 Pr AC H Pr, Uroporphyr~in ~ (Uro I) Uropoophyrinogen I (lJ~o- 9en 1) ~r' 11 AC _~/C~_ _ I 13omerasc -' ~ H2C ~ ~ Ch2 ~ _ ~C; Pr' H2 Pr, Uro-gen ]lI ldecanboxylase C2 C~3 ~ ~ N H3C <N ~CH3 ~ ~2 P., Coproporphyr~inogen III (copro-gen ]11) Pr, ~ AC _ Ac / ~pc Methenc t1c ~ N C _ Ac¢\ /;AC Pr~ t1 Pr~ Uro ]lI ~ethene ~_ n:, ~3C¢\ ~C~3 Pr, ~ P~ Copro ]]I FIG. 3.—Interpretation of steps in the conversion of bAL to COPRO-gen by soluble enzymes of erythrocytes separated by z,one electrophoresis.

88 PART II. BIOSYNTHESIS OF HEMOGLOBIN eluates from the starch which possessed tile highest activities. The maximum bAL-ase activity was 0.8 ,uMoles PBG formed per hour per ma. protein. The maximum PBG-ase activity was 0.44 Moles PBG decrease per hour per ma. protein. The maximum UD-ase activity was 0.009 Moles COPRO- gen formed per hour per ma. protein from URO-gen as substrate. The relative rates of migration toward the anode of the rabbit enzymes was bAL-ase > UD-ase > PBG-ase; of the chicken enzymes bAL-ase ~ PBG- ase > UD-ase; and of the human, UD-ase > PBG-ase > DAL-ase. COON COOH 1 1 COOH CH2 COOH CH2 I A I Ch2 CH2 C OCHER --CO ~ ~ H2N-H2C-CO B ~ H2 H~N-CH2~ iLH NH2 N FIG. 4. Hypothesis of action of bAL-ase at the active site. The properties of bAL-ase have been studied kinetically and with inhibitors. A hypothesis of the action of bAL-ase is presented in figure 4. The kinetic studies may be interpreted as indicating that one SAL is held more firmly than the second SAL, as judged from the slope of the curve of rate vs. log bAL concentration at low (10-4M) and at higher concentrations. From in- hibitor studies it is inferred that the attachment of bAL to the active site is relatively strong at the carbonyl group, relatively weak at the carboxyl group and negligible at the NH group. After both bAL molecules have been ad- sorbed on the active site, a l~etimine condensation should occur readily and spontaneously at B since pyrazine formation between two bAL molecules is known to occur readily.9 Ketimine formation occurs only with unionized NEIL groups; the decrease in bAL-ase activity below pH 6 may be due in part to the decrease in the unionized NH groups of SAL. (In addition there is a temperature-dependent denaturation below pH 6~. It is necessary to postulate some mechanism for the activation of the hy- drogen at C. A strong positive charge in the neighborhood of this atom might serve for activation. Such a positive charge might be imparted by a di- or trivalent metal ion; however no evidence for a metal ion activation has been obtained. (Studies of the inhibition of bAL-ase activity by EDTA in- dicate a reversible inactivation of a structurally-modified form of CAL-ase.) Thus the formation of the two bonds are interpreted to be due to a spon- taneous ketimine condensation at B and an enzyme activated condensation at A. PBG-ase. When a specific enzyme fraction prepared by zone electro- phoresis acts on PBG' the colorless compound URO-gen isomer type III is

BIOSYNTHESIS OF PROTOPORPHYRIN GRANICK 89 formed. However when this fraction is heated for an hour at 55°C. this heated fraction now converts PBG to URO-gen isomer type I. The result of the heat treatment is similar to that found in Chlorella.~° The erect of heat treatment suggests that the PBG-ase fraction consists (_7 r ot two enzymes. ()ne enzyme, "deaminase," may functior~ to form methane bonds between adjacent PBG molecules with the concomitant elimination of NH; (fig. 3~. If this action were to continue, the symmetrical URO-gen I should result, which is not further changed by the PBG-ase preparation. The second enzyme, "isomerase," would act on a product of the deaminase to convert it to URO-gen III. The isomerase enzyme is probably the one that is readily destroyed by heat. With spinach extracts Bogoradi~ has been able to separate two activities: one which results in the conversion of PBG to URO I, and the other which appears to act on some intermediate stage of the condensation to convert the intermediate to URO III. In the disease of chronic porphyria)'' which is due to a recessive gene, large amounts of URO I and COPRO I are excreted. :From the above con- siderations it is suggested that this metabolic lesion may be due to a diminu- tion in the active "isomerase" enzyme. Uroporphyrinogen decarboxylase (UD-ase). When a specific fraction prepared by zone electrophoresis acts on URO-gen then COPRO-gen is formed.~3 The enzyme fraction is incubated anaerobically in the presence of 0.005M GSH and URO-gen III (prepared from URO by reduction with sodium amalgam) at pH 6.8. at 38°C. in the dark. The results of the decar- boxylation are shown in figure 5. During the reaction the intermediate 7-, 6- and 5-carboxylic porphyrinogens remained at a low, roughly steady-state con- centration while the 4-carboxylic compound COPRO-gen continued to accum- ulate. The porphyrinogens were quantitatively assayed by titration with iodine. The oxidation of porphyrinogens with iodine and their autoxidative sensitivity to light were reported by Watsoni4 and are confirmed by the present quantitative studies. ~ ~ c: Q c' c' . . o ~ ~ ~ o 8 ~ ~ ~ UPO 6X10-5 6X10-5 6X10 5M 6X10-611 1hI? 4 hr. 23 Ire. 1 hr. 23 hr. NOenZYme~ 23 hr. 6X10-6~ 6X10-5 ~ CONDO FIG. 5. Action of UD-ase on URO-gen at indicated concentrations to form decar- boxylated porphyrinogens. The porphyrinogens were oxidized with iodine and the porphyrins separated by paper chromatography in a lutidine-aqueous NH3 system.

9o PART II. BIOSYNTHESIS OF HEMOGLOBIN The following properties of UD-ase from rabbit reticulocytes have been determined. The enzyme is an -SH enzyme whose activity is decreased by -SH-binding reagents and protected by GSTI. The apparent Michaelis con- stant for the enzyme is < 10-5M, and the optimum pH is 6.8. The enzyme decarboxylates all the four URO-gen isomers; the relative order of efficiency is III > IV > II -- I. Isomer III is decarboxylated about twice as fast as I. Only the fully reduced porphyrins and not the intermediate states of oxi- dation (pyrromethenes) are acted upon by this enzyme fraction. This con- clusion is based on kinetic studies and on the fact that the presence of either sulfite or dithionite, which combine with the pyrromethenes, does not affect the rate of decarboxylation. Leo isomerization of the porphyrinogens occurs either on reduction with sodium amalgam, on incubation with the enzyme fraction, or on oxidation with iodine. The enzyme fraction does not de- carboxylate bAL, PBG, URO or indole acetic acid. III. ENZYMES OF THE THIRD GROUP The conversion of COPRO-gen to PROTO is brought about by cell Articulates of the red cell and of Euglena but the intermediate steps are still unknown. Of appears to be necessary, but the reaction is not cyanide sensitive; therefore cytochrome oxidase is probably not involved. COPRO is not attacked but only the fully reduced COPRO-gen. Comparison of the behavior of COPRO and PROTO on reduction indicates that reduced PROTO is more readily autoxidizable than reduced COPRO under the . . same cone ltlons. One interesting problem in connection with studies on porphyrin biosyn- thesis is the localization of this biosynthetic chain in the cell. Two proto- plasmic organelles which contain porphyrin compounds are the mitochondria with their cytochromes, and the chloroplasts which, in addition to chlorophyll, also possess cytochromes. The fact that bAL synthesis from glycine requires mitochondrial enzymes and that cell Articulates are involved in the con- version of COPRO-gen to PROTO suggests that the synthesis of PROTO might be a function of such organelles. In this regard it would be interesting to know whether the soluble enzymes, which convert bAL to COPRO-gen, are also concentrated in these bodies. Another interesting problem is determining the control mechanisms and protective devices for maintenance of porphyrin biosynthesis. Suggestive of some control is the inhibitory action on SAL synthesis of certain amino and keto acids. For maintenance, an obvious mechanism is the high GSH content of the erythrocyte. Not only does GSH serve to maintain the activity of the -SH enzymes (bAL-ase and UD-ase) but GSH also serves to decrease the autoxidation of the porphyrinogens. It has also been observed that the high protein content of red cells would serve to stabilize bAL-ase, which is un-

BIOSYNTHESIS OF PROTOPORPHYRIN—GRANICK 91 stable in dilute solutions, and would also serve to protect PBG from under- going condensation and decompositior~ reactions. IV. SUMMARY The enzyme systems which take part in the conversion of glycir~e to PAL include the citric acid cycle, the electron transport system, and oxidative phosphorylation. These systems are presumably localized in mitochondria. In addition to the compounds required for the above systems, studies with inhibitors and red cell hemolysates implicate the following compounds in this synthesis, namely: pyridoxal phosphate, glutamine, inosine, and some as yet unidentified non-protein liver factors (fig. 1~. The conversion of bAL to COPRO-gen has been shown to take place via three soluble enzyme fractions which have been separated from red cells by zone electrophoresis (fig. 2~. One fraction unites two bAL molecules together to form PBG (fig. 3~. Studies on the mechanism of this reaction are reported (fig. 4~. Another fraction appears to contain two enzymes, one of which, a deaminase, is hypothesized to unite PBG molecules to form a pyrro-methane type intermediate which is then acted upon by another enzyme, isomerase, to form URO-gen III. The third fraction contorts a decarboxylase which acts on URO-gen to decarboxylate the acetic side chains to methyl groups, to result in the formation of COPRO-gen (fig. 5~. The conversion of COPRO-gen to PRC)TO requires cell Articulates arid Or. It is not poisoned by cyanide. REFEREN CES 1. For a review of the pertinent literature see: Wolstenholme, G. E. W., and Millar, E. C. P., eds.: Ciba Foundation Symposium on Porphyrin Biosynthesis and Metabolism. London, Churchill; Boston, Little, Brown and Co., 1955. 2. Granick, S.: Enzymes in the early steps of porphyrin biosynthesis to delta-amino levulinate. In preparation. 3. Shemin, D., and Kumin, S.: The mechanism of porphyrin formation. The forma- tion of a succinyl intermediate from succinate, J. Biol. Chem. 198: 827, 1952. 4. Schulman, M. P., and Richert, D. A.: An effect of pyridoxal-5-phosphate in vitro on heme synthesis and CO2 production from glycine-2-C-14, J. Am. Chem. Soc. 77: 6402, (December 5) 1955. 5. Wintrobe, M. M.: Factors and mechanisms in the production of red corpuscles, Harvey Lectures 45: 87, 1949-50, Academic Press, New York. 6. Cartwright, G. E., and Wintrobe, M. M.: Studies on free erythrocyte protopor- phyrin, plasma copper, and plasma iron in normal and pyridoxine-deficient swine, J. Biol. Chem. 172: 557, 1948. 7. Granick, S., and Manzerall, D.: Enzymes that convert delta-amino levulinate to coproporphyrinogen. In preparation. 8. Kunkel, H. G.: Zone electrophoresis, Methods of Biochem. Analyses 1: 141, 1954. 9. Gibson, K. D., Neuberger, A., and Scott, J. J.: The purification and properties of delta-aminolaevulic acid dehydrate, Biochem. J. 61: 618, 1955. 10. Bogorad, L., and Granick, S.: The enzymatic synthesis of porphyrins from por- phobilinogen, Proc. Nat. Acad. Sci. 39: 1176, 1953.

92 PART II. BIOSYNTHESIS OF HEMOGLOBIN 11. Bogorad, L.: Intermediates in the biosynthesis of porphyrins from porphobilinogen, Science 121: 878, 1955. 12. Rimington, C.: Haems and porphyrins in health and disease, I., Acta Med. Scand. 143: 161, 1952. 13. Mauzerall, D., and Granick, S.: Uroporphyrinogen decarboxylase. In preparation. 14. Watson, C. G., deMillo, R. P., Schwartz, S., Hawkinson, V. E., and Bossenmaier, T.: Porphyrin chromogens or precursors in urine, blood, bile, and feces, J. Lab. and Clin. Med. 37: 831, 1951. DISCUSSION Dr. Herbert C. Schwartz: We have heard Dr. Shemin's, Dr. Bogorad's and Dr. Granick's interesting accounts of their studies on the enzymes in- volved in biosynthesis of heme. I would like to tell you briefly about some of our studies. Dr. Cartwright, Dr. Wintrobe and I have studied the biosynthesis of heme from protoporphyrin and iron in a chicken hemolysate system. The per cent of added radioiron incorporated into heme (per cent uptake of Fe59) was used as a measure of heme synthesis. Our studies have suggested that this reaction is enzyme dependent. In a typical experiment, the incubation of hemolysate + protoporphyrin + Fe59 gave 13.5 per cent uptake of Flew, whereas pro- toporphyrin + Fe59 (without hemolysate) or hemolysate + Few (without protoporphyrin) gave less than 1 per cent uptake of :Fe59. Prom such a hemolysate an active, particle-free preparation was obtained by making the hemolysate isotonic with potassium chloride and homogenizing for 10 minutes in a Waring Blendor, as suggested by Dr. Shemin. The super- nate, after centrifugation at 100,000 x G. had two to three times the activity of the original hemolysate. Subsequent studies were performed on this soluble preparation. 50 4 30 A: 20 10 o 1 1 1 7.5 8.0 8.5 9.0 pH FIG. 1. Relation of uptake of Fe59 to pH. ?~ j i: \ ~ 10W / s _ Let/ 1 1 1 1 1 \t . 1. 4 5 6 7 8 9 10 P ~ FIG. Z. --Stability of iron uptake related to pH over 3-hour period.

DISCUSSION ~3 Optimal activity (fig. 1) was at pH 7.8. Optimal stability for three hours (fig. 2) was at pH 7.4. Time studies (fig. 3) showed the per cent uptake of Fe59 to be maximal in three to four hours and to be a linear function of time over the first 30 minutes. The rate of heme synthesis at 30 minutes was pro- portional to enzyme concentration. The preparation was not storable at 5° C., - 3(~° C., or lyophilized and was found to be inactivated after heating at 56° C. for 30 minutes. 35: 30 25 2C _ A ;~1^ _ 0 1 1 1 2 3 TIME tnourl) 1 / 1 1 4 S FIG. 3. Uptake of Fe5'0 with time. The effect of thiol inhibitors is shown in table I. There was marked in- hibition with 1 x 10-M p-chloromercuribenzoate (PC~:B) and 1 ~ 10-2M iodoacetamide, and slight inhibition with ~ x 10-3M p-chloromercuribenzoate and ~ x 10-3M iodoacetamide. TABLE I EFFECT OF THIOL INHIBITORS do Uptake Fe59 RIO Inhibition Control 3 S PCME ( 1 x 10 - 2M) 7 80 PCME (1 x 10 - 3M) 33 7 PCMB (1 x 10 - 4M) 35 0 lodoacetamide ( 1 ~ 10 - 2M) 22 37 Todoacetamide ( 1 x 10 - 3M) 32 9 fodoacetamide ( 1 ~ 10 - 4M) 3 S 0 When dialyzed against water for 22 hours (table II), there was a 70 per cent loss of activity. The original activity could be restored by reconsti- tution of the dialyzed sample with a heat-inactivated preparation or by the addition of 1 x 10-~M glutathione. While these studies were in progress, Krueger, ~:elnick and Klein inde- pendently published similar studies on the biosynthesis of heme from iron and protoporphyrin. Except for the dialysis data, their findings are essentially in agreement with ours,

94 PART II. BIOSYNTHESIS OF HEMOGLOBIN TABLE II % Uptake Few Experiment A Experiment B Control 29 52 Dialyzed (INTO) 9 16 Reconstituted 27 Glutathione ( 1 ~ 10 - 3M) 19 Glutathione ( 1 x 10—~M) 52 REFERENCES 1~ Goldberg, A., Ashenbrucker, H., Cartwright, G. E., and Wintrobe, M. M.: Studies on the biosynthesis of heme in vitro by Adrian erythrocytes, Blood 11: 821, 1957. 2. Schwartz, H. C., Cartwright, G. E., and Wintrobe, M. M.: Studies on the syn- thesis of heme from protoporphyrin, Clin. Research Proceedings 5: 29, 1957. 3. Shemin, D., Abramsky, T., and Russell, C. S.: The synthesis of protoporphyrin from delta-amino levulinic acid in a cell-free extract, J. of the Am. Chem. Soc. 76: 1, 1954. 4. Krueger, R. C., Melnick, I., and Klein, J. R.: Formation of heme by broken-cell preparations of duck erythrocytes, Arch. of Biochem. and Biophys. 64: 302, 1956.

