A Novel Continuous Enzyme Coupled Colorimetric Assay for Phospholipase A(2) and its Application in the Determination of Catalytic Activity of Oil-Body-Associated Oleosin Protein

Abstract

The present work reports a novel, continuous, specific, and colorimetric coupled method for the assay of phospholipase A(2) (PLA(2)), which involves use of acyl-CoA synthetase (ACS) as coupling enzyme and Dilinoleoyl Phosphatidylcholine (DLPC) as substrate. This method is based on the principle that free fatty acids (FFA) produced by PLA(2) are acted upon by ACS, which requires Coenzyme A (CoA) as co-substrate. The PLA(2) activity was measured in terms of amount of CoA utilized in the coupled reaction, determined indirectly by measuring unreacted CoA using 5,5'-dithiobis (2-nitrobenzoate). The PLA(2) assay was in agreement with Michaelis-Menten equation (Vm = 11.9 nmole. min(-1) mg(-1), Km = 5.3 nmole, Vm/Km = 2.2 min(-1) mg(-1)). This method provides a true measure of PLA(2) in comparison to other available methods, wherein PL analogs were used rather than the natural PL used in our study. The embedded advantages of this method would have wider acceptability and applicability.