Bovine Pepsinogen and Pepsin: I. ISOLATION, PURIFICATION, AND SOME PROPERTIES OF THE PEPSINOGEN

Abstract

Abstract As the first step in the investigation of the structure and action of little known gastric zymogens and enzymes, pepsinogen has been isolated from the mucosa of the fourth stomach (abomasum) of the cow. The pepsinogen was purified by ammonium sulfate fractionation, batch absorption on diethylaminoethyl (DEAE) cellulose, recycling gel filtration on Sephadex G-100, and finally chromatography on DEAE-cellulose. The preparation appears to be homogeneous by chromatography, amino acid analysis, ultracentrifugation, and amino-terminal analysis, and nearly homogeneous by disc electrophoresis. Amino acid analysis by ion exchange chromatography indicates the presence of 362 ± 2 amino acid residues, which account quantitatively for the total nitrogen of the preparation. Compared to swine pepsinogen (Rajagopalan, T. G., Moore, S., and Stein, W. H., J. Biol. Chem., 241, 4940 (1966)), which has 363 amino acids, the bovine pepsinogen shows considerable similarity in composition, especially with respect to the large number of acidic and small number of basic residues. However, there are at least 22 amino acid substitutions. In both proteins there are five amino acids that occur in equal numbers of residues per molecule; these are 4 methionine, 6 half-cystine, 6 tryptophan, 15 phenylalanine, and 35 glycine. In addition, the sum of the residues of certain similar amino acids is constant for bovine and swine pepsinogens; this is true for the pairs lysine and arginine (14 residues), aspartic and glutamic acids (72 residues), and leucine and isoleucine (57 to 58 residues). Significant differences occur in some other amino acids, notably histidine (2 residues in bovine pepsinogen, 3 in swine) and proline (15 and 19, respectively). The molecular weight of the bovine pepsinogen is 38,900 by amino acid analysis and 37,500 by ultracentrifugation.