Physicochemical studies on cytochrome b2. Sedimentation, diffusion and electrophoresis of the crystalline deoxyribonucleoprotein

Abstract

The paper describes sedimentation, diffusion and electrophoresis studies on the crystalline, enzymically active deoxyribonucleo-protein, Type I cytochrome b2 [L(+)-lactate-cytochrome c oxido-reductase of backer''s yeast]. Enzymic activity of this labile flavohaemoprotein was substantially retained during the experiments by rigorous exclusion of oxygen and of heavy metals. Cytochrome b2 behaves as an apparently monodisperse protein as indicated by boundary analysis of sedimentation patterns according to Baldwin (1957) and by electrophoresis at I 0.63 and at pH 5-8. Sedimentation-velocity experiments gave an intrinsic sedimentation coefficient, S020,w, of 9.17 x 10-13 sec. There is marked concentration-dependence of the sedimentation coefficient, described by the equation Sc020 w = S020,w (1-kc0), where c0 is [mu]M-cytochrome b2 haem, k is 1.40 X 10-3 1./[mu]mole of cytochrome b2 haem. Variable molecular weights for cytochrome bo were obtained by approach -to-equilibrium experiments, which also gave unsatisfactory sedimentation coefficients. The cause of this discrepancy is unexplained. Satisfactory values of 129,000 and of 14,600[plus or minus]200 were obtained for the molecular weights of yeast alcohol dehydrogenase and of horse-heart cytochrome c by similar procedures. From equilibrium-sedimentation data, a molecular weight of 177 300 was obtained by Lansing and Kramer''s method, but other values were obtained by the equilibrium differential equation and the integrated form of the same equation. Possible reasons for this discrepancy are discussed; there appears to be a rapid dynamic equilibrium between various polymeric forms of the enzyme. By static free diffusion, the diffusion coefficient. D20,w, of cytochrome b2 (Type I) at 0.4% of protein is 3.80 X 10-7 cm.2/sec. The partial specific volume as calculated from the composition is 0.71. The molecular weight derived from these values and the sedimentation coefficient is 186 400. The minimum molecular weight from analyses of flavin and of heme is 82 300 - 900, and the apparent molecular weight from physical measurements is 183 400[plus or minus] 3900. At the concentrations studied (5-110 [mu]M-haem) cytochrome b2 thus appears to exist predominantly as a dimer, containing two flavin and two heme groups/molecule. The calculated frictional ratio is 1[image]18, indicating that the molecule is approximately spherical.