THE ROLE OF IRON IN HEMOGLOBIN SYNTHESIS CLEMENT A. FINCH In comparison with the preceding elegant chemistry, this discussion cannot help but seem both diffuse and obscure. Having made the obvious statement that iron is essential for heme formation and after a few random observations on the behavior of the cell in accomplishing this synthesis, I find that most of my own thinking on this subject extrapolates to hemoglobin production in the intact animal. An attempt will be made to summarize briefly the information relating to the role of iron in hemoglobin synthesis at both a cellular level and as it functions in the total erythropoiesis of the intact organism. At a cellular level the pathway of iron incorporation into hemoglobin may be visualized as starting with the presentation of iron to the cell. In vitro various soluble ferrous or ferric salts of iron are easily assimilated by immature erythrocytes. Some iron chelates such as iron ascorbate or citrate permit uptake, whereas others such as ethylenediamine tetraacetate do not. In viva the transport vehicle is an iron-binding plasma protein called trans- ferrin or siderophilin.~ While this transport globulin may be considerably reduced in such diseases as nephrosis and infection, this decrease has never been demonstrated to be a limiting factor in the delivery of iron to the marrow. All immature red cells through the reticulocyte stage are able to take up iron, while mature erythrocytes do note 3 The pronormoblast and baso- philic normoblast have the greatest capacity to assimilate iron as determined by autoradiograph.4 5 This uptake of radioiron continues in situations where heme synthesis within the cell is arrested by lowering the temperature, or by addition of cyanide or deoxypyridoxine.6 Iron transfer from transferrin to the immature erythrocyte in vitro is reported to be decreased when the per cent of saturation of the iron binding protein drops below 30~.7 The iron assimilated by the developing red cell is either converted to heme, temporarily stored, or remains permanently as a non-heme f faction within the erythrocyte. Storage iron may be demonstrated as specific granules within the developing red cell. Such cells have been referred to as sidero- blasts when nucleated, and siderocytes when non-nucleated.S 9 These iron stores may be useful to insure adequate available iron during the phase of maximal heme synthesis within the cell. There is evidence that their amount may be influenced by the level of plasma iron, and that they may in turn in- fluence the amount of residual non-heme iron found in the adult circulating erythrocyte.6 In avian and mammalian red cells this fraction may be as much asSor10~.6 it The red cell nucleus would seem to be intimately concerned with hemo- globin synthesis. Appreciable amounts of radioiron are found in the nucleus as

96 PART II. BIOSYNTHESIS OF HEMOGLOBIN shortly after its uptake by the cell.)' i~ i4 At the time when the nucleus dis- appears from the cell, heme synthesis is nearly complete. The biochemical aspects of heme synthesis have already been discussed at some length from the standpoint of the porphyrin moiety. Granick has pre- sented evidence from studies of porphyrin synthesis by bacteria that vinyl groups of the porphyrin ring are essential for insertion of iron.~5 Goldberg et al. from studies employing a hemolysate system of erythrocytes suggest that iron enters at the protoporphyrin stage of pyrrole synthesis. General observations of conditions required for iron incorporation into heme in vitro with intact cells have been presented by Sharpest and by fensen and asso- ciates: which serve to distinguish this process by its enzymatic character from the uptake of iron by the cell. In man there are both hereditary anomalies and acquired defects in red cell production which, if understood, should do much to clarify the mechanics of heme synthesis and the role of iron in this process. Most helpful in ap- praising heme synthesis in To has been the cell content of hemoglobin pre- cursors either in the marrow or in circulating erythrocytes. Normally these are nicely regulated so that stockpiles of porphyrin, iron and globin become depleted as hemoglobinization of the cell is completed (fig. 1~. In specific NORMOBLAST ~ RETI CU LOCYTE Pro Baso Eosino Nucleic Acid Iron Granules Porphyri n Globin Hemoglobi n FIG. 1.- Diagram showing depletion of hemoglobin precursors and nucleic acid with normal formation of hemo- globin. abnormalities a deficiency or excess of these may be found. For example, ashen the supply of iron to the marrow is restricted, iron granules (storage irony within the normootast disappear, protoporpnyr~n accumulates and the total hemoglobin production is reduced, resulting in a hypochromic, microcytic erythrocyte. One interesting aspect of chronic iron deficiency anemia is that erythroid hyperplasia is less marked than with acute blood loss anemia or with hemolytic anemias. This raises the possibility that iron may be of importance in multiplication of erythroid cells as well as in heme syn- thesis. In the intact animal a consideration of the role of iron includes a considera- tion of iron supply to the marrow. Normally, this is largely derived from a O 1 1 , At' . 1 · 1 . 1

IRON IN HEMOGLOBIN SYNTHESIS FINCH 97 recircuiting of iron from senescent red cells processed by the reticulo-endo- thelial cells and returned through the plasma to the marrow. Studies of the reticulo-endothelial cell indicate that not only is this done within a matter of minutes or a few hours, but also that in the event of an increased need for iron above that provided by erythrocyte catabolism, additional iron is mobilized from body cells. Thus, following acute hemorrhage plasma iron rises at a time when marrow uptake is increasing. While infection would appear to impair reticulo-endothelial mobilization of iron, even here we have been unable to obtain convincing evidence that erythropoiesis is impaired by inadequate iron supply. Thus we conclude that if adequate iron exists in the body, transport to the marrow will be effected. One of the most useful techniques for measuring hemoglobin synthesis in the intact animal or man involves the use of radioiron. The plasma iron turnover, i.e., the amount of plasma iron being fed to tissues, has been shown largely to reflect marrow uptake.~7 The amount of iron appearing in circu- lating erythrocytes indicates how much of this iron was synthesized into hemoglobin within viable erythrocytes. The marrow transit time of radioiron indicates the time required for the process of iron incorporation and red cell maturation.iS Studies of this type indicate that in certain anemias—such as thalassemia, pernicious anemia and some "refractory" anemias with cellular marrow— the iron uptake by the marrow far exceeds the production of viable red cells.~'3 Further study is required to determine to what extent this "ineffective" ery- thropoiesis reflects a defect in fabrication of the cell as compared to abnormality in heme synthesis. Marrow iron transit time is also greatly shortened in certain hemolytic anemias and marrow abnormalities. Part of this is due to the premature delivery of young cells into circulation. Indeed, the total reti- culocyte pool of the marrow may be shifted to the blood under certain con- ditions. It is not known whether or not an acceleration in the rate of heme synthesis within the individual cell also contributes. We might consider what clinical diseases may be associated with abnormali- ties in heme synthesis. This is well documented in lead poisoning where i'2 vitro impairment of porphyrin synthesis has been demonstrated, and where red cells contain unused precursors of heme synthesis, i.e., porphyrin and iron. Likewise, these are isolated case reports in which excess non-heme iron deposits and red cell hypochromia have been demonstrated,20 which strongly imply a primary disturbance in heme synthesis. On the assumption that a decreased hemoglobin concentration within the erythrocyte indicates specifically impaired hemoglobin synthesis, attention has been directed toward thalassemia, pyridoxine and copper deficiencies. Hypochromia may also be seen in patients with hemolytic anemia, adequate iron stores, low plasma iron, but rapid marrow iron turnover. We have encountered this in acquired Coombs-positive hemolytic anemia, sickle-cell anemia and myelofibrosis with myeloid metaplasia.

98 PART II. BIOSYNTHESIS OF HEMOGLOBIN Erythropoiesis does not appear to be influenced in these patients by iron ad- ministration, but heme synthesis may be relatively curtailed by limited red- cell iron stores in similar fashion to that observed in v~tro.6 7' 21 Some caution, however, should be exercised in considering hypoctiromia as synonymous with impaired heme synthesis. Ire recent studies carried out in collaboration with Dr. Phillip Sturgeon, it has been found that iron turn- over, presumably reflecting for the most part heme synthesis, reached maximal levels in thalassemia despite the marked hypochromia of this diseased The relation of microcytosis without hypochromia to heme synthesis is even less clear. In these remarks no attention has been directed to the abundant informa- tion relating to pathogenesis and recognition of the iron-deficient state which after all is the one condition in which the role of iron in heme synthesis is clearly illustrated. This would seem to represent secure ground from which we should look to more unsettled areas. At present we cannot discern any Fraternal breakdown in iron metabolism, either of iron transport or of cellular handling of iron. There are, however, indications of block in heme synthesis in the accumulations of precursors of the heme molecule in a number of diseases. The ferrokinetic studies which have done much to characterize marrow iron turnover in the intact animal now need to be extended to the specific chemical reactions of the cell. Only then will the full significance of the gross chemical and morphologic abnormalities in else red cell be under- stood. REFEREN CES 7. 8. Laurell, C. B.: What is the function of transferrin in plasma? Blood 6: 183-187, 1951. Hahn, P. F., Ross, J. F`., Bale, W. F`., Balfour, W. M. and Whipple, G. H.: Red cell and plasma volumes (circulating and total) as determined by radio iron and by dye, J. Exper. Med. 75: 221, 1942. 3. Lash, R. J., Thomas, E. D., Chow, S. K., F`luharty, R. G. and Finch, C. A.: Iron metabolism. Heme synthesis in Vitro by immature erythrocytes, Science 110: 396-398, 1949. 4. La jtha, L. G. and Suit, H. D.: Uptake of radioactive iron (Fe59) by nucleated red cells in Vitro' Br. J. Haemat. 1: 55-61, 1955. 5. Kraus, L. HI. and Morrison, D. B.: In Vitro incorporation of iron59 into hemo- ~lobin S visualized by autoradiography, Proc. Soc. for Exper. Biol. and Med. 89: 598-602, 1955. 6. Jensen, W. N., Ashenbrucker, H., Cartwright, G. E. and Wintrobe, M. M.: The ur!tal~e in Vitro of radioactive iron by avian erythrs~cytes, J. Lab. Clin. Med. 42: 833-846, 1953. . Jandl, J. H., Inman, J. K. Sirr.mons, R. L.: Transfer of iron and cobalt from serum iron-binding protein to human reticulocytes, Clin. Res. Proc 5: 144, 1957. Douglas, A. S., Dacie, J. V.: The incidence and significance of iron-containing granules in human erythrocytes and their precursors, J. Clin. Path. 6: 307-313, 1953.

IRON IN HEMOGLOBIN SYNTHESIS FINCH 99 9. Kaplan, E., Zuelzer, W. W. and Mouriquand, C.: Sideroblasts. Study of stainable nonhemoglobin iron in marrow normoblasts, Blood 9: 203-213, 1954. 10. Jensen, W. N., Bush, J. A., Ashenbrucker, H., Cartwright, G. E. and Wintrobe, M. M.: The kinetics of iron metabolism in normal growing swine, J. Exper. Med. 103: 145-159, 1956. 11. Sharpe, L. M., Krishnan, P. S. and Klein, J. R.: Uptake by duck erythrocytes of iron added to blood, Arch. Biochem. and Biophys. 35: 409, 1952. 12. Mills, fI., Huff, R. L., Krupp, M. A. and Garcia, J. F.: Hemolytic anemia sec- ondary to a familial (hereditary) defect in hemoglobin synthesis, Arch. Int. Med. 86: 711-726, 1950. 13. Metcalf, W. K.: A simplified technique of spectrography and its application to the study of intracellular hemoglobin, Blood 6: 1114-1122, 1951. 14. Allfrey, V. and Mirsky, A. E.: The incorporation of Ni5-glycine by avian ery- throcytes and reticulocytes in vitro, J. Gen. Physiol. 35: 841-846, 1952. 15. Granick, S.: The structural and functional relationships between heme and chlorophyll, Harvey Lectures 44: 220-245, 1948, Academic Press, New York. 16. Goldberg, A., Ashenbrucker, H., Cartwright, G. E. and Wintrobe, M. M.: Studies on the biosynthesis of heme in Vitro by avian erythrocytes, Blood 9: 821-833, 1956. 17. Bothwell, T. H., Hurtado, A. N1., Donohue, D. M. and Finch, C. A.: Erythro- kinetics IV. Plasma iron turnover as a measure of erythropoiesis. Blood (in press ) .- 18. Finch, C. A.: Marrow iron turnover. Int'l Cong. of Hemat., 1954. 19. Giblett, E. R., Coleman, D. H., Pirzio-Biroli, G., Donohue, D. M., Motulsky, A. G. and :Finch, C. A.: Erythrokinetics: Quantitative measurements of red cell pro- duction and destruction in normal subjects and patients with anemia, Blood 4: 291-309, 1956. 20. Garby, L., Sjolin, S. and Vahlquist, B.: Chronic refractory hypochromic anaemia with disturbed haem-metabolism, Brit. J. Haemat. ]: 55-67, 1957. 21. Krueger, R. C., Melnick, I. and Klein, J. R.: Formation of heme by broken-cell preparations of duck erythrocytes, Arch. Biochem. and Biophys. 64: 302-310, 1956. 2Z. Sturgeon, P. and Finch, C. A.: Erythrokinetics in Cooley's anemia, Blood 73, 1957. · 64-

THE ROLE OF COPPER IN ERYTHROPOIESIS~ GEORGE E. CARTWRIGHT, CLARK J. GUBLER AND MAXWELL M. WINTROBEt Introduction. For the past eight years studies or the role of copper In erythropoiesis have been conducted in our laboratory in collaboration with Drs. Gubler and Wir~trobe. We have been assisted in this work by M. E. Lahey, \1. S. Chase, .r. A. Bush, W. N. Jensen, l. W. Athens, Helen Ashenbrucker and H. Markowitz and the results of our work have been published in detail in a series of articles.~~S The purpose of the present paper is to summarize these studies. Our research confirms in great part and e~cter~ds the much earlier observations of the Wisconsin group.9 A Pertinent literature on this subject has been reviewed ire previous publications.~~S . A deficiency of copper has heen nrodllced in swine. by feeding a diet of . . . ... . . . ... . homogenized evaporated milk to which a liberal amount of "copper-free" iron was added (30 mg./kg. body weight daily). Control animals were given 0.5 ma. of copper/kg. of body weight daily in addition to iron. The animals were two to tern days of age at the start of the experiment. Description of the Anemia. During the first month of the copper-defic- ient dietary regime, there is generally little or no decline in the hemoglobin of- volume of packed red cells (V.P.R.C.) (fig. 1~. Thereafter, a precipitous fall in these values occurs. If the animals are not treated, the hemoglobin de- creases from 15 to 2 gm./100 ml. and the N7.P.R.C. from 40 to 8 ml./100 ml. in about 40 to 50 days. The animals become extremely pale and weak, the respiratory rate increases, and death supervenes, apparently as a result of . . tissue anoxla. The type of anemia which develops is microc~rtic and hypochromic, as in- dicated by a substantial decrease in the mean corpuscular volume (M.C.V.) and in the mean corpuscular hemoglobin concentration (M.C.fI.C.) as well as by marked microcytosis and hypochromia of the erythrocytes in the blood smear. The anemia is not accompanied by a significant reticulocytosis (table I). Examination of the marrow of copper-deficient pigs reveals hyperplasia of erythroid elements. The normoblasts are predominantly polychromatophilic. Thus, the anemia associated with a deficiency of copper in the pig is morpho- logically similar in all respects to that due to a deficiency of iron (table I). Blood and Tissue Copper and Response to Copper. Within 14 days of the start of the experiment the plasma copper level of the copper-deficient These investigations have been supported in part by a research grant (C-2231) from the National Institutes of Health, Public Health Service, and in part by a con- tract (AT (11-1)-82, Project 6) between the United States Atomic Energy Com- mission and the University of Utah. This paper was presented by Dr. Cartwright. 100

COPPER IN ERYTHROPOIESIS CARTWRIGHT ET AL. 1Ol ~ _ _ 70 64D >35 ~ ~ .C.V 30 ~~ tM.~,_~ ~ id_ 20 40 60 ways Q{^ge BE 100 FIG. 1. Showing the development of microcytic, hypochromic anemia, leukopenia and neutropenia in a pig deficient in copper and the response of the blood to an oral administration of 0.5 ma. of copper/kg. of body weight/day. M.C.~., mean corpuscular volume; M.C.H.C., mean corpuscular hemoglobin con- centration; V.P.R.C., volume of packed red cells; R.B.C., red blood cells; Hb, hemo- globin; W.B.C., total leukocytes; P.M.~., polymorphonuclear leukocytes. (Lahey et al., Blood 7: 1053, 1952. By permission.) swine decreases from the normal value of about 186 ~g.~100 ml. to 0 to 25 ~g.~100 ml. and remains in this range until the animals are treated with copper (fig. 2). The copper in the red cells is depleted somewhat more slowly and to a lesser degree than is plasma copper (table II). Normal corpuscles of swine contain about 61 Agog of copper per red cell. Erythrocytes from anemic copper-deficient swine contain about 26 Agog of copper per cell. As

loo PART II. BIOSYNTHESIS OF HEMOGLOBIN- 300 t00 ~9 0~ ~ 60t ~,60 `5 40 H] ~3 S 20 CUT '13 §13 8 2D ~ ID Pi9 22-3 ~ Pi . PFe ~ \_._- ~CU ~ ~ ._ · -'---_ , relics ' , I `t 1 ~ ~1 a 40 60 80 100 110 Days olAge FIG. 2. Showing the development of hyp~oferremia, hypocupremia, microcytic, hy- pochromic anemia, leukopenia and neutropenia in a copper-deficient pig and the re- sponse to the oral administration of 0.5 ma. of copper/kg. of body weight/day. PFe, plasma iron; PCu, plasma copper; M.C.V., mean corpuscular volume; V.P.R.C., volume of packed red cells; W.B.C., total leukocyte count; P.M.N., polymorphonuclear leukocytes. (Lahey et al., Blood 7: 1053, 1952. By permission.) would be expected, the amount of copper in the tissues is greatly reduced (table III). The anemia responds rapidly and completely following the addition to the diet of 0.5 ma. of copper/kg. body weight/day. Three to eight days following the initiation of such therapy, the reticulocyte increase ranges between 18 and 45 per cent. Simultaneously with the increase in reticulocytes, there is an increase in the mean corpuscular volume to values within or above the normal range. A rapid increase in the erythrocyte count, hemoglobin and

