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

OCR for page 199
200 PART III. ABNORMAL HEMOGLOBINS of the salting-out curves and electrophoresis diagrams I presented should not be accepted, in view of the following facts. In view of the electrophoresis diagrams of 1 per cent normal adult hemo- globin in very low ionic strength cacodylate buffer at pH 6.5, it is very dif- ficult to deny the individuality of the two major constituents 3 and 4 of Hb A. These begin separating within two hours and the process of their separation as a function of time does not show any convection disturbances. In accordance with Dr. Svensson, with whom I discussed this problem, the electrophoretic individuality of these two constituents has to be considered as certain under the experimental conditions. The difference in net charge seems to be only Q.3, which would exclude the possibility of the faster moving component (No. 4) being an intermediate form of oxidation. Furthermore, our experimental results are not in favor of the hypothesis that: discontinuities in salting-out curves represent changes in the solid phase of the same hemoglobin. As I said earlier the solubility curves of crystals show exactly the same discontinuities as the salting-out curves of solutions (precini- tation of solid amorphous phases>. Therefore, I think that if it were a matter of transition between different crystalline forms, it would be surprising that we should get exactly the same pictures in both types of experiments.