A fluorimetric assay for acyl-CoA synthetase activities

Anal Biochem. 1991 Sep 2;197(2):384-8. doi: 10.1016/0003-2697(91)90408-l.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acyl-CoA Oxidase
  • Adenosine Triphosphate / chemistry
  • Animals
  • Coenzyme A Ligases / analysis*
  • Flavin-Adenine Dinucleotide / chemistry
  • Fluorometry / methods*
  • Homovanillic Acid / chemistry
  • Hydrogen Peroxide / chemistry
  • Hydrogen-Ion Concentration
  • Kinetics
  • Liver / enzymology
  • Oxidoreductases
  • Rats
  • Sensitivity and Specificity
  • Time Factors

Substances

  • Flavin-Adenine Dinucleotide
  • Adenosine Triphosphate
  • Hydrogen Peroxide
  • Oxidoreductases
  • Acyl-CoA Oxidase
  • Coenzyme A Ligases
  • Homovanillic Acid