A GAS-CHROMATOGRAPHIC MASS-SPECTROMETRIC METHOD USING A PORAPLOT COLUMN FOR THE DETECTION OF HYDROPEROXIDE LYASE IN CHLORELLA-PYRENOIDOSA

A gas chromatographic-mass spectrometric (GC-MS) method using a PoraPL OT Q column was developed for the analysis and identification of the v olatile products produced by the action of hydroperoxide lyase (HPLS) upon 13-hydroperoxylinoleic or 13-hydroperoxylinolenic acids. The deve loped procedure required no derivatization, was not affected by the pr esence of water, did not require cryogenic conditions to be maintained during injection, and allowed for the quantitation of most products. An acetone powder preparation of Chlorella pyrenoidosa cells was tritu rated with berate buffer pH = 8.0, and the mixture centrifuged at 12,0 00 x g. The supernatant and pellet were assayed for HPLS activity by G C-MS analysis of the volatile products given by linoleic acid hydroper oxide. The data showed that the majority df HPLS activity resides in t he pellet fraction, and that the primary volatile component was pentan e, with smaller amounts of 2-(Z)-pentene and 1-pentene being produced. The fact that HPLS activity resides in the water-insoluble fraction o f the acetone powder suggests that HPLS from Chlorella is a membrane-a ssociated enzyme. This investigation also determined that a spectropho tometric assay using alcohol dehydrogenase for measuring HPLS activity was not specific, but measured enzymatic activity other than HPLS.