COPPER IN ERYTHROPOIESIS CARTWRIGHT ET AL. 103 TABLE I SUMMARY OF THE MORPHOLOGIC CHARACTERISTICS OF THE ERYTHROCYTES OF CONTROL, COPPER-DEFICIENT AND IRON-DEFICIENT SWINE V.P.R. C. ml 1100 ml. M.C.V. ,u3 M.C.H.C. per cent Reticulocytes per cent Bone Marrow L:E Ratio Copper Iron Control Deficient Deficient 42 55 33 5 1.8 21 29 4 0.6 21 36 28 9 0.8 V.P.R.C. refers to volume of packed red cells M.C.~. refers to mean corpuscular volume M.C.X.C. refers to mean corpuscular hemoglobin concentration L :E ratio lenl~ocyte-erythroid ratio TABLE II A COMPARISON OF THE BIOCHEMICAL CHARACTERISTICS OF COPPER \NTD OF IRONS D EFICIENC, Plasma Cu g/100 ml. R.B.C. Cu g/100 ml. Plasma Iron g/100 ml. T.I.B.C. pcg/100 ml. F.E.P. g/100 ml. Copper Iron Control Deficiency Deficiency 15 207 186 110 175 511 118 67 38 628 110 30 864 127 T.I.B.C. refers to total iron-binding capacity of the plasma. F.E.P. refers to free erythrocyte protoporphyrin. TABLE III TISSUE COPPER IN CONTROL, CCPP ER- D EFICIENT AND IRON -D EFICIENT SWIM E Copper Iron Control Deficient Deficient Liver 8.1 0.6 31.6 Spleen 0.04 0.02 0.04 Kidney 0.7 0.3 1.2 Heart Q.4 0.2 0.5 The values are expressed in ma. per organ.

104 PART II. BIOSYNTHESIS OF HEMOGLOBIN V.P.R.C. ensues, and by three weeks after the initiation of therapy the blood has returned to normal. In general, the hemoglobin level increases more slowly than does the V.P.R.C., with the result that the microcytosis disap- pears before the mean corpuscular hemoglobin concentration returns to normal. The plasma copper level increases significantly within 24 hours and reaches the normal level in about five days. Iron Metabolism. Because of the morphologic similarities between the anemias of copper and of iron deficiency, various aspects of iron metabolism have been investigated in copper-deficient pigs. In spite of the fact that the copper-deficient pigs received 30 ma. of iron/kg. body weight/day from the beginning of the experiment, the level of iron in the plasma was reduced to levels comparable to those observed in iron-deficient swine (table II). Maxi- mal reduction in the plasma iron level occurs early in the course of the ex- periment and persists throughout the duration of the copper deficiency (fig. 2~. The hypoferremia is accompanied by an increase in the total iron-binding capacity of the plasma, with the result that there is a marked reduction in the per cent saturation of transferrin with iron (table II). Analyses of the livers and kidneys of copper-deficient pigs for iron reveal that there is a distinct decrease in the amount of iron in these organs (table IV). The amount of iron in the spleen is increased; the amount in the heart TABLE IV TISSUE IRON MG/ ORGAN Copper Control D efficient Iron Deficient Blood 961 189 IS5 Liver 87 27 5 Kidney 7 3 1 Spleen 6 12 3 Heart 4 9 3 ~ _ Total (mg/kg.) 40 15 9 is not significantly altered. Since most of the iron in the animal is normally contained in the hemoglobin in the circulating erythrocytes, and since there is a great reduction in the amount of circulating hemoglobin in the copper-de- ficient animals, it is apparent that the total amount of iron in the body is Greatly reduced. Since the corner-deficient animals were fed the same amount < ~ , . ~ r of iron over the course of the experiment as were the litter-mate control pigs, this observation suggests that the absorption of iron by copper-deficient . . . . pigs IS 1mPaire( .. In order to demonstrate more conclusively that the absorption of iron is impaired by a deficiency of copper, two copper-deficient and one control pig were given oral radioiron daily for 12 days. The animals were sacrificed 14 days later and the amount of radioactivity in the liver, blood, spleen, kidney

COPPER IN ERYTHROPOIESIS—CARTWRIGHT ET AL. 105 and heart was determined. Six per cent of the radioactivity administered was recovered in these tissues of the control pig and only taco per cent in the tissues of each of the copper-deficient animals. A similar type of experiment has been performed in rats, and again it was possible to demonstrate that in the absence of an adequate amount of copper, iron is not absorbed at the normal rate.3 That the anemia associated with a deficiency of copper is not due to failure to absorb iron can be readily demonstrated by the fact that the development of the anemia is neither prevented nor alleviated by the intravenous adminis- tration of large amounts of iron (tables NT and VI). Although it has been suggested in the past by ourselves' and by others I" ii TABLE V FAILURE OF INTRAVENCUS IRON TO PREVENT THE DEVELOPMENT OF ANEMIA IN COPPER-DEFICIENT SWINE Plasma Plasma Liver V.P.R.C. Copper Iron Iron Group ml./100 ml ~g/100 ml ~g/1OO ml ma. - Contro l 175 Copper- 19 8 15 27 D efficient Copper- 13 14 144 631 Deficient +I.V. Iron* * These animals were given one gram of iron intravenously at the beginning of the experiment, prior to the development of anemia. TABLE VI 186 FAILURE OF IRON ADMINISTERED INTRAVENOUSLY TO INDUCE A HEMOPOIETIC RESPONSE IN ANEMIC, COPPER-DEFICIENTT SWINE After Deficient V.P.R.C. ml/100 ml Reticulocytes per cent Iron* 21 10 M.C.V. 46 48 ,u3 M.C.H.C. 30 28 per cent Plasma Cu 14 18 g/100 ml Plasma Iron 48 140 ~g/100 ml T.I.B.C. 627 578 * 200 ma. of venously. ~g/lOO ml iron in the form of colloidal saccharated oxide of iron were administered intra- 1 efers to volume of slacked red cells. refers to mean corpuscular volume. refers to mean corpuscular hemoglobin concentration. refers to total iron-bin ding capacity of the plasma. ~ .~.~.C. M.C.V. M.C.H.C. T.I.B.C.

106 PART II. BIOSYNTHESIS OF HEMOGLOBIN that the mobilization of iron from tissue stores is impaired in copper deficiency, recent studies in our laboratory do not substantiate this suggestion (table VII). TABLE NIII FERROKINETIC STUDIES IN CONTROL AND COPPER-DEFICIENT SWINE AND IN SWINE WITH HEMCLYTIC ANEMIA Copper Hemolytic Control Deficient Anemia* Number of Pigs 20 4 3 Plasma Iron, ~g/100 ml 166 39 159 T-~/2 hours ~ 1.2 0.5 0.3 Plasma Iron Turnover Rate 1.1 I.7 4.9 mg/kg/24 hours To Injected Dose Incorporated 91 67 86 Into RBC RBC Iron Turnover Rate 0.6 1.1 4.1 mg/kg/24 hours Erythrocyte Survival 61 13 5 days * Induced with Phenylhydrazine. ~ Time required for one-half of the isotope to disappear from the plasma. The plasma iron turnover rate in copper-deficient pigs is even greater than in normal pigs and the amount of iron turned over through red cells per day was about twice as great in the deficient pigs as in the normal control animals. Furthermore, if the mobilization of iron were impaired by a deficiency of copper, then it might be expected that the activity of all heme-containing er~zymes would be reduced. Such is not the case (table VIII). Although the TABLE VIII HEMIN CHROMOPROTEINS % OF NORMAL VALUE Copper Iron Deficiency Deficiency Cytochrome C ( Heart) 124 46 Cytochrome Oxidase (Heart) 10 Catalase (Kidney) Myoglobin (Muscle) 65 121 104 99 25 Cytochrome oxidase activity of heart is greatly reduced, the Cytochrome C activity of heart muscle, the catalase activity of renal tissue, and the myoglobin content of muscle are not reduced, even in severely deficient animals. Thus, it would seem that although copper is involved in the absorption of iron, it is not concerned directly with the movement of iron between the body com- partments. Erythrocyte Survival Shoddies. Calculation of erythrocyte survival time from ferrokinetic data indicates that the survival time of erythrocytes from cGpper-deficient pigs is shorter than normal time (table VII). Measurement of the erythrocyte survival time by the use of radioactive chromium con-

COPPER IN ERYTHROPOIESIS—CARTWRIGHT ET AL. 107 firms this observation (table IX). The fact that erythrocytes from a normal pig, when transfused into a copper-deficient pig, do survive a normal period of time suggests that the shortened life-span of the copper-deficient cells is not due to an extracorpuscular abnormality. TABLE IX SURVIVAL OF RED CELLS TAGGED WITH RADIOACTIVE CHROMIUM Life Span Donor Recipient No. Pigs Mean T 1/2 (Calc.) - Normal Normal 4 17 + 3.6 64 Cu-Deficient Cu-Deficient 10 9 + 1.9 33 Normal Cu-Deficient 4 16 + 3.6 58 Cu-Deficient Normal 5 13 + 2.2 49 Fe-Deficient Fe-Deficient 3 18 + 4.2 69 On the other hand, if the decreased life span were due to an intracorpuscular cause, one would expect that the copper-deficient cells would not survive for a normal period when transfused into a normal recipient. Such is not the case. When cells from a copper-deficient pig are transfused into a normal pig, the survival time approaches normal. An explanation for this observa- tion is that copper may enter the "copper-deficient" red cells from normal plasma and correct the intracorpuscular defect. In support of this explana- tion, it has been demonstrated that radiocopper, when added to plasma either in vitro or in vivo, is taken up by erythrocytes within several hours.7 Role of Copper in Erythropolesis. The vital role of copper in erythro- poiesis is confirmed by these studies. However, the manner whereby copper so profoundly influences erythropoiesis is obscure. Since the daily hemoglobin (or red cell) production of normal pigs may be increased fourfold under the stimulus of anemia, and since the rate of hemo- globin ~ or red cell ~ production in copper-deficient pigs is only 1.1 to 1.3 times greater than in normal animals, it is apparent that the ability to pro- duce hemoglobin is greatly impaired in copper-deficient swine. Furthermore, both ferrokinetic studies and chromium erythrocyte survival studies indicate that the life-span of the erythrocyte in copper deficiency is shortened. It seems, therefore, that anemia develops in the absence of copper because of a limitation of the capacity of the marrow to produce cells and because of a shortened erythrocyte survival time. A possible explanation for the decreased survival time of the erythrocytes is that the copper is an essential component of adult red cells and when the copper concentration of the erythrocyte is below a certain minimal, critical level, the survival time of the cells is shortened. There are several observa- tions which are compatible with this hypothesis. Copper is a normal con- stituent of the adult red cell (table II). In copper-deficient pigs, the con- centration of copper in the erythrocytes decreases from the normal value of 100 ,ug/ 100 ml. of packed cells to 67. Furthermore, when the anemia is

PART II. BIOSYNTHESIS OF HEMOGLOBIN severe there is a tendency for the concentration of copper within the red cells to increase slightly (fig. 3~. One possible explanation of the latter ob- servation is that the cells with the least amount of copper have been selectively destroyed. 120 Cod To 50 ~ 80 40 :~O :30 Y4D ~ 20 10 \ VP.~.C. ~ .. . . _ x\: ,x - ax - An<- _ _` 20 40 60 SD DID Days FIG. 3. Showing the development of anemia (V.P.R.C., volume of packed red cells), and decrease in plasma copper (PCu) and red cell copper (RBC Cu) in copper-de- ficient swine (mean of 7 piers). The role of copper within the erythrocytes is unknown. Dr. Harold Marko- . . ~ . . . . . WltZ In our I erythrocytes.1~ A few of the physical characteristics of this protein are given in table X. We have chosen to call this compound erythrocuprein, rather than hemocuprein, the name originally used by Mann and Keilin.43 Its physical characteristics seem to be slightly different than those of the protein isolated by them and the name, erythrocuprein, clearly distinguishes this red cell protein from another "hemocuprein" ceruloplasmin, the copper protein aboratory has recently isolated a copper protein from human TABLE X Property Color Absorption Max. Mol. Wt. % Cu Atoms Cu / Mol. Isoelectric Point PHYSICAL AND CHEMICAL PROPERTIES OF ERYTHROCUPREIN AND CERULOPLASMIN Erythrocuprein Colorless None 3 5,000 .32 - .34 2 5.3 Ceruloplasmin Blue 605 my 151,000 .32 - .34 8 4.4

COPPER IN ERYTHROPOIESIS—CARTWRIGHT ET AL. 109 . ~ . Ot plasn~.li At least 80 per cent of the copper in erythrocytes is accounted for in the erythrocuprein fraction. It is possible that the shortened erythro- c~te survival time of copper-deficient red cells is due to deficiency of this copper protein. Studies on tile precise function of erythrocuprein in the me- tabolism of red cells are now- under way in our laboratory. Because of the striking morphologic and biochemical similarities between the anemias of iron and of copper deficiency, we have suggested that copper may in some manner influence the metabolism of iron. It is apparent from our studies that copper profoundly influences the absorption of iron. This sug- gests that a copper protein may be involved in some manner in the mechanism by which iron is absorbed from the gastro-intestinal tract. However, copper does not exert its influence on erythropoiesis by virtue of this effect on iron absorption since the anemia is neither prevented nor alleviated by the intra- venous administration of iron. That the activity of all heme chromoproteins is not uniformly depressed suggests that not all of the metabolic pathways of iron in the body are impaired. Furthermore, our recent observation that the daily turnover rate of iron through the plasma and into the red cells is in- creased rather than decreased in copper-deficient swine suggests that move- ment of iron within the body is not curtailed. The relation of copper to iron metabolism must be in some way concerned with the role of copper in red cell synthesis. Seminary. Swine deficient in copper develop a severe anemia which is morphologically indistinguishable from the anemia of iron deficiency. It appears that anemia develops in the absence of copper because of the limited capacity of the marrow to produce cells. It is suggested that deficiency of the erythrocyte copper compound erythrocuprein, is in some way related to these alterations in erythrocyte production arid survival. REFERENCES 1. Lahey, M. E., Gubler, C. J., Chase, M. S., Cartwright, G. E., and Wintrobe, M. M.: Studies on copper metabolism. II. Hematologic manifestations of copper deficiency in swine, Blood, 7: 1053, 1952. 2. Gubler, C. J., Lahey, M. E., Chase, M. C., Cartwright, G. E., and Wintrobe, M. M.: Studies on copper metabolism. III. The metabolism of iron in copper-de- ficient swine, Blood, 7: 1075, 1952. 3. Chase, M.S., Gubler, C. J., Cartwright, G. E., and Wintrobe, M. M.: Studies on copper metabolism. IV. The influence of copper on the absorption of iron, J. Biol. Chem., 199: 757, 1952. 4. Chase, M. S., Gubler, C. J., Cartwright, G. E., and Wintrobe, M. M.: Studies on copper metabolism. V. Storage of iron in liver of copper-deficient rats, Proc. Soc. Exper. Biol. and Med., &0: 749, 1952. Cartwright, G. E., Gubler, C. J., Bush, J. A., and Wintrobe, M. M.: Studies on copper metabolism. XVII. Further observations on the anemia of copper de- ficiency in swine, Blood, 11: 143, 1956. 6. Follis, R. H. Jr., Bush, J. A., Cartwright, G. E., and Wintrobe, M. M.: Studies

110 PART II. BIOSYNTHESIS OF HEMOGLOBIN on copper metabolism. XVIII. Skeletal changes associated with copper deficiency in swine, 97: 405, 1955. 7. Bush, J. A.. Jensen, W. N., Athens, J. W., Ashenbrucl~er, H., Cartwright, G. E., and Wintrobe, M. M.: Studies on copper metabolism. NIX. The kinetics of iron metabolism and erythrocyte life-span in copper-deficient swine, J. Exp. Med., 103: 701, 1956. 8. Gubler, C. J., Cartwright, G. E., and NVintrobe, M. M.: Studies on copper metab- olism. XX. Enzyme activities and iron metabolism in copper and iron deficiencies, J. Biol. Chem., 224: 533, 1957. 9. Elvehjem, C. A.: The biological significance of copper and its relation to iron metabolism, Physiol. Rev., 15: 471, 1935. 10. Schultze, M. O.: Metallic elements and blood formation, Physiol. Rev.'20: 37, 1940. 11. Marston, H. R.: Cobalt, copper and molybdenum in the nutrition of animals and plants, Physiol. Rev., 32: 66, 1952. 12. Markowitz, H., Cartwright, G. E., and Wintrobe, M. M.: Unpublished obser- vations. 13. Mann, T., and Keilin, D.: Haemocuprein and hepatocuprein; copper-protein com- pounds of blood and liver in mammals, Proc. Royal Soc., London, Series B. 126: 303, 1938. 14. Holmberg, C. G., and Laurell, C. B.: Investigations in serum copper. II. Isolation of the copper containing protein, and a description of some of its properties, Acta Chem. Scandinav., 2: 550, 1948. DISCUSSION Dr. I. H. Scheinberg: I was curious, Dr. Cartwright, as to the difference between erythrocuprein and the hemocuprein that was reported about 20 years ago which, I think, had the same physical characteristics. Dr. Edsall: It certainly had a very similar molecular weight. Dr. Cartwright: The molecular weight and copper concentration of ery- throcuprein are the same as for hemocuprein, the erythrocyte copper protein isolated by Mann and Keilin from ox blood. We have chosen to call our compound erythrocuprein for the following reasons. Hemocuprein from ox blood is blue and erythrocuprein is colorless; the isolation procedure used by us was quite different from the procedure used by Mann and Keilin; their protein was isolated from ox blood and not from human blood. Finally, Mann and Keilin used the term hemocuprein for both the serum copper protein and the red cell copper protein. The serum copper protein has since been isolated by Holmberg and Laurell, as well as by others. It is a distinctly different protein from the erythrocuprein which we have described. Therefore, two blood copper proteins (hemocupreins) have now been isolated, namely, cerulo- plasmin and erythrocuprein. Thus, it would seem advisable to use the term hemocupreins to refer to all blood copper proteins collectively.

