The Structure of Human Hemoglobin V. THE DIGESTION OF THE OL CHAIN OF HUMAN HEMOGLOBIN WITH PEPSIN*

Pepsin has been employed successfully in conjunction with other proteolytic enzymes for the degradation and structural determination of insulin (1, 2), corticotropin (3), ribonuclease (4), and tobacco mosaic virus (5). Because of the difference in specificity between this enzyme and chymotrypsin (6), it was thought that pepsin would hydrolyze some bonds not attacked by chymotrypsin and perhaps leave other chymotrypsin-sensitive linkages intact. We hoped that the peptides resulting from the peptic digestion of the CY chain would enable us to derive the order of the tryptic peptides unequivocally and independently of the results of the chymotryptic digestion. Such a derivation would lend added support to the structure reported in Paper IV of this series (7). It was also of interest to try to account for the entire cr chain in terms of t,he peptic peptides in order to insure further, and again independently, that no part of the molecule had escaped detection during the prior studies of the tryptic and chymotryptic digests. A 16-hour digestion period was used since it was thought that the total number of peptides would be less than what could be expected from an intermediate digest. In the 16.hour digest, it was hoped that the “all or none” specificity of pepsin acting on bonds in the o( chain could be determined. The complex mixture of peptides obtained by peptic digestion of the (Y chain for 16 hours was first fractionated by countercurrent distribution and then chromatographed on Dowex l-X2 as reported before (7). In some cases the peptic peptides were redigested with trypsin, and, if necessary, sufficient sequence data were accumulated to permit their placement in a unique position. Results of these procedures are shown in Fig. 1.