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The determination of phospholipase D activity in emulsion systems
I Aurich1, F Hirche, R Ulbrich-Hofmann
1Department of Biochemistry/Biotechnology, Martin-Luther University Halle-Wittenberg, Kurt-Mothes-Strasse 3, Halle, D-06120, Germany.
Abstract:
Although phospholipase D (PLD) is often used in emulsion systems consisting of buffer and a nonpolar organic solvent, most activity assays have been designed to work in purely aqueous milieu. Here a method is described for the determination of PLD activity in emulsion systems. The assay is based on the transphosphatidylation of phosphatidylcholine with 1-butanol in dichloromethane/buffer with the subsequent densitometric quantification of the products after their separation by HPTLC and staining with a CuSO4/H3PO4 reagent. The method is particularly appropriate for the determination of enzymes such as PLD from Streptomyces sp. that prefer the exchange of the head group in glycerophospholipids to their hydrolysis. Since the application of an organic solvent in the PLD assay allows the determination of the enzyme in analytes insoluble in aqueous media, the method can also be used to determine PLD activity in the presence of high concentrations of phospholipids.