The determination of phospholipase D activity in emulsion systems

Although phospholipase D (PLD) is often used in emulsion systems consisting of buffer and a nonpolar organic solvent, most activity assays have been d esigned to work in purely aqueous milieu. Here a method is described for th e determination of PLD activity in emulsion systems. The assay is based on the transphosphatidylation of phosphatidylcholine with l-butanol in dichlor omethane/buffer with the subsequent densitometric quantification of the pro ducts after their separation by HPTLC and staining with a CuSO4/H3PO4 reage nt. The method is particularly appropriate for the determination of enzymes such as PLD from Streptomyces sp. that prefer the exchange of the head gro up 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 determ ine PLD activity in the presence of high concentrations of phospholipids. ( C) 1999 Academic Press.