HEMOGLOBIN SYNTHESIS IN VITRO IN RABBIT RETICULC)CYTES~ HENRY FORSOOK Rabbit reticulocytes in vitro rapidly incorporate labeled amino acids into their proteins.) Nearly all the trichloroacetic acid precipitable material is hemoglobin. In experiments with labeled glycine, histidine, leucine or lysine the results were substantially the same whether the incorporation was measured in the mixture of proteins precipitated by trichloroacetic acid or in the iso- lated hemoglobin. The hemoglobin, as measured by the method of DraLkin and Austin,- in the reticulocytes as prepared by us, is about 1 j per cent, and constitutes nearly all of the dry weight of the material precipitated by tri- chloroacetic acid. These facts and the results of experiments in which the hemoglobin was isolated,3 warrant referring to amino acid incorporation in the proteins of rabbit reticulocytes as protein or hemoglobin synthesis. In the first experiments it was found that amino acid incorporation into the proteins of the reticulocytes was accelerated by the plasma of every mammal investigated and also by extracts of normal erythrocytes, rabbit reticulocytes, liver, spleen and yeast. We undertook the identification of the accelerating factors. The total accelerating effect, potential as well as actual, of plasma and tissue extracts has been accounted for by known substances they contain. Production of Reticalocytosis. Reticulocytosis was produced in adult rabbits by a modification of the method of London et al.4 One milliliter of a neutralized 2.5 per cent aqueous solution of phenylLydrazine hydrochloride divas injected subcutaneously each day for a week; over 90 per cent of the circulating red cells were then reticulocytes. Labeled amino acids. Methyler~e-labeled C~'~-glycine was obtained from Tracerlab, Inc.; as used, its specific activity was 5.5 x 106 c.p.m. per mmole. L-histidine, L-leucine and L-lysine were prepared labeled with C2'' in their carboxyl groups;' 5 their specific activities were respectively, 2.21, 2.29 and 1.56 ~ 106 c.p.m. per mmole. Their initial concentration in the reaction mix- ture was always 0.001 M. Incubation Procedure. The incubation was carried out in 20 ml. beakers in the DuLnoff apparatus6 under 95 per cent 02 arid 5 per cent COP at 37.5° C. for lengths of time indicated below. with a rocking rate of 100 cycles Per O , These studies were aided by a contract between the Atomic Energy Commission and the Division of Biology, California Institute of Technology. They were also sup- ported by a research grant from the National Institutes of Health, United States Public Health Service, and by a grant-in-aid from the American Cancer Society upon recommendation of the Committee on Growth of the National Research Council. The following were collaborators in the work described here: Drs. A. Abrams C. L. Deasy, E. H. Fischer, G. Keighley, J. Kruh, and R. H. Lowy. 111

112 PART II. BIOSYNTHESIS OF HEMOGLOBIN minute. Each beaker contained 0.5 ml. packed reticulocytes in 4 ml of re- action mixture. The solvent was either Krebs-Henseleit solution; or the latter solution with omission of calcium and phosphate salts (see below the reasons for the omission) as indicated. All the glassware and all the solutions except the amino acid mixture were sterilized in the autoclave. Since repeated autoclaving was found to destroy phenylalanine, the amino acid mixture was boiled once and then kept in the deep freezer in small flasks with just enough, usually 30 ml., for one experi- ment per flask. Preparation of Mixed! Proteins. At the end of the incubation the contents of each beaker were added to 80 ml. of water and the reticulocyte protein precipitated with 20 ml. of 35 per cent trichloroacetic acid. If the reaction mixture contained plasma or some other proteins the cells were first separated by centrifugation and washed twice with saline, before the trichloroacetic acid precipitation. After the precipitated protein had settled, leaving a clear su- pernatant solution, usually in about three hours, the supernatant solution was decanted off so as to leave a well drained paste of precipitated protein, which was then dissolved in 1 N NaOH and then reprecipitated by 100 ml. of 7 per cent trichloroacetic acid. After it had settled well, the precipitated protein was transferred to a centrifuge tube, in which it was washed twice with 7 per cent trichloroacetic acid, twice with a mixture of equal volumes of acetone and ether, once with acetone, twice with ether, and then dried at 85~. Preparation of Hemoglobin. The hemoglobin divas isolated and purified by a modification of the method of Roche et al.S~~° Equivalent amounts of KH~PO4 and K2HPO4 were added to give 2.9 M total phosphate. All the plasma and cellular proteins were salted out, whereas rabbit hemoglobin, which is extremely soluble, remained in solution. After standing overnight at room temperature, the salted-out proteins were removed by filtration. The hemoglobin was then salted out by further addition to the filtrate of equiva- lent amounts of the two salts to give 3.5 M phosphate. The hemoglobin was collected by filtration on a Buchner funnel and washed on a filter with 3.5 M phosphate solution. The washed hemoglobin was then dissolved in about 50 ml. Of water and dialyzed at 4° for 3 days against repeated changes of water total volume ~ 0 liters ~ . Preparation of Heme. The heme was isolated from the hemoglobin by an adaptation of the method of Anson and ~lirsky.~i Ten volumes of 1 per cent HC1 in acetone were added to the hemoglobin solution. The globin, which precipitated, was removed by filtration, an equal volume of water was added to the filtrate, and the diluted acetone solution was evaporated under a lamp. After a few hours the heme precipitated; it was washed by decantation four times with water, dried overnight at 100° C, dissolved in 0.5 ml. pyridine, and filtered onto a circle of lens paper that fitted exactly in a circular alum- inum cup, 19 mm. in diameter, and dried under a lamp. With 8 to 15 ma.

HEMOGLOBIN SYNTHESIS IN RETICULOCYTES—BORSOOK 1~ 3 of heme, a uniform spread was obtained for the measurement of the radio- activity. Preparation of Globin. The globin which had been filtered from the heme was washed on the filter with 1 per cent HC1 in acetone until the washings were colorless. It was then suspended in 80 ml. of 7 per cent tri- chloroacetic acid. The remainder of the procedure whereby it divas obtained in a dry state for the measurement of its radioactivity was identical to that of the mixed proteins described above. Measurement of Radioactivity. The material --'lose radioactivity divas to be measured was spread uniformly on an aluminum plate 19 mm. in diameter and counted in a Geiger-Muller end window counter. An empirical self- absorption curve was used to correct for the thickness of the sample. Calculations of Heme. Heme-pyridine hemochromogen contains 2 resi- dues of pyridine per heme;i2 8 molecules of glycine enter into the synthesis of 1 of home.: On this basis the number of millimoles of heme synthesized per I/4 molecule of hemoglobin (or per mole of heme attached to hemoglobin' isolated is given by ~ (counts per minute per ma. of heme-pyridine/(counts per minute per millimole of labeled glycine ~ 8) ~ Y 774 ~ 103. Incorporation of Amino Acids into Globin. In order to compare the rates of incorporation of four different amino acids into globin it is neces- sary to take into account the amounts of these amino acids in the globin. There are no reliable analytical data on rabbit hemoglobin. Accordingly, we have used the following data of Schroeder et aI.14 on human hemoglobin expressed as residues per molecule of hemoglobin: glycine 43.1, histidine 36.3, leucine 76.0, and lysine 44.0. Tristram i~ gives the following (as residues per molecule of hemoglobin) for horse hemoglobin: glycine 48, histidine 36, leucine 75, and lysine 38.0. In view of the similar amino acid composition of human and horse hemoglobins it seems likely that the values obtained by Schroeder et al. for human hemoglobin can be used for rabbit hemoglobin for purposes such as ours, without significant error. The number of millimoles of labeled amino acid incorporated into hemo- globin per residue of that amino acid in the protein let glycine be an example- is given by ~ (counts per minute per ma. of globin/ (counts per minute per millimole of glycine x 43.1 ) ~ x 6.6 ~ 104. Results. Table I is a summary of the main factors we have found which accelerate protein synthesis in vitro in rabbit reticulocytes. Not included are fructose-amino acids whose effects are referred to below in discussing the effect of iron. It is seen that iron and glucose, separately or together, had little accelerating effect. The amino acid mixture alone caused an increase of 70 per cent; amino acids and iron, but not amino acids and glucose, acted synergistically; glucose acted synergistically when added with amino acids and iron. Transferrin, added with amino acids, iron and glucose increased the rate still further. These results indicate that for a high rate of synthesis the

114 PART II. BIOSYNTHESIS OF HEMOGLOBIN TABLE I FACTORS ACCELERATING PROTEIN SYNTHESIS IN RABBITT RETICULOCYTES IN VITRO. Amino acid mixture Iron Glucose Trans- Cam. 1 ma. per 1errin per ml. ml. 50 Ham. per ml. _ _ + _ _ ~ + _ + _ _ _ ~ + _ _ + _ + _ + + _ + - Rate of protein synthesis 100 100 110 111 170 351 170 497 597 TABLE I.—The composition of the amino acid mixture is given in the text. The iron was in the form of ferrous ammonium sulfate. The transferrin was a crystalline metal-free preparation. constituents of hemoglobin, i.e. amino acids and iron, and an energy source, i.e. glucose, must be available in adequate amounts. The result with trans- ferrin points to the usefulness of an iron-chelating agent even though it may not be strictly necessary. Amino Acid Mixture The amino acid mixture used had the following composition, expressed as Am. per ml. of reaction mixture: L-alanine 45, L-arginine 21, L-aspartic acid 95, L-cysteine 12.5, glycine 100, L-glutamine 70, L-histidine 90, L-hydroxyproline 37.5, L-isoleucine 10, L-lysine 65. L- methionine 12.5, L-phenylalanine 65, L-proline 40, DL-serine 90, L-threonine 50, L-tryptophane 15, L-tyrosine 37.5, and L-valine 90. L-leucine was pro- vided in the carboxyl-C7; form and at a concentration of 10-3 M (131 Am. per ml.~. Only nine of the above nineteen amino acids were found to be in any degree limiting during a four-hour experiment. These, except leucine, are given in table II. In other experiments) it had been found that leucine is severely limiting. Three points may be made regarding results such as those in table II. Amino acids vary in the degree to which they are limiting; histidine is the most limiting. In a reaction mixture from which one of the limiting amino acids is withheld, the rate is at a characteristic suboptimal level from the beginning and persists so. Throughout the four hours glutamine had an accelerating effect, whereas added glutamic acid had no effect. In separate experiments it was found that C~'~-labeled glutamic acid Was not incorporated at all, whereas Ct4-glutamine was extensively incorporated. Evidently gluta- mic acid is unable to penetrate into the reticulocyte whereas glutamine can.

HEMOGLOBIN SYNTHESIS IN RETICULOCYTES BORSOOK TABLE II EFFECT OF AMINO ACID COMPOSITION RESULTS EXPRESSED AS PER CENT OF THAT IN SALINE ALONE WITHOUT ADDED A MINO ACIDS. 11; Hours Amino Acid Composition Complete " without histidine ,, " valine ,, ,. .. ,. .. ,, .. ,, ,, ,, 1 447 94 185 176 215 235 257 262 304 2 3 450 489 87 94 170 181 183 181 227 220 229 237 267 264 280 295 339 353 4 475 96 187 200 228 222 285 297 366 TABLE II. The complete reaction mixture contained the amino acid mixture de- scribed in the text, 5 hum. per ml. of iron as ferrous ammonium sulfate and 1 ma. per ml. of glucose. It did not contain transferrin. It has been observed that glutamic acid penetrates the cells of liver slices slowly. The limiting amino acids in rabbit reticulocytes include both those which are dispensable and indispensable. Isoleucine, methionine and threonine were not limiting. Although reticulocytes synthesize serine from glycinei and pre- sumably also synthesize glutamic acid, they do not make enough for a maximum rate of protein synthesis. These findings illustrate that the amino acid re- quirements of different tissues may be quite different from that of the animal as a whole. TABLE III EFFECT OF PARTIAL FIISTIDINE DEFICIENCY. RESULTS EXPRESSED AS PER CENT OF THAT IN COMPLETE REACTION MIXTURE. Amount of histidine added to otherwise complete reaction mixture gm./ml. molal O O 9.25 l8.S 27.75 37.0 46.25 74.0 Rate in successive hours 0.575 x 10-4 1.15 " " 1.72 " " 2.30 2.87 5.75 ,. .. ,. .. ,. .. Q-1 1 1-2 2-3 18 27 30 33 77 39 98 93 98 88 97 98 100 100 23 81 104 100 100 100 100 3-4 23 24 25 47 80 102 100

116 PART II. BIOSYNTHESIS OF HEMOGLOBIN Tables III, IV and V show in more detail how the quality of the amino acid mixture may determine the rate of protein synthesis. With suboptimal concentrations of the limiting amino acids the rate is at first nearly maximal, then declines, but may remain well above the minimal rate. Of course all the amino acids in hemoglobin are needed for its synthesis. The amino acid requirement in the reaction medium for a maximum rate of synthesis is inversely related to the amino acid content of the cells. Accord- ingly, in the case of reticulocytes of a different species, e.g. avian cells, the amino acid mixture required to be added to the reaction mixture may be different from that necessary for rabbit reticulocytes. The literature on the relation between amino acids and blood formation TABLE IV EFFECT OF PARTIAL PHENYLALANINE DEFICIENCY. RESULTS EXPRESSED AS PER CENT OF THAT IN COMPLETE REACTION MIXTURE. Amount of phenylalanine added to otherwise com- plete reaction mixture. Am. / ml. o 6.5 13.0 19.5 26.0 32.5 65.0 Rate in . , successive nours 0-1 molal o 0.39 X 10 - 4 0.78 " " 1.18 " " 1.57 1.96 3.93 " " ,. .. ,. .. 35 88 90 92 93 94 100 -2 2-3 3-4 37 39 66 62 90 87 93 105 92 100 93 102 100 100 38 61 80 98 102 101 100 TABLE V EFFECT OF PARTIAL VALINE DEFICIENCY. RESULTS EXPRESSED AS PER CENT OF THAT IN COMPLETE REACTION MIXTURE. Amount of valine added to otherwise complete reaction mixture ,ugm./ml. o 9.25 18.5 27.75 37.0 46.25 92.5 Rate in successive hours 0-1 molal o 0.78 X 10 - 4 1.57 " " 2.3 6 I' 7, 3.15 3.94 7.89 ,. .. ,. .. ,. .. 81 98 100 95 95 100 1-2 1 2-3 1 3-4 29 61 85 97 99 99 100 50 71 84 92 94 100 32 53 73 86 98 97 100

