Novel tritium release assays for isovaleryl-CoA and butyryl-CoA dehydrogenases.

An assay method for isovaleryl- and butyryl-CoA dehydrogenase activities that utilizes (2,3-/sup 3/H)isovaleryl- and (2,3-/sup 3/H)butyryl-CoA as substrates is described. The tritium removed enzymatically from carbons 2 and 3 of the substrate equilibrates with solvent, and the tritiated water thus formed is separated quantitatively from unreacted substrate by anion exchange chromatography. Detritiation of the 2,3-/sup 3/H-labeled acyl-CoA substrates by both rat liver mitochondrial sonicates and purified pig liver enzymes demonstrated substrate saturability and Michaelis-Menten kinetics. The apparent K/sub m/ values of the enzymes preparations for the same 2,3-/sup 3/H-labeled acyl-CoA were similar. Free (2,3-/sup 3/H)isovaleric and (2,3-/sup 3/H)butyric acids were not detritiated. With the purified dehydrogenases from pig liver, the assay measures enzyme-catalyzed tritium exchange from substrate to solvent in the absence of electron transfer agents, as well as transfer of tritium to solvent mediated by intermediary electron acceptors. The amount of tritium released from assay mixtures containing rat liver mitochondria was not significantly increased by termination of the reaction with acidic permanganate; however, in a system in which the purified pig liver dehydrogenases were incubated with appropriate tritiated substrates in the presence of electron-transferring flavoprotein or phenazine methosulfate and dichlorophenol indophenol, a major part of tritium removed from themore » substrate was sequestered on the dye. Using the purified pig liver enzymes, apparent K/sub H//K/sub T/ ratios of 4 and 8 were found for isovaleryl- and butyryl-CoA dehydrogenases, respectively, when the rates obtained by the tritium release assay were compared to those measured by the dye reduction method.« less