TITRIMETRIC TEST FOR LIPASE ACTIVITY USING STABILIZED TRIOLEIN EMULSIONS

A common assay system for lipase (triacylglycerol acylhydrolase, EC 3. 1.1.3) activity is based on the measurement of free fatty acids, liber ated from triacylglycerols, such as triolein, by enzymatic hydrolysis. This enzymatic reaction involves, in addition to substrate concentrat ion, the size of the surface area of the oil-water interface in the as say system. Only sufficiently dispersed and stabilized oil-water emuls ions are suitable for reproducible determination of lipase activity, e .g., in subcellular fractions of the oil storing cotyledons from rape seedlings. Stabilization of the emulsified substrate by both gum arabi c (Acacia) and the detergent, desoxycholate, was found to be crucial t o the measurement of the lipase activity in a pH-stat. Furthermore, co mplete ionization of free fatty acids is achieved at pH 9.0 and, conse quently, highest lipase activity was found in a test medium adjusted t o this pH value. Inclusion of CaCl2 into the assay medium leads to a d ecrease in lipase activity, addition of NaCI to an increase. Preincuba tion of enzyme preparations with different detergents activates the li polytic breakdown of triolein emulsions. Producing a large surface are a in the triolein emulsion and stabilizing it for hours, increases the sensitivity of the titrimetric assay method. Using such conditions it was possible to determine, for the first time, the low lipase activit y in non-fatty tissues, such as hypocotyls of rape seedlings.