Interface Specificity of Phospholipase Activity using a Novel Surface Dilution Kinetic Assay

Abstract

A novel assay using D-enantiomers of phospholipids as diluents for characterizing surface kinetics of lipid hydrolysis by phospholipases was employed to investigate interfacial phospholipase activity. The rationale of the method, verified in previous work, are: (i) D-enantiomers resist hydrolysis because of the stereoselectivity of the enzymes toward L-enantiomers and (ii) mixtures of L+D-lipids at various L:D ratios but constant L+D-lipid concentrations yield a surface dilution series of variable L-lipid concentration with constant medium properties. Kinetic characterization of bee-venom phospholipase A2 activity at various types of interfaces including mixed (L+D)-lipid vesicles and bile salt + phospholipid aggregate-water interfaces was performed. The data were fit to a kinetic model and interface kinetic parameters were obtained. Activity was measured by fluorescence as well as pH-Stat methods. In the fluorescence method, the free fatty acid (FFA) binding protein, ADIFAB, was used. FFA is produced upon lipid hydrolytic breakdown. The fluorescence emission from the bound FFA-ADIFAB complex occurs at a longer wavelength than that from the unbound ADIFAB. The rate of hydrolysis is determined from the rate of increase of FFA bound ADIFAB fluorescence and decrease of unbound ADIFAB fluorescence. Activity data show excellent agreement with a kinetic model derived with D-enantiomers as diluents. Interface kinetic parameters show clear differences between different interfaces. The significant outcomes are (i) the novel assay itself; (ii) its ability to determine interface specific kinetic parameters and thereby (iii) characterization of interface specificity of lipolytic enzymes.