A sensitive spectrophotometric assay for peroxisomal acyl-CoA oxidase.
A simple spectrophotometric assay was developed for peroxisomal fatty acyl-CoA oxidase activity. The assay, based on the H2O2-dependent oxidation of leuco-dichlorofluorescein catalysed by exogenous peroxidase, is more sensitive than methods previously described. By using mouse liver samples, cofactor requirements were assessed and a linear relationship was demonstrated between dye oxidation and enzyme concentration. By using this assay on subcellular fractions, palmitoyl-CoA oxidase activity was localized for the first time in microperoxisomes of rat intestine. The assay was also adapted to measure D-amino acid oxidase activity, demonstrating the versatility of this method for measuring activity of other H2O2-producing oxidases.
D042964:Acyl-CoA Dehydrogenase; D000640:Amitrole / Q000494:pharmacology; D000818:Animals; D002374:Catalase / Q000378:metabolism; D003067:Coenzymes / Q000494:pharmacology; D044943:Fatty Acid Desaturases / Q000378:metabolism*; D005452:Fluoresceins / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D066298:In Vitro Techniques; D007413:Intestinal Mucosa / Q000201:enzymology; D008830:Microbodies / Q000201:enzymology; D051381:Rats; D013053:Spectrophotometry / Q000379:methods*; D013347:Subcellular Fractions / Q000201:enzymology
D003067:Coenzymes; D005452:Fluoresceins; C029569:diacetyldichlorofluorescein; D006861:Hydrogen Peroxide; D002374:Catalase; D044943:Fatty Acid Desaturases; D042964:Acyl-CoA Dehydrogenase; D000640:Amitrole