APPLICATION OF CAPILLARY ELECTROPHORESIS TO DETERMINATION OF ENZYME-ACTIVITY AND OTHER PROPERTIES OF PHOSPHATIDYLINOSITOL-SPECIFIC PHOSPHOLIPASE-C

This paper describes a method for monitoring enzymatic activity using micellar electrokinetic chromatography (MEKC). MEKC is used to analyze the activity of phosphatidylinositol-specific phospholipase C (PI-PLC ) with convenience and precision. PI specific PLC enzyme converts phos phatidylinositol (PI) to diacylglycerol (DAG) and inositol 1,2-cyclic phosphate (IF). The assay system developed is based on monitoring both the breakdown of substrate and the formation of products simultaneous ly. To obtain the best separation for the substrate PI and product DAG , we investigated changes in concentration of electrolyte buffer and S DS as well as in pH of the electrolyte buffer. Since the structures of the substrate and products are different, the reaction could be monit ored easily by MEKC mostly based on hydrophobicity. Under the separati on conditions developed, we investigated the enzymatic activity of PI- PLC depending on the concentrations of phosphatidylinositol, various d ivalent cations and the reaction temperature. Matrix-assisted laser de sorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) wa s used to confirm the molecular masses of the substrate PI and product s DAG and IF. The results of this study show that capillary electropho resis can be widely applied to analyze and characterize many other enz ymes since capillary electrophoresis has several advantages as follows , simplicity, speed, no need for radiolabelled substrate, small sample volumes and sufficient accuracy. (C) 1997 Elsevier Science B.V.