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

OCR for page 162
DISCUSSION 163 published in 1955 and described in more detail in my paper later in this Conference. ~.~ ~ :::: I.,< .. :::~:~::~:::~:~:::~:~:~::::::::~:~:~:~:~:~::: ,. ,,, f . ..H,.~,,..h,.,.,. Act... FIG. 1. I;lectrophoresis of normal adult hemoglobin (I) and "protein X" (II). As can be seen in the upper part of the illustration (fig. 1), paper elec- trophoresis of normal adult hemoglobin in veronal buffer of pH 8.8 and ionic strength 0.025 always shows a small component in front of the major spot of Hb A and another one slightly slower than Hb C. This slow minor component, only visible after staining and corresponding to a colorless pro- tein we called "protein X" in 1955, is not removed by a single crystallization of the hemoglobin preparation nor by passing it through absorbents like celite. It is resistant to denaturation by bases and passes into the all~ali-resistant fraction of hemoglobin. Paper electrophoresis of this fraction (II) shows a large spot of protein X, a small spot of a slightly faster nonchromogenic com- ponent called X~ and the alkali-resistant hemoglobin. Protein X can be iso- lated by elusion of its spot and contains from 15 to 20 per cent hemoglobin, as determined by iron/nitrogen ratio and spectrophotometric analysis. I should like to ask Dr. Kunkel if it is to be excluded that his As minor component corresponds to hemoglobin A adsorbed on the so-called proteins X. This hypothesis is suggested by the additional fact that, as is A2, proteins X are always present in the hemoglobin of adults and of patients with Cooley's anemia but are never detected or only in extremely small amounts in newborn hemoglobin. For normal subjects proteins X appear from 11 to 12 months after birth. Dr. Keel: I did not have time to mention the non-hemoglobin compo- nents that orate observes following electrophoresis of red cell hemolysates. There are at least three that are constantly present and can be observed as clear bands or peaks. One of these which migrates slightly slower than Hb As at pH 8.6 is present at a concentration of 1 to 3 per cent of the total protein. This may well be Dr. Derrien's ~ protein. It has no relation to Hb A2 and can be separated from it by electrophoresis. We know this protein rather well because it has caused contamination or the Hb As when separation was not completely optimal. There is no possibility that fIb As represents a complex between Hb A and some other protein because it has the same sedimentation

OCR for page 162
164 PART III. ABNORMAL HEMOGLOBINS coefficient as Hb A and also has a very similar heme protein and iron protein ratio. Dr. Jerome Vinograd: I Irish to ask Dr. Kunkel whether his results were obtained with recrystallized material or with the material as it came from the red cells. Dr. Kunkel: We have had the opportunity of studying two crystalline specimens and both of these showed the As component to be present at the same 2.5 per cent concentration that was in the original hemoglobin solution. However, most of our work was done with the whole hemoglobin solution.