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Analytical Letters Volume 42, 2009 - Issue 10
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CHROMATOGRAPHY

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]
Pages 1341-1351 | Received 13 Oct 2008, Accepted 02 Apr 2009, Published online: 18 Jun 2009
 
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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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