A fluorimetric assay for acyl-CoA synthetase activities.
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.
Lageweg
W
W
Department of Pediatrics, University of Amsterdam, The Netherlands.
Steen
I
I
Tager
J M
JM
Wanders
R J
RJ
D042962:Acyl-CoA Oxidase; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D003066:Coenzyme A Ligases / Q000032:analysis*; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry; D005470:Fluorometry / Q000379:methods*; D006719:Homovanillic Acid / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D010088:Oxidoreductases; D051381:Rats; D012680:Sensitivity and Specificity; D013997:Time Factors
D005182:Flavin-Adenine Dinucleotide; D000255:Adenosine Triphosphate; D006861:Hydrogen Peroxide; D010088:Oxidoreductases; D042962:Acyl-CoA Oxidase; D003066:Coenzyme A Ligases; D006719:Homovanillic Acid