Title : A novel continuous enzyme coupled colorimetric assay for phospholipase A2 and its application in the determination of catalytic activity of oil body associated oleosin protein
The present work reports a novel, continuous, specific and colorimetric coupled method for the assay of phospholipase A2 (PLA2), 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 PLA2 are acted upon by ACS, which requires Coenzyme A (CoA) as co-substrate. The PLA2 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 PLA2 assay was in agreement with Michaelis–Menten equation (Vm = 11.9 nmole/min/mg), Km = 5.3 nmole, Vm/Km = 2.2 min-1mg-1). Compared to other available methods, this method provides an exact measure of PLA2 as natural PLs are used as substrate in place of PL analogues. The embedded advantages of this method would have wider acceptability and applicability.