An octaethylene glycol monododecyl ether-based mixed micellar assay for determining the lipid acyl hydrolase activity of patatin

Patatin was extracted from potato tubers (Solanum tuberosum L. cv. Spunta) and purified to homogeneity by ammonium sulfate salt fractionation and one sole chromatographic step. A spectrophotometric mixed micellar assay for patatin lipid acyl hydrolase (LAH) activity was designed with the detergent octaethylene glycol monododecyl ether (C₁₂E₈). Patatin LAH used p-nitrophenyl butyrate (PNP-butyrate) as substrate when solubilized in (C₁₂E₈) micelles. In the mixed micellar system, patatin LAH responds to the PNP-butyrate surface concentration expressed as mol% (=[PNP-butyrate]·100/([detergent]-critical micellar concentration)) and not to the molarity of PNP-butyrate. The kinetic parameters were determinined; V _max was independent of the mixed micelle concentration, as was K _m, when expressed as mol%. However, K _m was dependent on C₁₂E₈ concentration when expressed in molar concentration. C₁₂E₈/PNP-butyrate proved to be a reliable system for assaying patatin LAH activity and is superior to the commonly used Triton X-100 and SDS methods. It permits investigation of the substrate requirements of patatin LAH activity because the concentration-independent K _m can be determined both in mol% and as the absolute number of substrate molecules per micelle. In addition, the detergent did not affect the enzyme activity.