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OBSERVATIONS OF THE AMINO ACID COMPOSITION OF HUMAN HEMOGLOBINS WILLIAM H. STEIN With the availability within the last ten years of more or less routine micro J methods for the amino acid analysis of proteins, it has become feasible to use these methods to compare proteins from different sources. A recent instance of such an application can be found in the work of Brown, Sanger, and Kitai1 and of Harfenist and Craig on the insulins of different animal species. It was clearly shown by amino acid analysis that the insulins of the cow, the pig, and the sheep were distinct chemical compounds, after which Sanger and his colleagues) were able to pin-point the exact nature of the structural dif- ferences. Amino acid analysis began to be applied in a similar way to the problem of the several hemoglobins very soon after the existence of the problem was appreciated, that is, soon after Pauling, Itano, Singer, and Wells3 showed that sickle cell and normal human hemoglobin could be differentiated electro- phoretically. Schroeder, Kay and Wells4 analyzed these two types of hemo- globin in 1950, but they were unable to correlate the electrophoretic proper- ties with any clear-cut differences in amino acid composition. More recently, hemoglobin preparations have also been analyzed by Rossi-Fanelli and his colleagues, by Huisman and his coworkers ~ and by Dustin, Schapira, Dreyfus, and Hestermans-~[edard.S As a result of these investigations it has become clear that fetal hemoglobin (Hb F) has an amino acid compo- sition markedly different from that of the adult hemoglobins. It has also be- come clear that normal adult hemoglobin and the various abnormal varieties are very similar in composition, although Huisman has claimed that hemo- globin C contains slightly more lysine than do the others. The work to be described in this brief communication has been done jointly with Dr. Darrel H. Spackman, Dr. R. David Cole, and Dr. Stan- ford Moore.9 All the hemoglobin samples studied were prepared by Dr. Henry Kunkel at the Rockefeller Institute by the use of zone electrophoresis on starch.~ In figure 1 is shown an effluent curve obtained from an acid hydrolysate of sample of hemoglobin ~ obtained f tom a normal adult white male. The amino acid analysis was performed by chromatography on columns of sulfonated polystyrene resin using the automatic recording equipment developed in our laboratory by Dr. Sparkman. Single analyses were performed on 22-hour and 70-hour hydrolysates of each sample of hemoglobin. It is of particular interest to note (fig. 1) that the base line is perfectly flat between methionine and leucine, the area in which isoleucine would show 220

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AMINO ACID COMPOSITION STEIN 221 ._ .g ~ ' '' 'C " "" ' 'a Cat..,,, -..0 ~3 ~C:':,,'' C''~. 'in ~ c:::: ~ d a: _ C:: . ...... - ~-4 C:> ~ it ., o ~ tD C; I:: be, "_~ no _ -, - . . {:: , 8 ~ =;~ =; 3 ~ M~suap IvoTldo Id = 53 ~ , ~ d O L ~ ~ ~ - - ~ ~ ' in. . . . .,., ,, ,.~ , ,; O lo.< ~ is s c;, -c . ~2 4 ~ ,~ q' ._ cc: a' A: ._ "c <-. ~ lo: .. ............. . . -. . -.-.-. ~ t It i I I t t 1 1 1 8 A, A ~ ~ ~ _ ~2 A; .. .... .. c .. oT ~ ;~ ~ - o ~ ~ :c - ~ - oL ct ~ ~ ~ ~ _ . oo o _ Ct o cn C~ . _ ~ V s:; V ~ ~ V X ~ ~ o o.= ~ o r s =( 0 z 0 ~ O ;- \8 . 0 , c~ ~ 0 `> 2 b~ ~ . _ s" o ~ o ~ ~ V o Z; C C =- O Z o ~; ~ o ') ~ a, 0 - 0 := ~ ~-= ~ V - ~ X ~ == ~ = >~ O - s o ~ ._ ~ C C) ~ ~ ~ :- _ ~ s:: - ~ w~ 0 0 ~ C) ~ S ~ o C~ ~: o

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222 PART III. ABNORMAL HEMOGLOBINS up if it were present. Apparently, this sample of electrophoretically purified hemoglobin A is devoid of isoleucine, because the constancy of the base line, which is characteristic of the recorder procedure, coupled with the log scale of the plot, renders the method very sensitive to small quantities of amino acids. In fact, an amount of isoleucine equivalent to one residue per molecule of hemoglobin, less than 0.2 per cent, would appear as a peak about 173 of the size of the methionine peak. The absence of isoleucine was somewhat surprising, inasmuch as all the hemoglobin samples analyzed by Schroeder and by Huisman were reported to contain from 0.2 to 