ACYL-COENZYME A DEHYDROGENASE FROM PIG KIDNEY - PURIFICATION AND PROPERTIES

Comparatively large amounts of an acyl-CoA dehydrogenase have been obtained from pig kidney by a procedure which does not involve prior isolation of mitochondria. The pattern of substrate specificity and the extent of substrate-induced bleaching of the flavin chromophore, using butyryl-, octanoyl-, and palmitoyl-CoA, suggest that the enzyme be classified as a general acyl-CoA dehydrogenase. The purified flavoprotein exhibits absorbance ratios at 272, 373, and 446 nm of 5.7:0.65:1.0, respectively, with an extinction coefficient for bound flavin adenine dinucleotide (FAD) of 15.4 mM-1 cm-1 at 446 nm. Gel filtration, NaDodSO4 gel electrophoresis, and amino acid analysis indicate that the enzyme is a tetramer, comprised of subunits of about 42 000 molecular weight, containing 3-4 molecules of FAD as isolated. The blue, neutral flavosemiquinone is formed during anaerobic titration of the enzyme with dithionite (observed e560 = 2.8 mM-1 cm-1)- A similar level of semiquinone is formed during deazaflavin/light reduction, but no long-wavelength intermediates are observed on reduction with borohydride. Full reduction of the flavin requires 1.2 mol of dithionite. Back titration of the fully reduced enzyme with ferricyanide yields considerable levels of semiquinone (observed e560 = 4.1 mM-1 cm-1) and suggests that the extinction coefficient of this species is approximately 5.9 mM-1 cm-1. The disproportionation equilibrium between semiquinone, oxidized, and fully reduced flavin forms is attained very slowly in the absence of mediators. © 1979, American Chemical Society. All rights reserved.