Studies on the electron transfer system. LXVII. Polyacrylamide gel electrophoresis of the mitochondrial electron transfer complexes

A new method is described for the fractionation of the hydrophobic proteins of the mitochondrial electron transfer chain. The four constituent complexes of the chain, complex I (DPNH-coenzyme Q reductase), Complex II (succinic-coenzyme Q reductase), Complex III (reduced coenzyme Q-cytochrome c reductase), and Complex IV (cytochrome c; O₂ oxidoreductase E.C.1.9.3.1) and also succinic dehydrogenase (succinic; (acceptor) oxidoreductase, E.C. 1.3.99.1), after extraction with acetone, were completely soluble in a solvent system consisting of phenol-acetic acid-water (2:1:1, w/v/v). Zone electrophoresis of the solubilized mitochondrial complexes and of succinic dehydrogenase on polyacrylamide gel (7.5% in acrylamide, 35% in acetic acid and 5 M in urea) revealed, in every case, many protein bands with a specific and reproducible pattern for each enzyme complex. These bands include not only the proteins with known prosthetic groups but also other uncharacterized proteins. Bands were identified corresponding to (1) the flavoprotein of Complex II, (2) the flavine protein of the soluble succinic dehydrogenase, and (3) the cytochrome c₁ of Complex III. Bands were tentatively identified corresponding to the nonheme iron protein, to cytochrome b of Complex III, and to cytochrome a of Complex IV. The results support the concept that the electron transfer chain is composed of a series of proteins of small molecular weight.