A SPECTROPHOTOMETRIC ASSAY FOR HYDROPEROXIDE LYASE

Spectrophotometric assays for hydroperoxide lyase have traditionally measured the loss in absorption at 234 nm due to the disruption of conjugated diene in the fatty acid hydroperoxide. However, that assay does not distinguish between hydroperoxide lyase and hydroperoxide dehydrase activities, both of which cause a loss of conjugation in the substrate hydroperoxide. A new assay has been developed which is specific for hydroperoxide lyase. It is based on the ability of hydroperoxide lyase products, aldehydes and omega-oxoacids, to serve as substrates for yeast alcohol dehydrogenase. Thus, the new hydroperoxide lyase assay is a coupled enzyme assay, which is conducted spectrophotometrically at 340 nm and measures the oxidation of nicotinamide adenenine dinucleotide, reduced form (NADH). In addition to its specificity for hydroperoxide lyase, the coupled assay allows higher concentrations of both fatty acid hydroperoxide substrate and crude enzyme extracts than the assay conducted at 234 nm.