HPLC Assay of Phospholipase A₂ Activity Using Low-Temperature Derivatization of Fatty Acids

Vasil Atanasov Laboratory of Biocoordination and Bioanalytical Chemistry, Department of Analytical Chemistry, Faculty of Chemistry, Sofia University “St. Kl. Ohridski,”, Sofia, Bulgaria

Svetla Petrova Laboratory of Enzymology, Department of Biochemistry, Faculty of Biology, Sofia University “St. Kl. Ohridski,”, Sofia, Bulgaria

Mariana Mitewa Laboratory of Biocoordination and Bioanalytical Chemistry, Department of Analytical Chemistry, Faculty of Chemistry, Sofia University “St. Kl. Ohridski,”, Sofia, BulgariaCorrespondence[email protected]

Abstract

A modified isocratic reverse phase high-performance liquid-chromatographic (RP-HPLC) method for phospholipase (PLA) activity assay is developed. Natural lecithins and synthetic phospholipids are used as substrates, and released fatty acids are analyzed after single-phase derivatization (with p-bromophenacyl bromide) at low temperature. The procedure allows simultaneous determination of the total and specific phospholipase activity. This method was successfully applied to snake venom PLA₂ activity assay using natural (soybean and egg yolk lecithins) and synthetic (dipalmytoylphosphatydylcholine) substrates for quantitative determination of the enzyme activity.

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HPLC Assay of Phospholipase A₂ Activity Using Low-Temperature Derivatization of Fatty Acids

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HPLC Assay of Phospholipase A₂ Activity Using Low-Temperature Derivatization of Fatty Acids