An assay system for the detection of phospholipase C activity

Phospholipase C (PLC, EC 3.1.4.3) enzymes specifically hydrolyze the C-O-P-bond in phospholipids, yielding sn-1,2(2,3)-diglycerides and a phosphate residue bearing the corresponding head group. Biochemical characterization of PLC requires methods for determination of activity. During characterization and purification, proteins are separated by polyacrylamide gel electrophoresis (PAGE). For direct identification and visualization of PLC, a new assay for activity staining in native and renatured SDS-PAGE is described. Incubation of a gel containing an active PLC in the presence of alpha-naphthylphosphorylcholine leads to alpha-naphthol formation. This reacts with the diazonium salt Fast Red, forming a red dye which allows clear determination of PLC purity, molecular weight and substrate specificity. The assay was verified using commercially available PC-PLC and new PC-PLC-producing Bacillus cereus strains. The substrate alpha-NPC was prepared by chemical synthesis at an overall yield of 12%.