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A Spectrophotometric Microtiterplate Assay to Determine the Transphosphatidylation Potential of Phospholipase D
Author(s) -
Dippe Martin,
UlbrichHofmann Renate
Publication year - 2010
Publication title -
journal of the american oil chemists' society
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.512
H-Index - 117
eISSN - 1558-9331
pISSN - 0003-021X
DOI - 10.1007/s11746-010-1577-y
Subject(s) - phospholipase d , chemistry , hydrolysis , phosphatidic acid , phosphatidylcholine , glycerophospholipids , choline , phospholipid , ethanolamine , chromatography , lysis , biochemistry , enzyme , membrane
In addition to the hydrolysis of the terminal phosphate ester bond in glycerophospholipids, phospholipases D (PLDs) are able to catalyze the exchange of the polar head group. The biocatalytic potential of PLDs strongly depends on the ratio of the transphosphatidylation to hydrolysis rate which, therefore, is an important criterion in the screening for efficient PLDs from natural sources or combinatorial DNA libraries. Here, we present a fast spectrophotometric assay that allows the determination of the rate of both hydrolysis and transphosphatidylation of PLD‐containing solutions including cell extracts in one microtiterplate. The assay is based on the reaction of phosphatidylcholine solubilized in Triton X‐100 micelles with ethanolamine and the determination of phosphatidic acid (PA) and choline. PA is determined via Fe(III) complexation and represents hydrolysis, while choline is determined by the conventional choline oxidase/peroxidase assay and yields the total conversion. The difference between both values corresponds to the transphosphatidylation product. The method is suitable for measuring reactions rates as well as product yields after defined time periods. As shown for E. coli cells expressing PLD from cabbage, the assay can be applied to extracts of cells grown and lysed in microtiterplates.