0.4 per cent of this amino acid. The single sample ana- lyzed by Rossi-Fanelli, however, like the one referred to in figure 1, did not appear to contain any isoleucine. Although a difference of a few tenths of a per cent in an analytical value is not normally considered to be very significant, when this difference is between zero and one or two residues per molecule, it is no longer a question of the accuracy of the analytical figure but rather of the presence or the absence of an impurity. In view of the interest in comparing different hemoglobins, the question of purity is obviously of some moment. Accordingly, other hemoglobins were investigated. Samples of hemoglobin A from a normal Negro and from an individual exhibiting the thalassemic trait were analyzed, as were samples of hemoglobin C and hemoglobin E. All were prepared electrophoretically by Dr. Kunkel. All were devoid of isoleucine. To make sure that isoleucine was not being destroyed in some unexpected way, a sample of fetal hemoglobin was analyzed, and found to contain the anticipated 1.45 per cent of isoleucine. Finally, Dr. Schroeder was kind enough to send us a sample of a hydrolysate he had analyzed, and found to contain 0.2 per cent isoleucine. We were able to confirm his figure exactly. Thus, there can be no doubt; when isoleucine is present, we find it; but hemoglobin free of isoleucine can be obtained. Cysteine was another amino acid the content of which was low in the elec- trophoretically-prepared hemoglobin ~ table I ~ . According to Ingrami2 and to Benesch, Lardy, and Benesch,43 urea-denatured hemoglobin A contains 8 sulfhydryl groups per molecule by amperometric titration. Hommes, Drink- waard, and Huisman:4 found 8 sulihydryl groups in hemoglobins A, B. and C, and 8 half-cystine residues as well, determined as cysteic acid by the chroma- tographic method of Schram, Moore, and Bigwood.~5 As can be seen from table I, however, the electrophoretically-purified hemoglobins contain only 4 to 5 -SH groups per molecule by amperometric titration in 8 M urea. The total quantity of half-cystine, determined as cysteic acid,~5 is, in most cases, in acceptable agreement with the number of sulfhydryl groups determined amperometrically, indicating the absence of disulfide bonds in all of the samples. This was further confirmed with one sample of hemoglobin A which, when titrated after treatment with sodium sulfite, showed no increase

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AMINO ACID COMPOSITION STEIN 225 4. Schroeder, W. A., Kay, L. M., and Wells, I. C.: Amino acid composition of hemoglobins of normal negroes and sickle-cell anemics, J. Biol. Chem., 187: 221, 1950. 5. Rossi-Fanelli, A., Cavallini, D., and De Marco, C.: Amino-acid composition of human crystallized myoglobin and haemoglobin, Biochim. Biophys. Acta, 17: 377, 1955. 6. van der Schaaf, P. C., and [Iuisman, T. H. J.: The amino acid composition of human adult and foetal carbonmonoxyhaemoglobin estimated by ion exchange chromatography, Biochim. Biophys. Acta, 17: 81, 1955. 7. Huisman, T. H. J., van der Schaaf, P. C., and van der Saar, A.: Some charac- teristic properties of hemoglobin C, Blood, 10: 1079, 1955. 8. Dustin, J. P., Schapira, G., Dreyfus, J. C., and Hestermans-Medard, O.: La com- position en acides amines de l'hemoglobine foetale humaine, Compt. rend. sac. biol., 148: 1207, 1954. 9. Stein, W. H., Kunkel, H. G., Cole, R. D., Spackman, D. H., and Moore, S.: Ob- servations on the amino acid composition of human hemoglobins, Biochim. Bio- phys. Acta, 24: 640, 1957. 10. Kunkel, H. G., and Wallenius, G.: New hemoglobin in normal adult blood, Sci- ence, 122: 288, 1955. 11. Spackman, D. H., Stein, W. H., and Moore, S.: Automatic recording apparatus for use in chromatography of amino acids, Federation Proc., 15: 358, 1956. 12. Ingram, V. M.: Sulphydryl groups in haemoglobins, Biochem. J., 59: 653, 1955. 13. Benesch, R. E., Lardy, H. A., and Benesch, R.: The sulihydryl groups of crys- talline proteins, J. Biol. Chem., 216: 663, 1955. 14. Hommes, F`. A., Drinkwaard, J. S., and Huisman, T. H. J.: The sulfhydryl groups of four different human hemoglobins, Biochim. Biophys. Acta, 20: 564, 1956. 1 5. Schram, E., Moore, S., and Bigwood, E. J.: Chromatographic determination of cystine as cysteic acid, Biochem. J., 57: 33, 1954. 