The Structure of Human Hemoglobin

Abstract

The recent progress in the x-ray crystallography, biosynthesis, and chemical genetics of the heme proteins (l-7) has generated considerable interest in the primary structure of hemoglobin. Clearly, knowledge of the amino acid sequence of this protein would aid in the interpretation of the x-ray and biosynthetic data and would provide a framework for comparison of the abnormal and normal hemoglobins. These considerations, as well as the possibility of contributing to the general methodology of protein sequence determinations, prompted us to undertake this investigation. Work along these lines had been in progress in several other laboratories (8-10). In the preceding paper (II), it was shown that globin could be separated into Q( and /3 chains containing 141 and 146 amino acid residues, respectively. The present study describes in detail the separation, amino acid composition, and the NH2and COOH-terminal determinations of the peptides obtained by tryptic hydrolysis of the Q( (Val-Leu) chain (12) and of the p (Val-His-Leu) chain, on both the preparative and the analytical scale. Particular significance was placed on the amino acid composition of the peptides, as nonintegral numbers of two amino acid residues would indicate the presence of two inherited variants of hemoglobin A in the individual being studied (6). As in the sequence determination of a number of other proteins (13-15), trypsin was the reagent of choice for the initial cleavage of the chains of hemoglobin into peptide fragments, the main reason being the narrow specificity exhibited by this enzyme (16). The observation that trypsin acting on synthetic substrates hydrolyzed peptide bonds where the amide carbonyl group originated from cr-NHz-blocked arginine and lysine residues (17) has been extended to many proteins, and the same specificity has been found (13-15). There is the complication that a low order of chymotryptic-like activity is associated with even the most carefully purified samples of trypsin (18, 19). In our studies, trypsin prepared from chromatographically purified trypsinogen was used, but a small amount of cleavage at residues usually most susceptible to chymotrypsin was still observed. Nevertheless, reasonably high yields of pure peptides were obtained from tryptic digests of o( and @ chains. As reported previously (12), a fractionation of the tryptic peptides according to size was performed first. Dialysis by the procedure of Craig and King (20) was used initially for the QI