HEMOGLOBIN SYNTHESIS IN RETICULOCYTES BORSOOK 117 in animals is extensive, i7~39 but the results reported are unsystematic and some are conflicting. Nevertheless, the evidence from in Gino experiments tend to extend the significance of the relationship found between labeled amino acid incorporation into reticulocyte proteins and hemoglobin synthesis. For example Sebrell37 found that omission of some of the essential amino acids from the diet of rats made anemic by bleeding handicapped red cell regeneration more than the omission of others. The amino acids found to be most necessary for red cell and hemoglobin regeneration, stated in order of their effectiveness, were histidine, valine, lysine, phenylalanine, and leucine. The list agrees surprisingly well with the amino acids accelerating incorpora- tion of labeled amino acids into rabbit reticulocyte proteins. Nizet and Robscheit-Robbins3S found that dog reticulocytes did riot mature so quickly in vitro in the blood of dogs with hemorrhagic anemia and hypo- proteinemia as in normal blood, unless a mixture of the ten essential amino acids and glycine was added to the anemic blood. Variations from animal to animal were too great for any conclusions to be drawn regarding differences among the individual amino acids. Orten and Orten 3- studied the effects on hemoglobin regeneration of adding individual amino acids to the diet of young rats previously made anemic by a low protein diet. They concluded that there was no "key" amino acid; however, some amino acids appeared to be more effective than others in their experiments. Art apparent discrepancy had existed between feeding experiments on the one hand, and those on the incorporation of labeled amino acids on the other. In feeding experiments it was found that an indispensable amino acid is in- ~ r 1 effective for growth, for recovery from protein depletion, or for maintenance, unless it is fed or injected within a few hours of other necessary amino acids. The same applies to the carbohydrate in a meal with respect to the caloric re- quirement for growth or nitrogen balance.3S~~5i The interpretation placed on these Endings has been that all the amino acids must be present (presumably in the blood) at concentrations greater than the fasting levels for protein synthesis to occur. Against this interpretation stand the observations that a single amino acid, whether dispensable or indispensable, when injected or fed in labeled form can be seen to be extensively incorporated into the proteins of the animal in a few minutes. Extensive incorporation occurs whether the animal is normally fed or fasting. All the above (and similar) observations are in accord with the following interpretation. An animal's protein is, viewed in toto, always in a dynamic state. Growth, balance or loss in weight connote respectively more, equal, or less synthesis than breakdown of protein. For maximum synthesis all the necessary amino acids and an adequate source of energy Carbohydrate) need to be present at the same time (tables I and XIII). If orate amino acid is present in suboptimal amount the rate of protein synthesis is suboptimal in degree according to the degree of the deficiency. There is never a complete

llg PART II. BIOSYNTHESIS OF HEMOGLOBIN cessation of protein synthesis; hence under all conditions all amino acids are incorporated, and the rates of incorporation of all are affected to the same extent. Hence when in vivo an indispensable amino acid is withheld or given much later than the main meal, protein synthesis is too slow for growth or maintenance. Feeding the ~-vitheld amino acid later does not correct the deficiency because amino acids, whether inj ected or fed, are too rapidly cleared from the blood. Correspondingly a single amino acid, whether in- jected or fed, will be extensively incorporated because protein synthesis never stops in Volvo. In this sense the results in tables II - ~ and XIII are analogues of experiments in which animals are maintained on suboptimal amounts of indispensable amino acids (or incomplete proteins) or inadequate calories. Iron. The effect of the addition of iron to the reaction mixture is shown in table VI. F`eSO~ (NH~), SO4.6H`,O was used mostly; ferrous chloride divas equally effective but less convenient; ferric chloride was almost as effective. To obtain consistent and maximal effects it was necessary to add the iron to the reaction mixture after the ret~culocytes, as the last ingredient before incubation. The reaction mixture divas somewhat alkaline (pH about 8 ~ before the reticulocytes there added and before being placed under 95 per cent oxygen and 5 per cent carbon dioxide, and the iron may be pre- _ipitated when added at this point. FABLE VI EFFECT OF IRON Reaction mixture except for iron and transferrin Saline alone Complete 77 Concentration of iron molal x 10— A 0.35 0.89 1.79 (1 ~gm./m1.) 3.58 8.95 17.90 Leucine incorporated <moles/gm. protein 3.9 6.8 9.3 12.1 17.7 19.1 19.6 19.6 With enough iron added to an otherwise complete reaction mixture about three times as much leucine was incorporated in four hours. If one assumes that all the leucine incorporated represents newly synthesized hemoglobin (a warrantable assumption) data such as those in table VI indicate that about three times as much iron needed to be added to the reaction mixture than was incorporated into hemoglobin. The calculation is as follows. Hemoglobin contains 75 residues of leucine per 4 atoms of iron. )5 Each beaker contained G.5 ml. packed cells in 4 ml. reaction mixture, therefore, about 75 ma. of protein, most of which was hemoglobin. An increased incorporation of 12

HEMOGLOBIN SYNTHESIS I)l RETICULOCYTES BORSOOK 119 TABLE VII I NCREASE INT IRON BOUND AS HEMCGLOBIN WITH INCREASING AMOUNTS OF IRON- ADDED TO THE REACTIONS MEDIUM. Iron added to reaction 1' n~ealum concen- tratlon: ham. per ml. o 1.0 2.5 5.0 10.0 20.0 40.0 Total iron bound as h~moglob.n clueing periods of 1 to 4 hours. ,umoles >; 10 total: Moles x 10 o 0.17 0.44 1 hr. .092 0.16 0.17 0.89 0.18 1.79 0.17 3.58 0.18 7.16 0.18 2 furs 0.14 0.21 0.25 0.28 0.30 0.30 0.30 Increase in bound iron in 4 furs. consequent on addition of iron ,umoles x 10 4 furs. 0.16 0.24 0.30 0.34 0.38 0.38 0.38 .08 .14 .18 .22 .22 .22 oracles of leucine per gram of protein corresponds per beaker to an in- creased incorporation of about 0.5 x 10-1 Moles of iron into hemoglobin. To obtain this increase the addition of 1.6 ~ 10-i Moles of iron per beaker alas needed. Evidently there is very little iron in reticulocytes available for hemoglobin synthesis. The data indicate that some of the iron added to the reaction medium - was rendered unavailable. This is the interpretation we have placed on data such as those in table VII. It is seen that during the first hour the addition of a very small amount of iron, between 0.017 and 0.044 Insoles, had a pearl', maximal effect; whereas over a four-hour period between 0.089 and 0.179 Moles were required for a maximum increase in hemoglobin iron of only 0.022 ~moles. The sensitivity of rabbit reticulocytes in vitro to such low concentrations of iron raises the question whether or not iron is necessary for hemoglobin synthesis in addition to its participation in the structure of heme. Speakers at this symposium have stated that iron is needed by at least one of the enzymes required for the synthesis of heme at a stage prior to addition of iron to protoporphyrin. We have no information on whether iron may be needed by the enzymes involved in globin synthesis. The addition of cobalt ~ as CoCl~ ~ also accelerated protein synthesis in. reticulocytes, table VIII, but less than did the addition of iron. The minimum amount giving a maximum effect was of the same order of magnitude in both, 2-5xI0-,:\~. Table IX shows that when cobalt was added with iron at comparable con- centrations, only the accelerating effect of the iron was obtained.

120 PART II. BIOSYNTHESIS OF HEMOGLOBIN TABLE VIII RELATIVE ACCELERATING EFFECTS OF COBALT AND OF IRON. RESULTS EXPRESSED AS PER CENT OF VALUE WITHOUT EITHER METAL. Concentration of added metal molal 1 x 10 - 4 5 x 10 - 5 2 X " 1 x " 5 X 10— Cobalt 151 156 113 110 100 Iron 229 223 ~ 1 ~ lg8 154 * The cobalt was added as CoCl.,. TABLE IX NON-ADDITIVITY OF THE ACCELERATING EFFECTS OF COBALT AND IRON. RESULTS EXPRESSED AS PER CENT OF VALUE WITHOUT EITHER METAL. Concentration of iron molal 1 X 10— 5 x " 2 x " 1 x " 5 X 10 - 6 Leucine incorporated when, in addition to iron, cobalt was added at a 2 x 10— M 1 ~ 10—~ M 229 223 218 222 200 183 1Si 141 212 188 154 5 x 10—c M 208 189 141 Experiments were carried out also with aluminum, manganese, molybdenum and zinc at concentrations of 0.2-1.0 x 1O-4 M with and without added iron, table X. They were all slightly inhibitory when the iron was 1O-4 M; at lower concentrations of iron they had no effect. No transferrin was used in any of the above experiments with metals. 1 S' 40 ~ 30 z ~ 20 z 0 10 c, / ~ ~ rat 2 3 4 HOU R S FIG. 1. Effect of medium on heme syn- thesis in rabbit reticulocytes. /\ —Plasma ~ amino acids + iron + glucose O - Non-protein plasma filtrate dialyzed + amino acids + iron + —Amino acids + iron ~ glucose · —Blank

HEMOGLOBIN SYNTHESIS IN RETICULOCYTES BORSOOK 121 TABLE X EFFECT OF METALS RESULTS EXPRESSED AS PER CENT OF BLANK Metal added None Aluminum Cobalt Copper Manganese Molybdenum Zinc Additional Iron molar concentration I mol21 a ~1 0.2 X 10 - 4 1.0 '' ,' 0.2 1.0 0.2 1.0 0.2 1.0 0.2 1.0 0.2 1.0 ,' ~7 100 100 102 123 239 109 116 116 116 97 102 109 93 0.9 x 10—5 1 0.2 X 10—4~ 1 0.9 X 10— 186 186 206 174 283 316 300 293 237 293 TABLE XI COMPARISON OF THE ACCELERATING EFFECTS OF PLAS.MA (RABBIT) AND OF TRANSFERRIN. RESULTS EXPRESSED AS PER CENT OF VALUE IN- OTHERWISE COMPLETE REACTION MIXTURE. Protein added None Plasma ,, i, ,, ,' Transferrin ,' ,' ,, ,, Rabbitt serum albumin Bovine " " Human ~~ i, . Transferrin Amount concentration added added to reaction mixture . per ml. reaction Bum. per ml. mixture O 0.05 ml. 125 (estimated) 0.025 " 62.5 '' 0.005 " 12.5 " 0.001 " 2.5 " 0.0005 " 1.25 " 200 60 20 4 2 500 ,t4gm. O 500 Bum. 0 500 Bum. 0 Incorporation of leucine 100 141 135 125 118 100 141 134 132 110 100 97 98 97

122 PART II. BIOSYNTHESIS OF HEMOGLOBIN Transferrin causes an increase above that obtained in a reaction mixture optimal with respect to amino acids, glucose and iron. A similar effect had been obtained with whole plasma (rabbit and human) (fig. 1~; in twenty- three experiments rabbit plasma caused an increase of 25 +12 per cent over that in an otherwise optimal reaction mixture. The active principle was not dialyzable, and disappeared on boiling; fractionation suggested that it might be transferrin. As table XI show-e this surmise appears to have been correct. The estimate in table XI of the transferrin in the plasma added is based ore DraLkin's value '~ of 0.25 am. per 100 ml. plasma. The transferrin used was kindly provided by Dr. l. L. Oncley of Harvard University.* The specificity of transferrin is attested to further by the absence of any effect of rabbit, bovine or human serum albumin. Presumably the eFectiveness of transferrin comes from its capacity to chelate and so transfer iron. It is surmised that the accelerating effects of fructose- amino acids and citrate are for the same reason, table XII. The maximum accelerating effect is obtained with about 10-6 M transferrin and 5 x 10-4 NI fructose- or tagatose-amino acids or citrate. The different fructose- and tagatose amino acids are Amadori rearrange- TABLE XII ACCELER.~\TING EFFECTS OF FRUCTOSE- AND TAGATOSE-AMINO ACIDS, AND OF CITRATE. RESULTS EXPRESSED AS PER CENT OF VA! UE LIT OTHERWISE COMPLETE REACTION MIXTUR E. Substance i Fructose-Alanine " -Arginine -Aspartic acid " - Glutamic " -Glycine -Histidine . -Leuclne -Lysine -Phenylalanine -Serine i, ,, ,' ,, ', -Threonine ,? -Valine l a~,atose-Alanine Citrate ', -Glyci ne 120 135 125 113 118 130 123 129 125 125 121 120 127 128 108 i The initial concentration of all of the above substances was 5 ~; 10—I- M. Iron Concentration 0~5 ,u~m/ml | 5~0 ,ugm/ml. 105 102 104 :> It was prepared from human plasma, crystalline, metal-free, and consisted of 96 per cent 0-1 metal-binding protein, 3 per cent p-2 and a- and 1 per cent of oc-globulins.

HEMOGLOBIN SYNTHESIS IN RETICULOCYTES BORSOOK 123 ment products of N-glycosyl-amino acids,53~~;' made respectively from glucose and galactose; glucose and mannose give the same product. It is seen that the effect was approximately the same with all the sugar-amino acid compounds, and that they are relatively more effective at suboptimal concentrations of iron, 0.5 mu gm/ml, than at optimal concentrations, ~ 2 ~gmiml. Transferrin on the other hand is more effective at optimal iron concentrations. Citrate was somewhat less effective than the sugar-amino acid compounds. Table XIII shows the accelerating effect of glucose added to the reaction mixture. It is seen that the effect was little in the first hour of incubation and became progressively greater, presumably as the carbohydrate initially in the reticulocytes was consumed. TABLE XIII EFFECT OF GLUCOSE. Incorporation c/m. ma. Differential effect of glucose c/m. my. ~ i Time: Hours 1 Saline + glucose ~ amino acids + glucose + amino acids Per cent of total . . Incorporation 3.32 3.12 14.59 15.01 2 3 4 1 2 4.75 6.38 7.43 5.03 6.89 7.72 -0.20 0.38 22.94 25.78 26.01 26.15 34.05 36.84 0.42 3.21 2.7 1 12.2 3 0.51 8.27 4 0.29 10.83 24.2 1 29.3 TABLE XIII. The added glucose was 1 ma. per ml.; the amino acids were the complete mixture described above, the iron was 5 Am. per ml. TABLE XIV EFFECTS OF SODIUM AND OF POTASSIUM. Nature of saline solution Leucine incorporated ~moles/gm. protein Krebs-Henseleit saline solution All-sodium " " All-potassium " " ~/2 sodium + ~/2 potassium " ', 20.1 19.8 10.9 19.0 TABLE XIV. "All-sodium" and "all-potassium" saline solutions: the potassium and sodium salts of the Krebs-Henseleit solution were replaced by the corresponding sodium and potassium salts respectively.

124 PART II. BIOSVIN'THESIS OF HEMOGLOBIN TABLE XV EFFECTS OF MAGNESIUM AND OF PHOSPHATE. Leucine Mg. PO4 incorporated molal molal ~moles/gm. protein 0 0 18.2 0.008 0 18.6 0.0016 0 18.6 0 0.008 1 1.0 0 0.016 17.6 0.008 0.008 16.8 0.008 0.0016 18.6 0.0016 0.008 15.2 0.0016 0.0016 17.4 TABLE XV. The basic saline solution was the Krebs-Henseleit mixture from which the calcium, magnesium and phosphate salts were omitted. TABLE XVI EFFECTS OF CALCIUM AND OF CITRATE. Leucine CaCl~, Citrate incorporated molal molal ~moles/gm. protein 0 0 18.4 2.8 x 10 - 3 0 11.6 0 0.25 x 10 - :, 19.4 0 1.0 " 19.1 2.8 x 10 - 3 0.25 " 17.3 2.8 " 1.0 " 19.7 TABLE XVI. The basic saline solution was as in Table XIII. Tables XIV, XV, and XVI show some results of varying the electrolyte composition of the saline solution in which the cells were incubated. An all- potassium, but not an all-sodium saline solution was inhibitory; the inhibition by potassium was neutralized by sodium, table XIV; phosphate was inhibitory arid its inhibitory effect was neutralized by magnesium, table XV; calcium was inhibitory, its inhibitory effect was relieved by citrate, table XVI. After these observations the Krebs-Henseleit solution we had used heretofore was modified by omission of the calcium and phosphate salts. Tables XVII, XVIII and XIX show the effects of a variety of inhibitors at a range of concentration. The most unexpected result was that lead (as lead acetate) was the most powerful inhibitor found; it was much more in- hibitory than antimony, gold or mercury, which last were about the same. Among the antibiotics tested, aureomycin alas the most inhibitory and chloram-

HEMOGLOBIN SYNTHESIS IN RETICULOCYTES—BORSOOK 125 TABLE XVII EFFECT OF SO,ME OXIDATION, PHOSPHORYLATION AND OTHER INHIBITORS. RESULTS EXPRESSED AS PER CENT INHIBITION. Concentration: moIal 0 - 3 Arsenate Arsenite 2,4-D initrophenol ~ -Ethylmaleimide Diisopropylfluorophosphate PCMB Hg-Phenylsulfonate Lead-acetate 90 99 97 6 o 95 95 98 0— 35 s 96 o o 9 21 96 0~ ~ 10— 4 2 1 1 3 o o o o 18 24 o o o 2 79 o TABLE ~VIII EFFECT OF SOME INHIBITORY METALS. RESULTS EXPRESSED AS PER CENT INHIBITION. Concentration: molal lo - 3 Ammonium Alum Potassium Alum Antimony Potassium Tartrate Cupric Chloride Gold Lead Acetate Mercuric Acetate 17 10 98 16 96 98 100 2 4 93 o 84 96 92 10—~ 1 10—~ 1 10 - '3 1- o 4 5 o 2 79 5 o o o o o 18 o TABLE XIX EFFECTS OF SQME AMINO ACID, PURINE AND PYRIMIDINE ANALOGS AND ANTIBrOTICS. RESULTS EXPRESSED AS PER CENT INHIBITION. (~,one~?n t.rs~ tinn ~ rn r~~n ~ Benzimidazole 2,6-Diaminopurine sulfate 8-Azaguanine 4-Phthalimido-2,6- dimethylpyrimidine Chloramphenicol Aureomycin 19 ~8 12 42 93 71 2 13 o o 13 56 o o o o 6 45 2.5 x 10—~ 1 x 10 o o o o 12 o o o o o o

