A fluorimetric assay for acyl-CoA synthetase activities

✍ Scribed by Wessel Lageweg; Ivar Steen; Joseph M. Tager; Ronald J.A. Wanders

• Publisher: Elsevier Science
• Year: 1991
• Tongue: English
• Weight: 415 KB
• Volume: 197
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(91)90408-l

✦ Synopsis

In this paper we describe a fluorimetric assay for the measurement of long-chain acyl-CoA synthetase activity in rat liver postnuclear supernatants. The method is based upon the use of acyl-CoA oxidase which catalyzes the dehydrogenation of acyl-CoA esters to yield enoyl-CoA esters and H2O2. H2O2 subsequently reacts with homovanillic acid in a horseradish peroxidase-catalyzed reaction to form a highly fluorescent dimer (see G. G. Guilbault, P. J. Brignac, and M. Zimmer (1968) Anal. Chem. 40, 190-196). The increase in fluorescence can be followed either continuously or discontinuously. The method described is able to detect acyl-CoA synthetase activities as low as 20 microU/ml which is almost as sensitive as the standard isotopic assay used in most laboratories. The method is applicable to measure the activation of a variety of fatty acids. Finally, the method provides a simple means of carrying out kinetic studies.