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OCR for page 233
THE CHEMICAL DIFFERENCE BETWEEN NORMAL HUMAN AND SICKLE CELL ANAEMIA HAEMOGLOBINS V. M. INGRAM Previous articles have told the history of sickle cell anaemia, the first- and best-studied of the "molecular diseases.") They have also detailed the chemical evidence on the difference between the haemoglobins A and S. One should add the important finding of Perutz and his colleagues' that the solubility of deoxygenated haemoglobin S is very low and that this causes tactoids to appear which distort the red cell into the characteristic sickle shape. They also noted that x-ray diffraction patterns from crystals of the two haemo- globins were indistinguishable. This indicates that the difference between them is a small one and is not likely to be a difference in folding of the polypeptide chains since this involves shifting many atoms and would probably have been detectable. To summarize, by 1956 the known chemical difference between the taco proteins was that haemoglobin S contains about two carboxyl groups3 fewer per molecule than does haemoglobin A. I can now report that these carboxyl groups belong to glutamic acid and that they are replaced by two valine resi dues in haemoglobin S. This appears to be the only chemical difference be- t~veen the two proteins. The determination of the particular amino acids involved is made very difficult by the large size of the haemoglobin molecule. Experiments4 were therefore begun in 1956 to degrade these protein molecules into a number of small peptide fragments. It was hoped that if a rapid method could be found for characterizing the chemical properties of these peptides, then perhaps a replacement of even a single residue for another might be easily detectable. Accordingly, trypsin was allowed to digest samples of heat-denatured haemo- g;lobin A and S. since it splits specifically those bonds in the polypeptide chains which are formed by the carboxyl groups of the amino acids lysine and arginine. It is known that the haemoglobin molecule of 66,700 is composed of two iden- tical half molecules.' 5 In each of these there are about 25 lysines and ar- ginines6 and hence approximately 25 peptides are expected to be formed. In- deed, under the conditions used mixtures of about 25 peptides, on the average less than 10 amino acids long, resulted from each of the two haemoglobins. This is additional proof that haemoglobin is composed of equal halves, for otherwise some 50 different peptides would result. The two mixtures were compared by a two-dimensional combination of paper electrophoresis and paper chromatography.4 As a result the peptide spots were spread out in a characteristic map or "fingerprint" (figs. 1, 2 ~ . By working under very rigorously standardized conditions it was easily possible to obtain fingerprints 233

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234 PART III. ABNORMAL HEMOGLOBINS o Y80 Hb A o ~0 o oU to Hb S o FIG. 1. -"Fingerprints" of tryptic digests of hemoglobins A and S. Of haemoglobin A and S in which all peptides occupied identical positions except for one, called peptide no. 4, which appeared in a new position in the haemoglobin S fingerprint. It must therefore have a different structure and will represent the portion of the polypeptide chains where the chemical dif- ference between the two proteins lies. The structures of these two peptides, the Hb A and Hb S no. 4 peptides, are shown in fig. 3. The structure of these two peptides has now been established, mainly by partial acid hydrolysis; the fragments are shown in fig. 3, separated on a finger- print. In addition, use was made of end group analyses, qualitative amino acid analyses, and Edman stepwise degradation of some of the fragments of

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CHEMICAL DIFFERENCE BETWEEN HE A AND HB S INGRAM 235 ~0~ tic OC o c: o - c' o H) I lib (a) (a) fib d lIb S (b) (b) E o :;.v L ~ ' ',l-PV TV L-L) ,' .~-V~ P~ vC, , ~ P vat L, Gt~6 g7+c{;~ fib A ~ S F`IG. 2. Further examination of tryptic digests of hemoglobins A and S. (a) Slowest moving positively charged fractions; (b) neutral fractions. (From Nature 178: 792, 1956.) ~ ~ ~ _ _ H's - Val -Leu-Leu-Thr-Pro-Glu -Glu -Lys 4, 4. . _ ~ _ H_ Hi s - Va I - Leu - Leu- Thr - Pr o- Val - Glu - Ly s FIG. 3.Acid degradation and structure of the no. 4 peptides from the hemoglobins A and S. partial acid hydrolysis. The indicated charge distribution was inferred from the electrophoretic behaviour of the peptides. Both peptides contain the same nine amino acids except for one; the first glutamic acid of the Hb A no. 4 carboxyl peptide, changes to another, valine, in the lob ~ peptide. l hus a group is lost. Since there are two identical half molecules, this change occurs twice in the whole haemoglobin molecule and the fact that haemoglobin S has about 2 carboxyl groups fewer is now explained. The two haemoglobins differ very little; only one out of nearly 300 amino acids in the half molecule changes. However, the present experimental results do not help to explain the abnormally low solubility of deoxygenated sickle cell haemoglobin,2 which is the cause of the anaemia. In particular, there is as yet no evidence to indi- cate the position of the no. 4 peptide along the haemoglobin peptide chains nor where this peptide is located when the chains are folded in the globular molecule of haemoglobin. In order to show that the two no. 4 peptides really do carry the only change

