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154 PART III. ABNORMAL HEMOGLOBINS 19. Itano, H. A., and Robinson, E.: Hemoglobin intermediates, Fed. Proc. 16: 199 (March) 1957. 20. Huisman, T. H. J., and Prins, H. K.: Chromatographic estimation of four dif- ferent human hemoglobins, J. Lab. Clin. Med. 46: 255 (Aug.) 1955. 21. Harfenist, E. J., and Craig, L. C.: Countercurrent distribution studies with in- sulin, J. Am. Chem. Soc. 74: 3083 (20 June) 1952. 22. Harfenist, E. J.: The amino acid compositions of insulins isolated from beef, pork, and sheep glands, l. Am. Chem. Soc. 75: 5528 (20 Nov.) 1953. 23. Ranney, H. M.: Observations on the inheritance of sickle-cell hemoglobin and hemoglobin C, J. Clin. Invest. 33: 1634 (Dec.) 1954. 24. Allison, A. C.: Notation for hemoglobin types and genes controlling their syn- thesis, Science 122: 640 (7 Oct.) 1955. DISCUSSION Dr. J. Steinhardt: It is worth mentioning that in our work with ferro- hemoglobin we have never been able to separate ferrocyanide ion from meth- emoglobin prepared with ferricyanide, no matter how long we dialyzed. I suppose this does have some effect on the net charge and mobility. Dr. Itano: I think the facts that the boundaries in the methemoglobin- methemoglobin cyanide solutions are not as sharp as those in the methemo- globin-carbonmonoxyhemoglobin solutions and that the proportions appear to change with time indicate that there is some dissociation of the cyanide from the me/hemoglobin. Actually, there has to be a finite rate of dissocia- tion since the equilibrium constant of the reaction is measurable. The other consideration is that the spectrophotometric method is not as sensitive as electrophoresis in the detection of these minor changes in heme. To be more specific, say that a preparation contains after storage 1 or 2 per cent me/hemoglobin. It is rather difficult to pick up this change by spectro- photometry except by the most careful work. However, at this low level of oxidation, probably only one heme out of four will be affected in any one molecule so that a orate to two per cent heterogeneity in methemoglobin corresponds to two to eight per cent heterogeneity by electrophoresis. Dr. F. Do. J. Roughton: I was greatly interested to read Dr. Itano's pre- liminary communication last December, and now to hear this fuller evidence - direct evidence of intermediates in the case of ferri compounds and of CO ferrohemoglobin-ferrihemoglobin mixtures. Yesterday I mentioned how slowly nitric oxide hemoglobin dissociates thousands of times more slowly than carbon monoxide hemoglobin. I wondered whether there might be a possibility of Dr. Itano and Miss Robinson demonstrating something with completely ferrous hemoglobin compounds if they used nitric oxide hemo- globin as one of the members. Dr. Itano: Since we are doing electrophoretic experiments one of the important considerations was a difference in charge. That is the reason for our using ferric compounds. Although ferrohemoglobin and its compounds
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DISCUSSION 155 differ in the pK values of their heme-linked groups, I am not sure whether the fractional differences in net charge that these heme-linked groups cause would be detectable by electrophoresis. The reaction between oxygen and hemoglobin is too fast. Unless the nitric oxide hemoglobin differs significantly in net charge from hemoglobin, I do not think we can do it by electrophoresis. Dr. Roughfo~z: But if you had a mixture of nitric oxide and hemoglobin and plain hemoglobin, the dissociation of the nitric oxide hemoglobin inter- mediates would be so frightfully slow. esoeciallv at 0°. it might take actually days to occur. D'. R. Ber~esch: Dr. Itano, you said that ferricyanide is known only to affect the heme and leaves the globin unaffected. There exists the possibility that ferricyanide also oxidizes -SH groups of the globin, and Dr. Remmer, working in Dr. Shemin's laboratory, a few years ago told me privately that much more ferricyanide was reduced by hemoglobin than could be accounted for on the basis of iron. Would you like to comment on this? Dr. Itano: I investigated this matter quite thoroughly before beginning the experiments. Anson and Mirsky studied this very phenomenon years ago and found that if one carried out the oxidation below about pH 6.8, which we did, no sulThydryl groups are oxidized. Also, I think the studies of Conant and of Wyman indicate a virtually stoichiometric reaction between ferricya- nide and the iron of hemoglobin. On the other hand, if the reaction is carried out at higher pH, some sulfhydryls are oxidized. I think also that sulihydryls in globin may be more susceptible to oxidation than those in hemoglobin. The other factor is that unless oxidation of the sulfhydryl groups pro- duces isomers or some aggregates, one would not expect an intramolecular oxidation of two sulibydryls to disulfide to affect electrophoretic mobility. I am not aware of any oxidation of sulfLydryl that would increase the positive charge of the molecule, which is the type of charge alteration we have observed. Dr. V. 211. Ingram: I would like to confirm what Dr. Itano said. If you oxidize oxyhemoglobin with ferricyanide very carefully at pH 6 and at low temperatures, there is no change of available -SH groups in the native horse or bovine hemoglobin. If you use alkaline pH, then you do lose -SH groups. Dr. John H. Taylor: Our results confirm and extend what has been brought out already. We have used the reaction with p-chloromercuribenzoate ~ PCMI3 ~ to measure available -SH groups in several native mammalian hemoglobins before and after treatment with ferricyanide, as well as with some other reagents. Oxyhemoglobin samples freshly prepared in the presence of EDTA show a definite number of PCMB-reactive sites, depending upon the species, e.g., human 2, bovine 2 and canine 4 -SH per mole (68,000~. If you oxidize any of these hemoglobins cautiously with ferricyanide at pH 7 you do not change the number of titratable -SH groups. If you oxidize with excess ferricyanide at pH 9, much as described by Ansor~ and Mirsky, ~ J ~ ~ A _ ~ J
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156 PART III. ABNORMAL HEMOGLOBINS you do abolish a definite number of -SH groups two being removed in all three instances mentioned and you do increase the negative charge on the molecule as shown by suitable electrophoretic measurements. We should like to suggest that the increased charge may arise from oxidation of -SH to the sulfinate or sulfonate level, rather than through dimerization or other structural change, but we have not yet proved this to be so. Samples of methemoglobin in which the number of PCMB-reactive groups has been decreased by alkaline ferricyanide treatment are less homogeneous than "normal" methemoglobin with unchanged -SH, as you can readily demonstrate by means of paper strip electrophoresis. The heterogeneity in- creases slowly as the material is kept in the cold and the visible absorption spectrum also changes. The faster moving fraction, isolated from a starch slab, shows the greatest difference in spectrum from normal me/hemoglobin. It would be of interest to compare this material with the heterogeneous fraction of human hemoglobin, described by Dr. Kunkel, which also appears to increase with the age of the preparation.
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