Pepsin from Pepsinogen: PREPARATION AND PROPERTIES

Abstract

Abstract 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 isoleucine 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 amino-terminal 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 pepsinogen. 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 NH4OH. Pepsin prepared in this manner in 95% yield is homogeneous on DEAE-Sephadex A-25 and on hydroxylapatite. It shows no evidence of heterogeneity upon reduction, carboxymethylation, and passage through Sephadex. It yields isoleucine as the sole amino-terminal and alanine as the sole carboxyl-terminal residue. Carboxypeptidase B does not liberate basic amino acid residues. Failure of the carboxypeptidases to liberate any amino acid other than alanine suggests that proline is the third residue from the carboxyl terminus of both pepsinogen and pepsin. A proline residue in this position would block further action by either carboxypeptidase A or B. The homogeneity of the pepsin formed depends critically upon the pH of the activation process. If activation is carried out at pH 3 or pH 3.9 instead of at pH 2, additional active species are obtained. Amino acid analyses have been carried out on pepsinogen and on the pepsin derived from it. The zymogen has been found to have 363 amino acid residues; the enzyme, 321. In the activation process, 42 residues, including 9 of lysine, 2 of histidine, and 2 of arginine, are cleaved off. These are almost exactly accounted for by the amino acid composition of the total peptide fraction obtained from the activation mixture. Pepsin freshly prepared from pepsinogen has about 1.3 times the activity of the commercial product against hemoglobin as a substrate, but somewhat less activity against acetylphenylalanyldiiodotyrosine. Preliminary evidence is mentioned which suggests that freshly prepared pepsin may differ in several properties from the commercial material. The investigation of these and other aspects of the behavior of pepsin should be facilitated by the availability of homogeneous, reproducible preparations of the enzyme.