Purification and properties of electron-transferring flavoprotein from pig kidney

Abstract

Electron-transferring flavoprotein was isolated from pig kidney by a simple procedure with a 7-fold higher yield over a previous method using pig liver. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration, amino acid analysis, peptide mapping, and measurement of flavin content indicate that pig kidney electron-transferring flavoprotein contains nonidentical subunits (MW 30,000 and 33,000) with 1 FAD/dimer. These data contrast with reports that the liver protein is a dimer of identical subunits containing 2 flavin molecules. Dithionite and ferricyanide titrations indicate that flavin is the only redox-active moiety in pig kidney electron-transferring flavoprotein. Disproportionation of the anionic semiquinone is very slow, requiring about 10 h for half-completion. In contrast to results obtained with the liver protein, pig kidney electron-transferring flavoprotein does not bind crotonyl-CoA significantly, and the semiquinone form is not reoxidized by crotonyl-CoA directly. These data do not support recent suggestions for a broader role of electron-transferring flavoprotein in .beta. oxidation.