126 PART II. BIOSYNTHESIS OF HEMOGLOBIN phenicol next; bacitracin, penicillin G and streptomycin caused less than 10 per cent inhibition at 5 x 10-3 M. The following amino acid, purine and pyrimidine analogs caused less than 10 per cent inhibition at 5 x 10-3 M: o- fluorophenylalanine, 3-fluoro-L-tyrosine, p-2-thienylalanine, 4-amino-5-imid- azolecarboxamide, isoguanine sulfate, 6-mercaptopurine, 6-aminouracil, 5- bromouracil, 4,6-diLydroxypyrimidine, 2-thiocytosine. TABLE XX COMPARISON OF RATES OF HEME SYNTHESIS AND CONCURRENT INCORPORATION OF GLYCINE INTO GLOBIN.3 Duration (1) furs. 1 2 4 Number of Heme Glycine experiments Synthes s incorporated (2) (3) (4) mM/M heme mM/gly residue In global 2 1.67-2.87 2.19- 2.80 21 2.48-7.51 2.71- 6.30 16 2.53-9.36 2.76-10.33 ( 3 ) (5) 1.14 + 0.24 0.98 + 0.15 1.06 + 0.14 In rabbit reticulocytes in vitro the rates of synthesis of heme and of glycine incorporation into globin per glycine residue were equal, table XX. This was observed with rates of hemoglobin synthesis which were made to vary widely by variatiorls in the iron fructose-amino acids in the reaction mixture. This result would not have occurred if there had been a pool of significant size of unlabeled intermediates of either heme or of globin, or of free heme or globinO The rates of synthesis of the two parts of hemoglobin under our experimental conditions, i.e. rabbit reticulocytes in vitro over periods not greater than hours, must have been nearly the same. This conclusion requires that the relative rates of incorporation of different amino acids into globin be the same. This was tested. The procedure was to add labeled glycine, histidine, leucine or lysine to different aliquots of cells containing otherwise the same reaction mixture, and then to incubate all the aliquots concurrently under identical conditions. Different conditions of stimulation were used in a number of different experiments. The results obtained, summarized ire table ~XI, were that per mole of amino acid in TABLE XXI RELATIVE RATES OF INCORPORATION IN GLOBIN OF GLYCINE, HISTIDINE, LEUCINE AND LYSINE.:3 His/Gly Leu/Gly Lys I Gly His /Leu 1.09 + 0.35 1.01 ~ 0.27 0.84 + 0.18 0.98 ~ 0.05

HEMOGLOBIN SYNTHESIS IN RETICULOCYTES BORSOOK 127 g;lobin, glycine, histidine, leucine, and lysine were incorporated at the same rate. All the deviations from 1 ire the different sets of ratios are within the experimental error and the uncertainties of the correction factor applied to the glycine incorporation for the change ire specific activity of the glycine within the cells. The foregoing observations indicate that in rabbit reticulocytes in vitro the rates of synthesis of heme and of globin are somehow regulated so that both proceed at or nearly at the same rate. The two processes are so different that large differences might have been expected. The above observations notwith- standing, with any marked pathological changes induced within the cells, it may be expected that the regulation would be broken down. Radiations and starvations appear to have such an effect; heme synthesis appears to be the more sensitive of the two processes. The two processes are more easily dislocated in avian than in rabbit cells.59 In disease (human) where there is interest in changes in the blood, the hemoglobin concentration is noted with such other hematological data as red cell count and hematocrit value. It might be that in some diseases the dis- location of heme and of globin synthesis is more pathognomonic than a possible anemia, i.e. suppression of the synthesis of both parts of hemoglobin or abnormally rapid destruction of red cells. REFERENCES 1. Borsook, H., Deasy, C. L., Haagen-Smit, A. J., Keighley, G., and Lo by, P. H.: Incorporation in vitro of labeled amino acids into proteins of rabbit reticulo- cytes, J. Biol. Chem. 196: 669, 1952. 2. Drabkin, D. L., and Austin, J. H.: Spectrophotometric studies. II. Preparations from washed blood cells; nitric oxide hemoglobin and sulfhemoglobin, J. Biol. Chem. 112: 51, 1935. 3. Kruh, J., and Borsook, H.: Hemoglobin synthesis in rabbit reticulocytes in ~vitro, J. Biol. Chem. 220: 905, 1956. 4. London, I. M., Shemin, D., and Rittenberg, D.: Synthesis of heme in vitro by the immature non-nucleated mammalian erythrocyte, J. Biol. Chem. 183: 749, 1950. 5. Borsook, H., Deasy, C. L., Haagen-Smit, A. J., Keighley, G., and Lowy, P. H.: Incorporation in vitro of labeled amino acids into rat diaphragm proteins, J. Biol. Chem. 186: 309, 1950. 6. Dubnoff, J. W.: An apparatus for the incubation of tissue slices and homogenates, Arch. Biochem. 17: 327, 1948. 7. Krebs, H. A., and Henseleit, K.: Untersuchungen uber die Harnstofibildung im Tierkorper, Z. Physiol. Chem. 210: 33, 1932. 8. Roche, J., Derrien, Y., and Moutte, M.: Solubility in concentrated saline solutions and specific characteristics of blood hemoglobins, Bull. Soc. chim. biol., Trav. Mem. 23: 1114, 1941. 9. Schapira, G., Dreyfus, J. C., and Kroh, J.: Recherches sur la biochimie de l'hemo$10bine a ['aide du fer radioactif. 1. Fractionnement des hemoglobines de lapin adulte par denaturation alcaline, Bull. Soc. chim. biol. 33: 812, 1951. 10. Kruh, J., Dreyfus, J. C., and Schapira, G.: Recherches sur la biochimie de

128 PART II. BIOSYNTHESIS OF HEMOGLOBIN 15. 17. l'hemoglobine a [' aide de fer ra dioactif. v. Biosynth es e de s hemoglobines in vitro, Bull. Soc. chim. biol. 35: 1181, 1953. 11. Anson, M. L., and Mirsky, A. E.: Protein coagulation and its reversal. The preparation of insoluble globin, soluble globin and heme, J. Gen. Physiol. 13: 469, 1930. 12. Schack, J., and Clark, W. M.: Metalloporphyrins VI. Cycles of changes in sys- tems containing heme, J. Biol. Chem. 171: 143, 1947. 13. a) Shemin, D., and Wittenberg, J.: The mechanism of porphyrin formation. The role of the tricarboxylic acid cycle, J. Biol. Chem. 192: 315, 1951. b ) Shemin, D., and Kumin, S.: The mechanism of porphyrin formation. The formation of a succinyl intermediate from succinate, J. Biol. Chem. 198: 827, 1952. 14. Schroeder, W. A., Kay, L. M., and Wells, I. C.: Amino acid composition of hemo- globins of normal negroes and sicl~le-cell anemics, J. Biol. Chem. 187: 221, 1950. Tristram, G. P.: The amino acid composition of proteins, in Neurath, H., and Bailey, K., The Proteins, 1: 180, Academic Press. 1953. 1 6. Cohen, P. P., and Hayano, M.: Urea synthesis by liver homogenates, J. Viol. Chem. 166: 251, 1946. Fontes, G., and Thivolle, L.: Le tryptophane et l'histidine vent des acides amines hematogenes, Compt. rend. Acad. 191: 1088, 1930; Recherches experimentales sur les processus chimiques de l'hematopo~ese et sur la pathogenic des enemies, Le Sang 4: 658, 1930. 1 8. Okugawa, Y., and Tatsui, M.: Uber die Wirkung des Tryptophans auf experi- mentelle Anamie und ihre Beziehung zur Milz, Z. Physiol. Chem. 195: 192, 1931. 19. Matsuoka, A., and Nakao, T.: Uber die Wirkung des Methyltryptophans auf kunstliche Anamie und Ernahrung, Z. Physiol. Chem. 195: 208, 1931. 20. a) Drabkin, D. L., and Miller, H. K.: Hemoglobin production, II. The relief of anemia due to milk diet, by feeding amino acids, J. Biol. Chem. 90: 531, 1931; b) Hemoglobin production III. The relief of anemia due to milk diet, by feed- ing amino acids and related compounds, J. Biol. Chem. 93: 39, 1931. 21. Elvehjem, C. A., Steenbock, H., and Hart, E. B.: Ineffectiveness of purified glu- tamic acid as a supplement to iron in the correction of nutritional anemia, J. Biol. Chem. 93: 197, 1931. 22. Keil, H. L., and Nelson, V. E.: The effect of oral administration of amino acids and intraperitoneal injection of various elements and hydrochloric acid on re- generation of hemoglobin, J. Biol. Chem. 97: 115, 1932. 23. Alcock, R. S.: The role of tryptophan in blood development, Biochem. J. 27: 754, 1933. 24. Kotake, Y.: Zum intermediaren Sto~wechsel des Tryptophans, Ergeb. Physiol. 37: 245, 1935. 25. Hamada, T.: Zur F`rage der hamatopoetischen Wirkung des Tryptophans, Z. Physiol. Chem. 243: 258, 1936. 26. Pearson, E>. B., Elvehj em, C. A., and Hart, E. B.: The relation of protein ~.o hemoglobin building, J. Biol. Chem. 119: 749, 1937. 27. Chin Kyu-sui: Uber den Einflusz des a-N-Methyltryptophans (Abrins) auf kunstliche Anamie und auf die Ernahrung, Z. Physiol. Chem. 257: 18, 1938-39. 28. Whipple, G. H., and Robscheit-Robbins, F. S.: a) Amino acids (natural and syn- thetic) as influencing hemoglobin production in anemia, Proc. Soc. Exp. Biol. and Med. 36: 629, 1937; b) Amino acids and hemoglobin production in anemia, J. Exp. Med. 71: 569, 1940.

HEMOGLOBIN SYNTHESIS IN RETICULOCYTES BORSOOK 129 29. Harris, H. A., Neuberger, A., and Sanger, F.: Lysine deficiency in young rats, Biochem. J. :'7: 508, 1943. 3 0. Albanese, A. A., Holt, L. E., Jr., Kaj di, C. N., and Frankston, J. E.: Observa- tions on tryptophane deficiency in rats. Chemical and morphological changes in the blood, I. Biol. Chem. 148: 299, 1943. 31. Gillespie, M., Neuberger, A., and Webster, T. A.: Further studies on lysine de- ficiency in rats, Biochem. J. 39: 203, 1945. 32. Orten, A. IJ., and Orten, ). M.: A study of hemoglobin formation following the administration of certain amino acids to rats fed a diet low in protein, J. Nutr. 30: 137, 1945. 33. Kornberg, A.: Amino acids in the production of granulocytes in rats. Federation Proc. 5: 142, 1946. 34. Yeshoda, K. M., and Damodaran, M.: Amino acids and proteins in haemoglobin formation. I. Tryptophan, Biochem. J. 41: 382, 1947. 35. Daft, F. S.: Pteroylglutamic acid ("folio acid"), liver extract and amino acids in treatment of granulocytopenia in rats, Pub. Health Rep., U.S.P.H.S. 62: 1785, 1 947. 36. Orten, J. M., Bourque, J. E., and Orten, A. U.: The inability of human or beef globin to support normal hematopoiesis in the rat without added isoleucine, J. Biol. Chem. 160: 43 5, 1945. 37. Sebrell, H.: Anemias caused primarily by malnutrition, Federation Proc. 8: 568, 1949. 38. Nizet, A., and Robscheit-Robbins, F. S.: Reticulocyte ripening in experimental anemia and hypoproteinemia. Effect of amino acids in vitro, Blood, 5: 648, 1950. 39. Chandran, K., and Damodaran, M.: Amino acids and proteins in haemoglobin formation. 2. Isoleucine, Biochem. J. 49: 393, 1951. 40. Cannon, P. R., Steffee, C. H., Frazier, L. J., Rowley, D. A., and Stepto, P. C.: The influence of the time of ingestion of essential amino acids upon utilization in tissue-synthesis, Federation Proc. 6: 390, 1947. 41. Geiger, E.: Experiments with delayed supplementation of incomplete amino acid mixtures, J. Nutrit. 34: 97, 1947. 42. Geiger, E.: The role of the time factor in feeding supplementary proteins, J. Nutrit. j'6: 813, 1948. 43. Geiger, E.: The importance of the time element in feeding of growing rats: Experiments with delayed supplementation of protein, Science 108: 42, 1948. 44. Geiger, E.: The role of the time factor in protein synthesis, Science 111: 594, 1950. 45. Geiger, E.: Extra caloric function of dietary components in relation to protein ... . _ ~ . ~ , ~ _ A utilization, Federation Proc. 10: 670, 1951. 46. Geiger, E., Hagerty, E. B., and Gatchell, lI. D.: Transformation of tryptophan to nicotinic acid investigated with delayed supplementation of tryptophan. Arch. Biochem. 23: 315, 1949. 47. Harte, R. H., Travers, J. J., and Sarich, P.: The effect on rat growth of alternated protein intakes, J. Nutrit. 35: 287, 1948. 48. Henderson, R. and Harris, R. S.: Concurrent feeding of amino acids, Federation Proc. 8: 385, 1949. 49. Sanadi, D. R., and Greenberg, D. M.: Effect of amino acid deficiencies on in- corporation of radioactive carbon-labeled amino acids into animal tissue pro- teins, Proc. Soc. Exp. Biol. and Med. 69: 162, 1948. 50. Schaeder, A. J., and Geiger, E.: Cataract development in animals with delayed supplementation of tryptophane, Proc. Soc. Exp. Biol. and Med. 66: 309, 1947. 5 1. Tarver, H., and Schmidt, C. L. A.: Radioactive sulfur studies. I. Synthesis of

130 PART II. BIOSYNTHESIS OF HEMOGLOBIN methionine. II. Conversion of methionine sulfur to taurine sulfur in dogs and rats. III. Distribution of sulfur in the proteins of animals fed sulfur or methion- ine. IV. Experiments in vitro with sulfur and hydrogen sulfide, J. Biol. Chem. 146: 69, 1942. 52. Drabkin, D. L.: Metabolism of the hemin chromoproteins, Physiol. Rev. 31: 345, 1951. 53. Borsook, H., Abrams, A., and Lowy, P. H.: Pructose-amino acids in liver: Stimuli of amino acid incorporation in rvilro, J. Biol. Chem. 215: 111, 1955. 54. Abrams, H., Lowy, P. H., and Borsook, H.: Preparation of 1-amino-1-deoxy-2- ketohexoses from aldohexoses and a-amino acids. I., J. Amer. Chem. Soc. 77: 4794, 1955. 55. Lowy, P. H., and Borsook, H.: Preparation of N-substituted 1-amino-1-deoxy-D- arabino-hexuloses of arginine, histidine and lysine, J. Amer. Chem. Soc. 78: 3175, 1956. 56. Altman, K. I., Casarett, G. W., Masters, R. E., Noonan, T. R., and Salomon, K.: Hemoglobin synthesis from glycine labeled with radioactive carbon in its u- carbon atom, J. Biol. Chem. 2~76: 319, 1948. 57. Salomon, K., Altman, K. I., Casarett, G. W., and Noonan, T. R.: Effect of x-ray radiation and of starvation on hemoglobin synthesis in the rat, Federation Proc. 8: 247, 1949. 58. Richmond, J. E., Altman, K. I., and Salomon, K.: The effect of x-radiation on the biosynthesis of hemoglobin, J. Biol. Chem. 190: 817, 1951. 59. London, I. M., et al. See these Proceedings. DISCUSSION Dr. Peratz: I should like to comment on Dr. Borsook's observation that globin synthesis is inhibited in the absence of a supply of RNA despite the very large reservoir of RNA present in the cell. I wondered whether one might think of two kinds of RNA in the cell- one which is structural, as it were, and forms the template for the formation of globin and is therefore not avail- able, and another part of RNA which is metabolic and which has to be con- tinuously resynthesized in order that globin synthesis can be carried on.

