DIRECT SEPARATION OF HYDROPEROXY-PHOSPHATIDYLCHOLINE AND HYDROXY-PHOSPHATIDYLCHOLINE DERIVATIVES - APPLICATION TO THE ASSAY OF PHOSPHOLIPIDHYDROPEROXIDE GLUTATHIONE-PEROXIDASE

We have developed a method for assaying the activity of phospholipid h ydroperoxide glutathione peroxidase (PHGPx) which is both more sensiti ve and specific than the spectrophotometric assay. The assay is based on the direct detection of the enzymatic product 1-palmitoyl-2-(13-hyd roxy-cis-9, trans-11-octadecadienoyl)-L-3-phosphatidylcholine by HPLC. Under the conditions used, baseline separation is achieved for produc t and substrate. The utility of the method is demonstrated by the meas urement of PHGPx activity in crude extracts from human lenses and from human Hep G2 hepatoma cells. This method is also suitable for measuri ng the specificity of PHGPx for cofactors apart from glutathione. The assay was used to demonstrate that cysteine alone at pH 7.4 mimics PHG Px activity. (C) 1995 Academic Press, Inc.