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, liberated from triacylglycerols, such as triolein, by enzymatic hydrolysis. This enzymatic reaction involves, in addition to substrate concentration, the size of the surface area of the oil-water interface in the assay system. Only sufficiently dispersed and stabilized oil-water emulsions 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 arabic (Acacia) and the detergent, desoxycholate, was found to be crucial to the measurement of the lipase activity in a pH-stat. Furthermore, complete ionization of free fatty acids is achieved at pH 9.0 and, consequently, highest lipase activity was found in a test medium adjusted to this pH value. Inclusion of CaCl2 into the assay medium leads to a decrease in lipase activity, addition of NaCI to an increase. Preincubation of enzyme preparations with different detergents activates the lipolytic breakdown of triolein emulsions. Producing a large surface area 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 activity in non-fatty tissues, such as hypocotyls of rape seedlings.