THE INCORPORATION OF GLYCINE INTO GLOBIN AND THE SYNTHESIS OF HEME IN DUCK ERYTHROCYTES AND RABBIT RETICULOCYTES~ IRVING M. LONDON,-: HELENA MORELL ID ANTON KASSENAAR-~ The studies which I should like to report were designed to investigate the formation of globin by duck erythrocytes in vitro and to compare the rates of synthesis of heme and of the incorporation or glycine into globin under different experimental conditions. The biosynthesis of heme has been extensively studied ire intact human, rabbit and avian erythrocytes,~ '' and in non-intact prepara- tions of these cells.3~5 Although evidence for the formation of peptide bonds in vitro in the hemoglobin of duck erythrocytes was obtained several years ago with the use of N75-labeled histidine,0 relatively little attention has been given to the usefulness of this system for the study of the formation of protein. Chicken erythrocytes and reticulocytes have been employed for the study of the rates of formation of heme and of hemoglobin.0 The incorporation in vitro or various isotopically-labeled amino acids into tile total protein of reticulocytes obtained from phenylLydrazine-treated rabbits leas been studied; and more recently these observations have been extended to the incorporation of glycine into globin and its utilization for the synthesis of heme in these cells.S As a system for the study of protein synthesis, or for the incorporation of an amino acid into a protein, the immature mammalian or avian erythrocyte affords the advantages of simplicity and ready availability. More important, however, is the fact that as the protein under study, hemoglobin can be iso- lated in relatively pure form and with highly reproducible analytic values. Materials and Procedures. In these experiments, the standard reaction mixture consisted of 4 ml of washed duel: erythrocytes suspended in 8 ml of isotonic sodium phosphate buffer, pH 7.4; an amino acid mixture such as described by Borsook and his associates,7 but without glycine; glucose in a concentration of 200 mg per 100 ml; penicillin G and streptomycin sulfate, 10 ma. of each per 100 ml of incubation mixture; and glycine-2-C2'' in a concentration of 1.2 mg of glycine (10 microcuries) per 12 ml of incubation mixture. In the standard incubation procedure, 50 ml Erlenmeyer flasks, con- taining 12 ml of reaction mixture, were incubated with shaking in a water bath at 37° C. for 4 hours. The reaction mixture was exposed to air and all experiments were done in duplicate or triplicate. After incubation, the samples were transferred to a cold room (4° C.) and This work was supported by grants from the Office of Naval Research (contract Nonr-1765 (00) ), the American Cancer Society and the Atomic Energy Commission. -i-This paper was presented by Dr. London. +~ Fellow of the Rockefeller Foundation 1956. Present address: Department of Endo- crinology, University of Leiden, Leiden, Holland. 131

132 PART II. BIOSYNTIIESIS OF HEMOGLOBIN were washed three times with 10 volumes of isotonic sodium chloride. The erythrocytes were lysed with 10 ml of distilled water and the lysate was mixed thoroughly by shaking with 2 ml of toluene until a firm emulsion was formed. After centrifugation the clear layer of hemoglobin solution was removed and filtered through paper. The hemoglobin solution was then added dropwise to 10 to 12 volumes of acetone, containing 1.2 ~ of con- centrated HC1, as in the Anson and Mirsky procedure.0 The precipitated "globin" was washed several times with acid-acetone until the supernatant solution after centrifugation was colorless. The globin was then redissolved in water and reprecipitated with acid-acetone. The precipitated material was dissolved in distilled water, precipitated with 14 To trichloroacetic acid, washed twice with 7 ~ trichloroacetic acid and once with distilled water. The globin was then dissolved in 2 ml of 1N NaOH and the precipitation and washing procedures were repeated. The final preparation was dried by washing with an ethyl ether-acetone mixture (1 :1) twice with acetone and, finally, twice with ethyl ether. The final preparation is a pure white powder. Care must be exercised to keep it dry for it takes up water readily and its radioactivity may become erroneously low. This procedure has yielded uniformly reproducible results. The precise nature of the protein, which is called "globin," is not definitively established, but from experiments which are reported below, it would seem reasonable to conclude that the protein which we isolate by this method is, indeed, the "globin" of hemoglobin. Hemin was isolated from the solution of acid- acetone and was recrystallized prior to counting its radioactivity. The radioactivity of the hemin and of the globin was determined in a thin end-window gas flow counter. The results are expressed as the specific ac- tivity, counts per minute per millimole of glycine in heme or in globin. The method of calculation and the assumptions on which the calculation is based are presented elsewhere.~° The "heme to globin ratio" represents the specific activity of the glycine in heme relative to the specific activity of the glycine in globin, i.e., counts per minute per millimole of glycine in heme relative to counts per minute per millimole of glycine in globin. The data represent the means of replicate samples with a maximal range of variation of + 5~. E~idence for Parity of the Protein Preparations. Protein samples iso- lated by the method described above were shown to be of constant specific activity when the material was redissolved in 1N sodium hydroxide and the purification procedures were repeated. Furthermore, protein samples iso- lated from lysed erythrocytes which had been incubated with labeled glycine at 37° C. or from intact erythrocytes which were incubated at 4° C. con- tained no measurable radioactivity. ~lable 1 presents a compar~son ot the rad~oact~v~ty ot prote~n prepared by this method with that of globin isolated from crystalline hemoglobin in ali- quot samples. No significant differences in radioactivity were found. It ~ · · r

GLYCINE INTO GLOBIN- LONDON, MORELL AND KASSENAAR 133 TA:E3LE I COMPARISON OF RADIOACTIVITY OF PROTEIN ISCLATED FROM "HEMOGLOBIN SOLUTION" AND OF GLOBIN ISOLATED FROM CRYSTALLINE HEMCGLOBIN Experiment Protein Isolated from Globin Isolated from Number "Hemoglobin Solution" Crystalline Hemoglobin c.p.m./mM c.p.m./mM 1 24,950 2S,750 2 22,300 23,250 seems reasonable, therefore, to refer to the protein isolated from the "hemo- globin solution" as globin. Incubation Medium. In preliminary experiments incubation in plasm resulted in a higher rate of incorporation of glycine into globin than incuba- t~on in 0.9C/o sodium chloride solution, isotonic sodium phosphate buffer pH 7.4 or Krebs-Ringer phosphate buffer plI 7.4. However, in order to elimi- nate unknown variable factors which might be present in plasma, isotonic phosphate buffer was chosen as the medium. There was little difference in the rate of incorporation of glycine into globin in a medium of isotonic phos- phate buffer, Krebs-Ringer phosphate buffer or 0.9 /7o sodium chloride. 7:onicity of Medium. The system for the incorporation of glycine into globin has a sensitivity to changes in the tonicity of the incubation medium as indicated in table II. On lowering the tonicity to 50% of isotonicity, the incorporation of glycine into globin is diminished by as much as 22 to 63~. Only slight hemolysis was observed at this level of tonicity. On raising the tonicity to 125570 of isotonicity, there was some diminution in the incorpora- tion of glycine into globin. TABLE II INFLUENCE OF TONICITY OF THE INCUBATION MEDIUM ON THE INCORPORATION OF GLYCINE INTO GLOBIN AND HEME Radioactivity of Glycine per cent per cent of control In Heme of control Tonicity of Medium Expt. No. per cent of Control In Globin c.p.m /mM c.p.m. /mM 1 Control 30,300 125 25,600 84 75 20,700 68 50 11,200 37 2 Control 52,000 50 27,300 52 3 Control 23,400 — 67,500 50 18,400 78 65,000 96 Isotonic sodium phosphate buffer, pH 7.4. and the same buffer diluted with distilled water svere used as the incubation Tnedia.

134 PART II. BIOSYNTHESIS OF HEMOGLOBIN' The synthesis of heme, however, is not significantly reduced in a medium 50~%o of isotonicity (table II). The differential effect of hypotonicity is con- sistent with the findings that heme synthesis proceeds in non-intact avian erythrocytes3-, and that the incorporation of amino acids into the protein of rabbit reticulocytes ceases if the cells are lysed.7 Influence of Amino Acids and Glucose. Addition of glucose or of a mix- ture of amino acids to the cell suspension results in only slight enhancement of the incorporation of glycine into globin during incubation periods of four hours. With longer periods of incubation, significantly greater incorporation occurs in those preparations to which glucose or amino acids and glucose have been added (Eg. ~ ~ . ~ 4xi04 _ L) - Z- 3x104 _ o Z 2x104 o ,_ 104 - ~_ O +AMINO ACIDS 2 - + GLUCOSE . _ = ~ 105 _ +AMINO ACIDS — ° _ . ~~ ' ~ . 10 .5 4 10 20 1.5 4 10 20 TIME IN HOURS Time in Hours FIG. 1. (left) The influence of amino acids and of glucose on the incorporation of glycine into glol~in in duck erythrocytes. FIG. 2. (right)—The effect of time of incubation on the biosynthesis of heme and on the incorporation of glycine into globin in duck erythrocytes. Time of Incubation. The rates of heme synthesis and of incorporation of glycine into globin are most rapid in the initial hours of incubation. With longer incubation both processes are decelerated, but the incorporation of glycine into globin is more markedly slowed. The changes in the rates of the two processes are reflected in the progressive increase in the heme-globin ratios (fig. 2). Temperature of Incubation Median. Both processes are sensitive to temperature, but the sensitivity of heme synthesis is more striking. At 40° C., the synthesis of heme was 100 times more active than at 10 °, while the incorporation of glycine into globin was increased only twenty-six times (table III). The Efects of Iron, Cobalt and Lead. Further evidence for the dissocia- tion of heme synthesis and of the incorporation of glycine into globin is pro- vided in the studies on the effects of iron, cobalt and lead or the two pro-

GLYCINE INTO GLOBIN- LONDON, MORELL AND KASSENAAR 135 TABLE III INFLUENCE OF TEMPERATURE OR SYNTHESIS OF HEME AND INCORPORATION OF GLYCINE INTO GLOBIN Temperature C. Specific activity of glycine (c.p.m./mM) "Eeme/ Globin Ratio" in gIol~.n in heme - 10° 800 1~070 1.3 20 ° 3~500 9~800 2.8 30° 14,900 48,500 3.2 40° 20,600 107,000 5.2 Incubation time four hours. Composition of incubation mixture as described in standard conditions. cesses. Previous studies have demonstrated the inhibitory effects of leader i' and cobalti3 on heme synthesis in duck and chicken erythrocvtes and in bone marrow. The inhibitory effects of these metals can also be demonstrated at an earlier stage of porphyrin synthesis, namely the conversion of delta-amino- levulinic acid to porphobilinogen.~4 The addition of iron on the other hand has been shown to enhance the formation of heme in vitro.5, :0 In table IV the effects of these metals on the incorporation of glycine into Gloria as well as on the synthesis of heme are presented. The enhancement of heme synthesis by iron and the inhibition by lead and cobalt are marked. The incorporation of glycine into globin, however, is unchanged on addition of cobalt or iron and is inhibited to a lesser degree than is heme synthesis by lead. The dissociation of the two mechanisms is reflected in the marked variations that are induced in the heme-globin ratios. Since the addition of iron did not increase the incorporation of glycine into globin of duck erythrocytes, whereas Kruh and Borsook had reported that iron increased the formation of globin in rabbit reticulocytes,S experiments TABLE IV THE EFFECTS OF LEAD, COBALT AND IRON ONT SYNTHESIS OF HEME AND INC~RPCRATION OF GLYCINE INTTO GLOBIN Specific activity of gIycine (c.p.m./mM) Metal Added in Robin per cent of control in heme per cent of control "Heme/Globin Ratio" Control Pb+~- FeCI2 CoCl 15,200 10,800 15,000 1 5,700 71 100 103 50,500 11,600 96,000 2,030 190 4 3.3 1.3 6.4 0.1 Ferrous chloride, lead acetate and cobaltous chloride were added in concentrations of 5 :; 10~ M. 4 ml. of cells were suspended in 8 ml. of previously boiled saline containing metal salts. Glucose, penicillin and streptomycin were added in the usual concentrations. The cells were preincubated with the metals for two hours before substrate was added. After addition of the usual amount of glycine 2-C1-i, the incubation was continued for four hours.

136 PART II. BIOSYNTHESIS OF HEMOGLOBIN were performed with the blood of rabbits in which reticulocytosis had been induced with acetylphenylhydrazine. The results indicate that the incorpora- tion of glycine into globin in rabbit reticulocytes is enhanced by iron, in con- firmation of the results of Kruh and Borsook, and that the enhancement is very similar in degree to that of heme synthesis (table V). TABLE V INFLUENCE OF IRON ON SYNTHESIS OF HEME AND ON INCORPORATION OF GLYCINE INTO GLOBIN OF RETICULOCYTES OF RABBITS TREATED WITH PHENY~HYDRAZINE Specific activity of glycine ( c.p.m./mM ) "Heme/Globin Sample Ratio" in globin in heme Control 96,800 10,100 1.1 Control 93,400 10,650 1.1 Control 94,200 10,550 1.1 Fe+ ~ 123,600 149,000 1.3 Few ~ 123,500 145,000 1.2 Few ~ 122,600 137,000 1.1 Each sample consisted of 2.5 ml. of cells suspended in 8 ml. of boiled 0.9 per cent NaC1 solu- tion, containing the amino acid mixture, glycine 2-C14 and antibiotics in the same amounts as in the standard incubation mixture. Iron was added as FeCl.~ in the concentration of 40 micro- grams of iron per sample (? x 10~ M). The incubation was carried out for two hours at 37° C. The Endings in rabbit reticulocvres differed from those in normal duck erythrocytes not only in terms of the effects of iron on glycine incorporation into globin, but also in the "heme-globin ratios." "Heme-globin ratios" of 1.1 and 1.2 in rabbit reticulocytes are similar to those previously described.S In the normal duck erythrocytes, however, the "heme-globin ratios" are generally much higher and more variable. To determine whether the "heme- globin ratio" might be closer to unity and whether iron might enhance glycine incorporation into globin in more immature duck erythrocytes, the cells of acetylphenylhydrazine-treated ducks were employed. The results in the more immature erythrocytes confirm the findings observed in the normal duck erythrocytes, namely that the incorporation of glycine into globin is not enhanced by iron, that the synthesis of heme is increased on addition of iron and that the "heme-globin ratio" is usually much greater than unity (table VI ~ . To determine whether incubation of duck erythrocytes in the more natural environment of duck plasma might result in a "heme-globin ratio" closer to unity, experiments were performed in duck plasma, but the "heme- globin ratios" obtained were even higher than those usually found in a phosphate buffer medium. The Effects of N~cleosides. Incubation with purine ribosides has been shown to prolong the viability of stored human and rabbit erythrocytes 17 to enhance the resistance of fresh human erythrocytes to osmotic lysisi8 and to increase the concentration of phosphate esters in human and rabbit erv- throcytes.~9 A Since the mechanism by which the purine ribosides exert these

GLYCINE INTO GLOBIN—LONDON, MORELL AND KASSENAAR 137 TABLE VI SYNTHESIS OF HEME AND INCORPORATION OF GLYCINE INTO G~os~N IN ERYTHROCYTES OF NORMAL AND OF ACETY~pHENy~HyDRAz~xE-TREATED DUCKS; EFFECT OF IRON IN ERYTHROCYTES OF ACETY~PHENy~HyDRAz~NE-TREATED DUCKS Expt. No. 1 2 __ ~ ~ Specific Activity of Glycine (c.p.m./mM) Incubation Incubation Time, Duck Medium Hours Erythrocytes in Globin in Heme NaCl 2.5 Normal 48,000 75,000 O.g~o ( boiled ) Acetylphenyl- hydrazine treated a) Control 182,000 406,000 b) Fe++ added 172,000 1,190,000 Isotonic 4 Normal 14,200 48, 5 00 Phosphate Buffer Acetylphenyl- 148, 500 645,000 hydrazine treated ,- "Heme/Globin Ratio" 1.6 2.2 6.9 3.4 4.3 When iron was added, it was in the form of Fecal at a concentration of 5 x 10—4- M. Standard experimental conditions were observed except as noted. TABLE VII EFFECTS OF PURINE RIBOSIDES AND RELATED COMPOUNDS ON HEME SYNTHESIS AND ON GLYCINE INCORPORATION INTO GLOBIN IN DUCK ERYTHROCYTES AND IN IMMATURE ERYTHRCCYTES OF RABBITS Expt. No. 1 2 3 Erythrocytes Acetylphenyl- hydrazine-treated rabbits Normal Ducks Acetylphenyl- hydrazine-treated ducks Specific Activity of Glycine (amp lmM) Nucleoside "Heme/ Globin Added in Globin in Heme Ratio" Control 10,700 Adenosine, 5 M/ml 7,800 Control 3 3,100 65,000 Adenosine, 10 M/ml 15,300 59,000 Inosine, 10 M/ml 17,700 56,000 Deoxyadenosine, 31,600 59,000 Deoxyguanosine, Cytidine and Thymidine Control 148,500 645,000 Adenosine, 5 M/ml 46,000 296,000 2.0 3.8 3.2 1.9 4.3 6.4