16. Hirs, C. H. W., Stein, W. H., and Moore, S.: The amino acid composition of ribonuclease, J. Biol. Chem., 211: 941, 1954. DISCUSSION Dr. [Y. J. Schroeder: I wish to add to Dr. Stein's remarks about the iso- le~cine problem. ()f the many samoles we have analyzed. only one have we found to be free of isoleucine and that was onta~ne~ oy co~umn e~ectro- phoresis. Yesterday, Dr. Edsall remarked that the work which Dr. Rhinesmith, Prof. Pauling and I reported some time ago gave 3.6 N-terminal residues to the hemoglobin molecule. The non-integral number is an absurdity. F`urther work leads us to believe that there are 4.0 N-terminal residues as Dr. Edsall had surmised. We come to this conclusion by a study of partial hy- drolysis of DNP-globin. It we partially hydrolyze DNP-globin for various periods of time, we find that very rapidly, and indeed, within 15 minutes, 90 per cent of two residues per molecule has been released in the form of DNP- valyl-leucine. On continued hydrolysis, the DNP-valyl-leucine simply de- creases in the way in which you would expect DNP-valyl-leucine itself to 1 - J ~ J 1 ~ 1 1 1

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224 PART III. ABNORMAL HEMOGLOBINS in the number of sulfhydryl groups. Apparently, electrophoresis not only removes art impurity that contains isoleucine, but also one that is rich in sulfhydryl groups. The value for the sulfLydryl groups seems to vary some- what from one hemoglobin sample to the next, and in some cases an integral molar quantity is not found. This may be a result of experimental error, or it may mean that the impurity has not been completely removed even from the electrophoretically-prepared samples. In agreement with Hommes, Drink- waard and Huisman, less half-cystine was toured in fetal hemoglobin than in hemoglobin A, but the value is three to four rather than six residues per molecule. Unlike these authors, however, we have not been able to secure any evidence for the presence of a disulEde bond in fetal hemoglobin. Except for isoleucine and cysteine, the analytical results obtained in the present studies (table I) tend to be slightly lower than, but are in general quite similar to, other values to be found in the literature. It should be emphasized, however, that the single analyses performed on each 22- and 70- hour hydrolysate are insufficient to provide definitive information relative to the amino acid composition of hemoglobin A. Nor have sufficient analyses been carried out to decide with assurance whether or not the various hemoglobins (table I) have the same amino acid composition, particularly in view of the fact that there is reason to suspect from the suliLydryl analyses that these preparations may not be completely pure. The nature of the impurity or impurities present in many of the hemo- globins analyzed heretofore cannot be decided at this time. Minor hemoglobin components are removed by electrophoresis, but it is doubtful whether they contain sufficient isoleucine or cysteine to account for the results. For exam- ple, there would have to be a 10 to 25 per cent contamination by fetal hemo- globin to contribute 0.2 to 0.4 per cent isoleucine, whereas only 1 per cent or so of fetal hemoglobin has been reported to be present in adult hemoglobin. Fetal hemoglobin, of course, could not account for the high cysteine values, since it contains fewer -SH groups than does adult hemoglobin. Unfortunately, this brief discussion has provided few final answers. The results have been presented simply to call attention to the fact that the absence of isoleucine is one criterion for evaluating the purity of hemoglobin, and that determination of sulThydryl groups and of half-cystine as cysteic acid may be useful in following the purification of this protein. REFEREN CES 1. Brown, H., Sanger, Flu., and Kitai, R.: The structure of pig and sheep insulins, Biochem. J., 60: 556, 1955. 2. Harfenist, E. J., and Craig, L. C.: Differences in the quantitative amino acid composition of insulins isolated from beef, pork and sheep glands, l. Am. Chem. Soc., 74: 4216, 1952. 3. Pauling, L., Itano, H. A., Singer, S. J., and Wells, I. C.: Sickle cell anemia, a molecular disease, Science, 110: 543, 1949.