Pepsin from Pepsinogen

Commercial samples of crystalline swine pepsin have been found to be heterogeneous by a number of criteria. Several active fractions can be obtained by chromatography on hydroxylapatite with phosphate buffers, pH 5.7, of increasing molarity as eluents. This has been found to be an effective procedure for evaluating the homogeneity of pepsin. End group analysis by the cyanate method yields 1 eq of isoleutine but as many as 9 other amino-terminal residues in fractional molar amounts. Treatment with carboxypeptidase A liberates 1 eq of alanine and fractional molar amounts of five other amino acids. These results suggest that commercial pepsin is a mixture of autodigested products, cleavage having occurred at various points in the pepsin chain. Evidence of fragmentation is also observed when reduced and carboxymethylated pepsin is passed through columns of Sephadex. It seems probable that autodigestion occurs during the industrial preparation of 1: 10,000 pepsin, the starting material from which crystalline pepsin has traditionally been prepared. In order to obtain pepsin more suitable for structural studies and for investigations of the active site of the enzyme, it is necessary to begin with pepsinogen. In agreement with the results of others, we find that commercial samples of the zymogen are essentially homogeneous. Chromatography on diethylaminoethyl Sephadex A-25 and on hydroxylapatite does not reveal heterogeneity. Leucine is the sole aminoterminal residue found by the cyanate method. Treatment with carboxypeptidase A liberates no free amino acids from the native zymogen, but after reduction and carboxymethylation, carboxypeptidase A liberates 1 eq of alanine as the sole carboxyl-terminal residue. Apparently the carboxyl terminus is masked in native pepslnogen. A relatively homogeneous pepsin may be readily obtained by the activation of pepsinogen at 14’ and pH 2 for 20 min. Separation of the enzyme from the peptides formed during the activation process is accomplished by passage through a column of sulfoethyl Sephadex C-25 at pH 4.4 and 0’. The acidic pepsin passes through unretarded, whereas the basic peptides are tenaciously held and may be eluted with 0.1 M NH,OH. Pepsin prepared in this manner in 95%