138 PART II. BIOSYNTHESIS OF HEMOGLOBIN effects is probably the introduction into the erythrocyte of phosphorylated ribose, its metabolism via the he.xose-monophosphate shunt and the Embden- Meyerhof cycle and the formation of high energy phosphate esters, we in- vestigated the effects of the purine ribosides on the incorporation of glycine into globin and on the synthesis of heme. The results of studies in duck erythrocytes and in immature erythrocytes of rabbits are presented in table NIII. In the erythrocytes of normal ducks and of acetylphenylhydrazine- treated ducks, the incorporation of glycine into globin is diminished under the influence of Cosine or adenosine; the degree of inhibition is greater than that noted on heme synthesis. In immature erythrocytes of rabbits the in- hib'2tory influence of adenosine on the incorporation of glycine into globin is observed. The deoxyribosides had no significant effect on either process in normal duck erythrocytes. The incorporation of glycine into the globin of intact erythrocytes repre- sents a composite effect of at least two processes: ~ ~ ~ the uptake of the amino acid by the cell and (2) the incorporation of the amino acid into the globin. The first process has been studied in duck erythrocytes by Christen- sen, Riggs and Ray who showed that normal duel: erythrocytes take up amino acids from a plasma or saline medium against a concentration gradient.") This concentrative activity is considerably less than that which has been observed for guinea pig brain,~'' rat diaphragm'':; or rabbit reticulocytes.~4 Coupled with the lesser concentrative activity of duck erythrocytes for amino acids is a relative insensitivity of the concentrative process to anoxia and to agents such as cyanide, 2,4 dinitrophenol and other metabolic in- hilaitors in high concentrations. a In experiments in normal duck erythrocytes in which the incorporation of glycine into globin has been markedly diminished while heme synthesis has been unimpaired (e.g., in hypotonic media, or under the influence of nucleo- sides) it seems likely that the primary effect is on the mechanism of incorpora- tion of glycine into the protein rather than on uptake of the glycine by the duck erythrocyte. Support for this interpretation may be derived from the finding that the synthesis of heme from glycine is not diminished under these conditions. This finding can be used to support this interpretation on the assumption that the same metabolic pool of glycine is utilized for the syn- thesis of heme and for incorporation of glycine into globin. Further experi- mental work is required to determine whether this assumption of a common metabolic pool is correct. The studies in duck erythrocytes demonstrate that the synthesis of heme and the incorporation of glycine into globin are readily dissociated in vitro by prolonging the time of incubation, by changing the temperature or tonicity of the medium, by the use of metals and by the use of nucleosides. The mechanism for the incorporation of glycine into globin appears more sensi- tive than the synthesis of heme to environmental changes which may induce

GLYCINE INTO GLOBIN LONDON, MORELL AND KASSENAAR 139 structural disorganization within the erythrocyte. It is, of course, not sur- prising that two processes which are so distinct are differentially affected by these various environmental and metabolic conditions. The Gradings serve to focus attention, however, on the need for determining in various disorders of hemoglobin metabolism the extent to which the synthesis of heme or of globin or of both may be disturbed. These techniques are readily applicable to the study of humeri bone marrows in normal and in disease states. This in vitro system of immature avian or mammalian erythrocytes also provides a suitable tool for the study of the relationship of nucleic acid syn- thesis to hemoglobin formation and of the influences of hormones and of other metabolites on these synthetic processes. Recent work by Mrs. Morell and Dr. Savoie ire our laboratory indicates that rabbit bone marrow in vitro may be used for the study of the temporal relations of heme and globir~ formation. Such studies should complement the investigation in vivo of these mechanisms and of their ir~terrelatior~s.';~~"7 REFERENT CES 1. London, I. NI, Shemin, D., and Rittenberg, D.: a) The i,' ditto synthesis of heme in the human red blood cell of sickle cell anemia, J. Biol. Chem. 171: 797, 1948; b) Synthesis of heme ill vitro by the immature non-nucleated mammalian ery- throcyte, J. Biol. Chem. lS]: 749, 1950. 2. Shemin, D., London, I. M., and Rittenberg, D.: a) The in vitro synthesis of heme from glycine by the nucleated red blood cell, J. Biol. Chem. 171: 799, 1948; b) The synthesis of protoporphyrin in vitro by red blood cells of the duel;, l. Biol. Chem. 181: 757, 1950. 3. Shemin, D., and Kumin, S.: Alpha ketoglutarate-succinate reaction; the forma- tion of a succinyl intermediate for succinate, Federation Proc. 11: 285, 1952. 4. London, I. M., and Yamasaki, M.: Heme synthesis in non-intact mammalian and avian erythrocytes, Federation Bloc. 11: 250, 1952. 5. Dresel, E. I. B., and Falk, l. S.: a) Haem and porphyrin formation from delta- aminolevulinic acid and from porphobilinogen in haemolysed chicken erythro- cytes, Biochem. J. 61: 80, 1956; b) Haem and porphyrin formation in intact chicken erythrocytes, Biochem. J. 63: 72, 1956. 6. Allfrey, V., and Mirsky, A. E.: The incorporation of Ni5-glycine by avian ery- throcytes and reticulocytes in Citron J. Gen. Physiol. 35: 841, 1952. 7. Borsook, H., Deasy, C. L., Haagen-Smit, A. J., Keighley, G., and Lowy, P. H.: Incorporation in Vitro of labeled amino acids into proteins of rabbit reticu- locytes, J. Biol. Chem. 196: 669, 1952. 8. Krnh, J., and Borsook, H.: Hemoglobin synthesis in rabbit reticulocytes in Vitro, J. Biol. Chem. 220: 90 5, 19 5 6. 9. Anson, M. L., and Mirsky, A. E.: Protein coagulation and its reversal. The prep- aration of insoluble Robin, soluble globin and heme, J. Gen. Physiol. 11: 469, 1930. 10. Kassenaar, A., Morell, H., and London, I. M.: The incorporation of glycine into globin and the synthesis of heme in vitro in duck erythrocytes, J. Biol. Chem. (Ir1 Press) 11. Eriksen, L. E.: Lead intoxication: 1. The effect of lead on the in vitro biosyn-

140 PART II. BIOSYNTHESIS OF HEMOGLOBIN thesis of heme and free erythrocyte protoporphyrin, Scand. J. Lab. CIin. Invest. 7: 80, 1955. 12. Goldberg, A., Ashenbrucker, H., Cartwright, G. E., and Wintrobe, M. W.: Studies on the biosynthesis of heme in vitro by avian erythrocytes, Blood 11: 821, 1956. 13. Laforet, M. T., and Thomas, E. D.: The effect of cobalt on heme synthesis by bone marrow in ~vitro, J. Biol. Chem. 218: 595, 1956. 14. Morell, H., and London, I. M.: Unpublished data. 15. Shemin, D.: Personal communication. 16. Gabrio, B. W., Donohue, D. M., and Finch, C. A.: Erythrocyte preservation. V. Relation between chemical changes and viability of stored blood treated with adenosine, J. Clin. Invest. 34: 1509, 1955. 1/. Gabrio, B. W., Donohue, D. M., Huennekens, F. M., and Finch, C. A.: Erythrocyte preservation. VII. Acid-citrate-dextrose-inosine (ACDI) as a preservative for blood during storage at 4° C., J. Clin. Invest. 35: 657, 1956. 18. Jaffe, E. R., Lowy, B. A., Vanderhoff, G. A., Aisen, P., and London, I. M.: Proc. VI Int. Cong. of Hematology, Boston, 1956; Abstract in Federation Proc. 15: 304, 1956. 19. Prankerd, T. A. J., Altman, K. I., and Young, L. E.: Abnormalities of carbohy- drate metabolism of red cells in hereditary spherocytosis, I. Clin. Invest. 34: 1268, 1955. 20. Rubinstein, D., Kashket, S., and Denstedt, O. F`.: Studies on the preservation of blood. IV. The influence of adenosine on the glycolytic activity of the ery- throcvte during storage at 4° C.. Canad. T. of Biochem. PhYsiol. 34: 61. 1956. 21. Christensen, H. N., Riggs, T. R., and Ray, N. E.: Concentrative uptake of amino acids by erythrocytes in vitro, J. Biol. Chem. 194: 41, 1952. 22. Stern, J. R., Eggleston, L. V., Heins, R., and Krebs, H. A.: Accumulation of glu- tamic acid in isolated brain tissue, Biochem. J. 44: 410, 1949. 23. Christensen, H. N., and Streicher, J. A.: Concentration of amino acids by the excised diaphragm suspended in artificial media. I. Maintenance and inhibition of the concentrating activity, Arch. Biochem. 23: 96, 1949. 24. Riggs, T. R., Christensen, PI. N., and Palatine, I. M.: Concentrating activity of reticulocytes for glycine, J. Biol. Chem. 194: 53, 1952. 25. Thorell, B.: Studies on the formation of cellular substances during blood cell pro- duction, Acta Med. Scand. 200: (Suppl.) 1-120, 1947, (accompanies vol. 129). 26. Hammarsten, E., Thorell, B., Aqvist, S., Eliasson, N., and Akerman, L.: Studies on the hemoglobin formation during regenerative erythropoiesis, Exp. Cell Res. 5: 404, 1953. 27. Muir, H. M., Neuberger, A., and Perrone, J. C.: Further isotopic studies on haem- oglobin formation in the rat and rabbit, Biochem. J. 52: 87, 1952. DISCUSSION Dr. Felix Hazlrowitz: I just want to comment briefly on what Dr. London said about Christensen's experiments on the uptake of glycine into red blood cells. Dr. Lietze in my laboratory has repeated and confirmed these experi- ments, using radioactive glycine. About 80 to 90 percent of the intracellular glycine is free; only 10 to 20 percent is bound to protein. Dr. Kurt Salomon: I would like to comment briefly on the findings of Dr. London concerning the fact that the biosynthesis of hemoglobin can be affected in different ways. In collaboration with [ones E. Richmond and Kurt I.

DISCUSSION 141 Aitman several years ago we investigated the ability of bone marrow prepara- tions of irradiated rabbits to incorporate the alpha carbon atom of glycine into hemin and globin.i The animals were irradiated with 800 r, and then killed at various time intervals after radiation, e.g., immediately after radia- tion, or to be more precise as soon as possible the preparation of the bone rr~arrow taking about half an hour 24 hours later, 48 hours later, etc. The bone marrow was removed from the long bones, homogenized and incubated ill the presence of alpha carbon labeled glycine. We then isolated hemoglobin and prepared from it hemin and globin. It was found that hemin synthesis decreased approximately 24 hours after radiation, and remained low for about three to four weeks. After this time period hemin synthesis increased. Globin synthesis on the other hand was not influenced markedly during the first week after irradiation, and remained on that level over the time period studied. In other words, one may say that in intact rabbits radiation affects hemin and globin synthesis differently, the biosynthesis of hemin being more radia- tion-sensitive than the biosynthesis of globin, but we do not know why. We concluded, as Dr. London does, that the biosyntheses of hemin and of globin are dissociated processes, a finding which has been confirmed by Neuberger and associates using somewhat different methods of approach. Dr. H. Borsook: I regret that in the rush of trying to get through my paper I did not refer to the much earlier work of Dr. Salomon and his associates. We knew that the two processes of heme and of globin synthesis could be separated. In the demonstration that the two processes are separable, one may overlook what to me, at any rate, is a remarkable fact that, at least in rabbit reticulocytes, the two go so closely together. There is evidently some kind of interaction. This interaction can be suspended or interfered faith. It becomes an interesting question: is this a mere coincidence in rabbit reticulocytes? I think not, because one can vary the rate of hemoglobin syn- thesis greatly and at the same time retain equal rates of synthesis of heme and oT globin. It becomes an interesting general question regarding the synthesis `~f conjugated proteins in general, namely: is there any relation between the synthesis of the protein part and of the conjugate? Dr. Salomo7~: May I add one more comment? My previous remark should be somewhat qualified for an adequate assessment of the radiation effects mentioned. It should be kept in mind that bone marrow after irradiation with the doses used is changed histologically and biochemically.3 Keeping this in mind we can only say that the ratio of the biosynthesis of hemin to globin is different from what it was before radiation of the animal, but we know noth- ing about the causes of this phenomenon. It might be that by using a better- 1.Richmond, J. E., Altman, K. I., and Salomon, K.: The effect of x-radiation on the biosynthesis of hemoglobin, J. Biol. Chem. 190: 817, 1951. 2. Muir, H. M., Neuberger, A., and Perrone, J. C.: Further isotopic studies on hemoglobin formation in the rat and rabbit, Biochem. J. 52: 87, 1952. 3. Lajtha, L. G.: Bone marrow cell metabolism, Physiol. Rev. 37: 50, 1957.

142 PART II. BIOSYNTHESIS OF HEMOGLOBIN defined in vitro system and carrying out simultaneous cytological studies an alternate interpretation may be necessary. Dr. Jean-Cla?lde Savoie: I should like to report on some studies concerned with the site of formation of heme within the duck erythrocyte. This work was done in collaboration with Mrs. Helena ~I:orell and Dr. Irving M. London. Duck erythrocytes were incubated for varying periods of time with glycine-2-C7~. After incubation, the erythrocytes were fractionated and hemin was isolated from each fraction and its specific activity determined. The re- sults indicate that heme is synthesized in close association with particulate matter of the cell and that, with time, the newly synthesized heme passes pro- gressively into the soluble portion of the erythrocyte. Samples of duck erythrocytes were suspended in 50 ml. of isotonic NaCl. I;~errous iron as FeCl~, was added in a concentration of 1 ~ 1O-4 Molar to enhance heme synthesis. The samples in duplicate were incubated in room air faith shaking in a water bath at 37° C. To each sample glycine-~-C~J' was added as the isotopic substrate. Immediately after incubation the erythrocytes were separated from the suspension medium by centrifugation and were washed four times with three volumes of cold, isotonic saline. The packed cells were lysed with distilled water and al ter hemolvsis; 10.6 gnu. of sucrose dissolved in 10 ml. of plater svas added to reconstitute the added water to isotonicity. The hemolysate was then centrifuged for 20 minutes at 4° C. and at 23,000 ~ G and the supernatant hemoglobin solution, fraction I, was separated from the residue by Recantation. In order to remove front the residue the hemoglobin which was not firmly bound to it, the residue was washed twice. The first washing divas performed with 100 ml. of distilled H,O and the suspension was then centrifuged at 23,000 x G for 20 minutes and the supernatant solution decanted. The residue was next extracted with isotonic sodium chloride and the extract re- moved by high speed centrifugation. The water and saline washes~were com- bined (fraction II) and the washed residue constituted fraction III. lIemin was isolated from each fraction by HCl-acetone extraction and crystallized. The hemin in crystalline form was plated at infinite thickness and counted in a thin end-~vindow gas flow counter. Table I presents the results of an experiment which divas performed on a ing periods of time. The data indicate that after one half hour of incubation, the specific activity in the hemin derived from the stroma is much higher than that derived from the hemoglobin solution, the ratio of the specific ac- iarge sample of duck erythrocytes which were pooled and incubated for vary- tivities in these two fractions being close to 6. With more prolonged incu- bation, there is a progressive decline in this ratio which indicates the pro- gressive increase in the specific activity of the hemoglobin in the supernatant solution. D ~ , 1 ~

DISCUSSION 143 Table II shows the results of separate experiments, each performed with ~ different pool of duck erythrocytes incubated with glycine 2-C for periods of time varying from one half to four hours. The results indicate that despite the different samples of blood and the performance of these experiments at different times, the ratio of the specific activity of the hemin of fraction I to specific activity of the hemin of fraction III at any given time is similar, and that there is ~ progressive decline in this ratio with time. Heme is formed in close association with the particulate matter of the erythrocyte. With longer periods of incubation, progressively larger amounts of the newly-formed and highly-labeled theme are released into the soluble portion of the cell. More precise fractionation of the cells will be performed in order to localize the sites of heme formation more definitely. TABLE I CHANGES IN SPECIFIC ACTIVITIES OF 'FLESIDUE BOUND HEMIN' AND OF IIEMIN FROM "[IEMOGLOBIN SOLUTION" WITH INCREASING INCUBATION TIMES FRACTION I FRACTION II FRACTION III Fr III RATI O Fr I 1/2 Fir. 1 Hr. A B A B 73 72 212 187 115 124 244 240 432 428 753 680 5.9 5.9 3.6 3.6 773 4 Hr. A B 792 822 860 1,510 1,630 2.0 2.0 (Counts are expressed as counts per minute per ma. of hemin). Fraction I - Hemin flom Hemoglobin Solution. Fraction II - Hemin from Combined Washes of Residue. Fraction III- Hemin from Water-insoluble Cell :Residue. A and B are duplicate samples. TABLE II RATIOS OF SPECIFIC ACTIVITIES OF RESIDUE-BOUND HEMIN (FRACTION III) TO SPECIFIC ACTIVITIES OF HEMIN OF HEMOGLOBIN SOLUTION (FRACTION I) Exp. 1 Exp. 2 Exp. 3 Exp. 4 ]/2 Hr. 1 Hr. S.8 5.5 3.2 3.4 2.9 2.8 5.9 5.9 3.6 3.6 2 Hr. 2.2 2.2 4 Hr. 1.5 1.5 2.0 2.0

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