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An Octaethylene Glycol Monododecyl Ether-Based Mixed Micellar Assay for Determining the Lipid Acyl Hydrolase Activity of Patatin

An Octaethylene Glycol Monododecyl Ether-Based Mixed Micellar Assay for Determining the Lipid Acyl Hydrolase Activity of Patatin

M Jiménez, J Escribano, M Pérez-Gilabert, S Chazarra, J Cabanes, F García-Carmona

Lipids. 2001 Oct;36(10):1169-74.

PMID: 11768162

Abstract:

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-butyratel x 100/([detergentl critical micellar concentration)) and not to the molarity of PNP-butyrate. The kinetic parameters were determined; Vmax was independent of the mixed micelle concentration, as was Km, when expressed as mol%. However, Km 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 Km 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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP5117191	Octaethylene glycol		5117-19-1	Price

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