Determination of peroxisomal fatty acyl-CoA oxidase activity using a lauroyl-CoA-based fluorometric assay

Abstract

A simple, sensitive fluorometric method for the determination of peroxisomal fatty acyl-CoA oxidase (EC 1.3.99.3) activity has been developed. Studies of enzyme activity relative to subcellular distribution and to clofibrate induction indicate that this assay is specific for peroxisomal fatty acyl-CoA oxidase. The lauroyl-CoA-dependent production of H 2O 2 is quantitated by measuring the oxidation of 4-hydroxyphenyl-acetic acid to a fluorescent product in a horseradish peroxidase-coupled assay. Assays can be performed in either a fixed time or continuous mode. In either mode, H 2O 2 production is related to a change in fluorescence intensity throguh use of standard curve generated with known amounts of H 2O 2. The use of lauroyl-CoA (12:0), rather than the more generally used substrate palmitoyl-CoA (16:0), provides significant advantages. Much of the substrate inhibition problem associate with palmitoyl-CoA has been avoided, and a greater than 4.5-fold higher specific activity has been achieved compared with palmitoyl-CoA-based assay. In the fixed-time mode, linarity relative to time and to the amount of enzyme added has been established without resorting to the use of the bovine serum albumin as a substrate binding medium. Sensitivity is estimated to be at least equal to that of the tmost sensitive methods reported, while reliability, versatility and range have been improved. Use of this method should greatly facilitate the study of perxisomal β-oxidation regulatory mechanisms in hepatocyte cell culture systems as well as in other circumstances where low activities or small samples must be assayed.