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 PoraPLOT Q column was developed for the analysis and identification of the volatile products produced by the action of hydroperoxide lyase (HPLS) upon 13-hydroperoxylinoleic or 13-hydroperoxylinolenic acids. The developed procedure required no derivatization, was not affected by the presence 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 triturated with borate buffer pH = 8.0, and the mixture centrifuged at 12,000 x g. The supernatant and pellet were assayed for HPLS activity by GC-MS analysis of the volatile products given by linoleic acid hydroperoxide. The data showed that the majority of HPLS activity resides in the pellet fraction, and that the primary volatile component was pentane, with smaller amounts of 2-(Z)-pentene and 1-pentene being produced. The fact that HPLS activity resides in the water-insoluble fraction of the acetone powder suggests that HPLS from Chlorella is a membrane-associated enzyme. This investigation also determined that a spectrophotometric assay using alcohol dehydrogenase for measuring HPLS activity was not specific, but measured enzymatic activity other than HPLS.
Nuñez
A
A
ERRC, ARS, USDA, Wyndmoor, Pennsylvania 19038, USA. anunez@arserrc.gov
Foglia
T A
TA
Piazza
G J
GJ
D000446:Aldehyde-Lyases / Q000032:analysis* / Q000378:metabolism; D002708:Chlorella / Q000201:enzymology*; D003577:Cytochrome P-450 Enzyme System; D008401:Gas Chromatography-Mass Spectrometry / Q000295:instrumentation / Q000379:methods; D007202:Indicators and Reagents; D008041:Linoleic Acids; D008042:Linolenic Acids; D008054:Lipid Peroxides
D007202:Indicators and Reagents; D008041:Linoleic Acids; D008042:Linolenic Acids; D008054:Lipid Peroxides; C025998:13-hydroperoxylinolenic acid; D003577:Cytochrome P-450 Enzyme System; D000446:Aldehyde-Lyases; C054540:hydroperoxide lyase; C032890:13-hydroperoxylinoleic acid