Spectrophotometric determination of phosphatidic acid via iron(III) complexation for assaying phospholipase D activity.

The ability of negatively charged phosphatidates to form complexes with Fe(3+) ions was used to design a simple spectrophotometric assay for the quantitative determination of phosphatidic acid (PA). In the reaction with the purple iron(III)-salicylate, PA extracts Fe(3+) ions and decreases the absorbance at 490 nm. Lower competition with salicylate for Fe(3+) ions was observed with single negatively charged phosphatidates such as phosphatidylglycerol (PG), whereas neutral phosphatidates such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE) showed no influence on the absorbance of the iron(III) complex. The detection limit of the method on a microplate scale was 10 microM PA. Based on these results, an assay for determining the activity of phospholipase D (PLD) toward natural phospholipids such as PC, PE, and PG was developed. In contrast to other spectroscopic PLD assays, this method is able to determine PLD activity toward different lipids or even lipid mixtures.