Hydrolysis of monomeric substrates by porcine pancreatic (pro)phospholipase A2. The use of a spectrophotometric assay

In the course of searching for specific chromogenic substrates which might be useful for the assay of phospholipase A2 at monomeric substrate concentrations, we tested short-chain lecithin analogues in which the oxygen ester bonds were replaced by thio ester. Spectrophotometric measurement of the hydrolysis of the thio ester in the presence of 5,5′-dithiobis(2-nitrobenzoic acid) or di-4-pyridyl disulfide provides a continuous and sensitive assay. Using this assay it was found that the zymogen expressed considerably higher enzymatic activities than phospholipase A2 which reinforces the conclusions of Pieterson et al. [Biochemistry 13, 1455 (1974)] that the zymogen, like the phospholipase, has a fully functional active site and that the fundamental difference between phospholipase A2 and the zymogen only becomes evident if the substrate is present in an organized lipid water interface. The use of the spectrophotometric assay allowed a determination of the pH-rate profile of both phospholipase A2 and its precursor. Phospholipase A2 shows an optimum at pH 8.5 whereas the maximal activity of the precursor remains constant between pH 6 and 9. Initial velocity patterns as a function of Ca2+ and substrate are consistent with a random addition of substrate and cofactor to the enzyme.