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236 PART III. ABNORMAL HEMOGLOBINS in the molecule, the other peptide spots in the fingerprints were compared for amino acid composition. No differences were found. Furthermore, since haemoglob~n has a tryps~n-res~stant core about 30 ~ of the molecule this large piece was in each case isolated and digested with chymotrypsin. This treatment readily yielded again two mixtures of peptides, one from the haemo- globin A core and the other from haemoglobin S. They were compared by fingerprinting and chromatographic ex~minntinn of the neutral oentides (fig. 4~. Again no differences could be detected. One is therefore led to con- clude that the only difference lies in the two no. 4 peptides. 1 1 O ~ O ~ I' ~ o ~0 :6 Q 0 o f:.~ Lo + (a) Hb A _ + Hb S FIG. 4. (a) "Fingerprints" and (b) chromatography of neu- tral peptides of the chymotryptic digests of the hemoglobins A and S "trypsin resistant cores." Hb A Hb S (b) It is widely believed that haemoglobin is the first protein product made by the gene; it follows that changes in the gene should be faithfully reflected by changes in the protein. Neel has shown that a single mutation of a haemo- globin gene produces the abnormal "sickle cell" gene.7 It appears now from the results briefly presented here that what is presumably an alteration of a portion of the gene results in an alteration of a portion of the polypeptide chain of the corresponding protein,- in this case haemoglobin. In the "sickle cell" mutation the change in the protein is very small indeed, indicating that this mutation is extremely localized in the gene. Perhaps this affects only a single base pair in the very long chain of the DNA of the gene. These ideas fit in very well with the demonstration by BenzerS and Streisinger,9 working with intact bacteriophage, that genes can be divided into hundreds of sub- units. Similar divisibility had also been shown for the genes of Aspergillusl and Ne2crospora.l1

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CHEMICAL DIFFERENCE BETWEEN HE A AND HB S INGRAM 237 It is also possible to report progress in similar investigations on haemoglobin C, carried out in collaboration with Mr. l. A. Hunt. This was the second abnormal human haemoglobin to be discovered)' and it results from another single mutation of the haemoglobin gene. As earlier papers indicated, it has even fewer net negative charges per molecule than does haemoglobin S and is therefore easily distinguishable electrophoretically. The solubility of reduced haemoglobin C is very near the normal. Haemoglobin C has been submitted to the detailed comparison with haemo- globin A outlined above for sickle cell haemoglobin. Trypsin digests of the whole protein and chymotrypsin digests of the resistant core were prepared and were examined by fingerprinting (fig. 5) and by chromatography. Again G t: ~ . + HbA - 1 1 j:~: MU HbC FIG. 5. Portion of the "finger- prints" of tryptic digests from the hemoglobins A and C. the only peptide affected by the mutation is the no. 4 peptide of the tryptic haemoglobin A digest, the same one that showed the "sickle cell" change. Its place in the corresponding haemoglobin C digest is taken by two new peptides, one neutral, the other positively charged. It is too early yet to speculate on the chemical changes underlying these observations; the structural analyses of the peptides are not far enough advanced. It is however, strikingly evident that the same very small region of the protein is affected by this second muta- tion. Genetic evidence showsi2 that the haemoglobin S and C mutations are allelic, i.e., on the same place in the gene, or at any rate closely linked. The chemical evidence to date on the primary protein products of these genes in- dicates that the same very small portion of the peptide chains is affected in troth cases. This is the exact chemical counterpart of the concept of allelic mutations. REFEREN CES 1. Pauling, L., Itano, H. A., Singer, S. J., and Wells, I. C.: Sickle cell anemia, a molecular disease, Science 110: 543, 1949. 2. a ) Perutz, M. F., and Mitchison, J. M.: State of haemoglobin in sickle-cell anaemia, Nature 166: 677, 1950. b) Perutz, M. F`., Liquori, A. M., and Eirich, i?.: X-ray and solubility studies of haemoglobin of sickle-cell anaemia patients, Nature 167: 929, 1951. 3. Scheinberg, I. H., Harris, R. S., and Spitzer, J. L.: Differential titration by means of paper electrophoresis and the structure of human hemoglobins, Proc. Nat. Acad. Sci. 0: 777, 1954.