Quantitative Determination of Clostridium welchii Phospholipase C Activity in Aqueous Reaction Medium

Abstract

1. A new method is described for determining Clostridium welchii phospholipase C [EC 3.1.4.3] in an aqueous reaction medium. The method consists of two steps of reaction; the 1st step is the hydrolysis by phospholipase C of lecithin sol dispersed with sodium deoxycholate in the presence of calcium ions and the 2nd step is the selective hydrolysis of the resulting phosphorylcholine by staphylococcal acid phospha-tase [EC 3.1.3.2] in the presence of magnesium ions. The amount of inorganic phosphate liberated in the 2nd step is determined. 2. By the use of [³²P]-labelled rat liver lecithin as substrate, the radioactive, water-soluble products of hydrolysis in the 1st and 2nd steps are identified as exclusively phosphorylcholine and inorganic phosphate, respectively. 3. The method is highly reproducible to an extent within ±6 per cent down to approximately 0.3 egg units of phospholipase C. The method is fairly simple and rapid to perform, and is suitable for use in processes of purification of phospholipase C