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OCR for page 48
48 PART I. STRUCTURE OF HEMOGLOBIN cussions of the Faraday Society, 20: 216, The physical chemistry of enzymes, 1955. 44. George, P., and Irvine, D. H.: A possible structure for the higher oxidation state of metmyoglobin, Biochem. J., 60: 596, 1955. 45. Riggs, A.: The metamorphosis of hemoglobin in the bullfrog, J. Ger,. Physiol., ]5: 23, 1951. 46. Wald, G., and Riggs, A.: The hemoglobin of the sea lamprey, petromyzon mari- nus, J. Gen. Physiol., 35: 45, 1951. 47. Keilin, D.: On cytochrome, a respiratory pigment, common to animals, yeast, and higher plants, Proc. Roy. Soc., Series B. 98: 312, 1925. DISCUSSION Dr. Felix Horowitz: There are two points on which I would like to comment. One of these is the crevice problem and the other the problem of imidazole residues as points of attachment of the heme groups. I think that we can state one thing definitely, namely that the hemes must form some bridges between the globin moieties. This is clear from the follow- ing simple experiment. The initial interfacial tension between p-xylene and an aqueous solution free of hemoglobin is 35 dynes/cm. As increasing quantities of hemoglobin are added this tension decreases to a minimum of about 24 dynes/cm., corresponding to a drop of 11-12 dynes/cm. Globin, in contrast to all other proteins, lowers the interfacial tension to about 12 dynes/cm, at pH 9. Evidently, the drop in interfacial tension is twice as high as that caused by hemoglobin.) On adding further amounts of globin, the interfacial tension remains unchanged at 12 dynes/cm. How- ever, if one equivalent hemin is added to this solution, the interfacial tension increases again to 24 dynes/cm. There are two interpretations which come to our mind. One of these is that globin has only one half of the molecular weight of hemoglobin and that, therefore, twice as many particles are present in the interface, resulting in high interfacial pressure and low interfacial tension. Another interpretation is that heme is bound to a lipophilic group of the globin molecule so that the affinity of globin to the organic solvent, p-xylene, is higher than that of hemo- globin. Both interpretations may be right. When hemin is added to the globin film, it seems to form bridges between globin molecules, thus causing the formation of the larger hemoglobin molecules; consequently both the number of molecules in the interfacial film and the interfacial pressure decrease. I conclude from these observations that the hemes form bridges between the globin moieties. However, I do not know whether the hemes are buried in crevices. They may be bound in such a fashion that there is ample space for the iron atoms to combine with other ligands. Ir1 a hemoglobin article written a few years ago~ I discussed the problem of the hemaffinic (heme-linked) groups and stated that imidazole was not very probable as a ligand. I am glad that Dr. George has also some doubts in this

OCR for page 48
DISCUSSION 49 respect. The principal reason for the general belief that imidazole groups are involved, is the high histidine content of mammalian globins. However, the Robins of invertebrates are very poor in histidine. My belief that imidazole groups are not involved in binding heme is based on the great stability of many hemoglobins over the whole range from pH 4 to pH 12. The imidazole groups undergo a change from the cationic to the uncharged form in this plI range. The great lability of all hemoglobin at pH values of less than 3.5 suggests rather an acidic hemaffinic group which, at lout pH values, gains a proton and, thereby, loses its negative charge. REFEREN CES 1. Haurowitz, F., Boucher, P., Dicks, M., and Therriault, D.: Interfacial pressure of proteins, Arch. Biochem.~ Biophys. 59: 52, 1955. 2. Haurowitz, F`., and Hardin, R. L.: Respiratory proteins, in Neurath-Bailey, Pro- teins, Vol. II, A, 279, 1954. Dr. Jacinto Steinharclt: This is a comment on Dr. Hauro~vitz's remarks on the surface effects of globin and their reversal on the addition of heme. His point would be more convincing if globin had a quarter of a molecular v~eight of hemoglobin instead of half, because the molecular weight of hemo- globin on acid denaturation under the mildest conditions (pH 2.8 at 0) appears to be reduced to a quarter. Since the globin itself has a molecular weight of half in the region of its stability, there is something else holding the pieces together besides heme. This is a very minor point and it does not exclude the possibility that there are heme bridges in the higher structure. Incidentally, the evidence for the molecular weight being reduced to a quarter its normal value is not absolutely conclusive. The evidence is merely that the kinetics of the regeneration of denatured hemoglobin to native hemo- globin is very accurately second order, i.e., the native molecule must have a higher molecular weight than the denatured molecule. However, at the pH ot regeneration native ferrihemoglobin has half the molecular weight it shows at pH 7. Of course, a second-order rate process does not unambiguously refer tc an association reaction, but it usually does for reactions in solution. Dr. Edsall: May I ask if there are any direct measurements by ultracen- trifuge or other determinations about the molecular weight of the acid material by which one could show directly that it was a quarter ? Dr. Steinhardt: I have not at present the facilities to make such measure- ments. They s~rould not be easy to make or to interpret. The interpretation of sedimentation measurements made in such acid solutions with a molecule as small as this would be a very troublesome one. The dissociation into half molecules at pH 5 has been observed at Upsala bsT Gralen.