Determination of enzymatic activity and properties of secretory phospholipase A(2) by capillary electrophoresis

Abstract

A capillary electrophoretic (CE) system coupled with a diode array UV detector was used for the assay of secretory phospholipase A(2) (sPLA(2)) activity. This method is based on monitoring both the breakdown of substrates and the formation of products simultaneously using micellar electrokinetic chromatographic techniques. Under our developed separation conditions, we analyzed the substrates and products quantitatively, and investigated enzyme activity as a function of reaction time and presence of enzyme activator or inhibitor. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was also utilized to confirm the phosphatidylcholine, a substrate of sPLA(2). In order to test the feasibility of the developed method for measurement of enzymatic activity, we compared it to the conventional radioactive assay method for sPLA(2). On the basis of our results, the conventional method can be complemented, or even replaced, by this new CE method which possesses the advantages of short analysis time, use of non-radiolabeled and inexpensive substrates, simple measurement of enzymatic activity, and exact quantitation of substrate and product. (C) 1999 Elsevier Science B.V. All rights reserved.