Study of the heterogeneity of bovine pepsin A by pseudo isoelectric focusing.

Pseudo isoelectric focusing (PIEF), i.e. focusing stopped before the equilibrium is reached, has been successfully applied to analyse bovine pepsins A obtained from abomasum mucosa extracts and from 3 individual abomasal juices. Whole bovine pepsin A from mucosa extracts, was resolved in PIEF, using an acidic pH gradient(2-4), into five major and active components. Its fractionation on hydroxyapatite only gave four components which were not homogeneous in PIEF, suggesting that interactions between the different components occur during the chromatographic procedure. After treatment with potato acid phosphatase, whole bovine pepsin A showed only one band in PIEF, displaying enzymic activity and with a mobility identical to that of the less anodic band in untreated pepsin. These results, together with organic phosphate determinations, obviously confirm that the heterogeneity of bovine pepsin A is due to the phosphate content which appears to range between 0 and 3 mol./mol. for the 5 components. Two of them, the phosphate content of which appears to be 1 mol./mol., seem to differ in the location of this phosphate group in the molecule. Whole bovine pepsin A preparations from abomasal juices shared the same pattern in PIEF, identical to that observed with bovine pepsin A from mucosa extracts, thus excluding that dephosphorylation might be involved in the secretory process, as well as disproving the theory that dephosphopepsinogen might be an intermediate in the synthesis of pepsinogen, as it was previously suggested.