A fluorometric assay of acyl-CoA oxidase activity by a coupled peroxidatic reaction: elimination of interfering side reactions.
We have developed a simple, reliable, and sensitive method for the assay of peroxisomal fatty acyl-CoA oxidase (FAO, EC 1.13.-) in subcellular fractions. It is based on a peroxidase-linked oxidation of 4-hydroxyphenylacetic acid to a fluorescent compound [M.S. Poosch and R.K. Yamasaki (1986) Biochim. Biophys. Acta 884, 585-593]. Our method eliminates the contribution of important interfering side reactions, notably those due to the presence of reducing agents, which function as competitive substrates to 4-hydroxyphenylacetic acid. Rapidly reacting thiol groups are of particular importance, notably CoASH present endogenously (e.g. in peroxisomes and mitochondria) or formed by enzymatic hydrolysis of acyl-CoA. Alkylation of the thiol compounds by N-ethylmaleimide eliminates this disturbing side reaction, and increases the amount of fluorescent product in the coupled peroxidatic reaction. The method is suitable for routine assay of FAO activity in a wide range of tissues, notably in those with a low specific peroxisomal beta-oxidation activity and/or a high content of reducing agents. As an example of this we have included data from rat heart peroxisomal fractions. The effect of alkylation of sulfhydryl groups in the incubation mixture also applies to other oxidase reactions based on H2O2-coupled peroxidatic reactions, if the oxidase itself does not contain functional sulfhydryl groups.
Kvannes
J
J
Department of Biochemistry, University of Bergen, Norway.
Flatmark
T
T
D042962:Acyl-CoA Oxidase; D000818:Animals; D002374:Catalase / Q000037:antagonists & inhibitors; D008099:Liver / Q000201:enzymology; D008297:Male; D008830:Microbodies / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D010088:Oxidoreductases / Q000032:analysis*; D009195:Peroxidase; D010648:Phenylacetates; D051381:Rats; D011919:Rats, Inbred Strains; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013438:Sulfhydryl Compounds / Q000032:analysis
D010648:Phenylacetates; D013438:Sulfhydryl Compounds; C008070:4-hydroxyphenylacetic acid; D010088:Oxidoreductases; D002374:Catalase; D009195:Peroxidase; D042962:Acyl-CoA Oxidase