LOW-TEMPERATURE SOLUTION BEHAVIOR OF METHYLOPHILUS-METHYLOTROPHUS ELECTRON TRANSFERRING FLAVOPROTEIN - A STUDY BY ANALYTICAL ULTRACENTRIFUGATION

The solution behaviour of electron transferring flavoprotein (ETF) fro m Methylophilus methylotrophus was investigated at low temperature (4 degrees C) by analytical ultracentrifugation. The concentration depend ence of the apparent weight average molecular weight, M-w,M-app, estab lished the existence of the protein in heterodimeric state (M = 63,700 Da), but also signified the possible dissociation of the heterodimer at lower concentrations into its constituent subunits (M = 28,900 Da a nd 33,700 Da, together with FAD and AMP cofactors of collective M = 11 20 Da). This similarity in subunit size allows approximate quantificat ion of the dissociation in terms of expressions for a monomer-dimer eq uilibrium. The dissociative behaviour was confirmed by determination o f the point average molecular weight, M-w,M-app(r), as a function of t he ETF concentration, c(r), throughout the sedimentation equilibrium d istributions obtained with loading concentrations of 0.4 and 0.7 mg/ml . By means of the recently formulated ''psi'' procedure for direct ana lysis of solute self-association a value of (1.5+/-0.1) mu M has been obtained for the dissociation constant K-d. Sedimentation velocity exp eriments yielded an estimate of the heterodimer sedimentation coeffici ent, s(20,w)(0) of (4.5+/-0.2) S which for M = 63,700 Da suggests a gl obular structure.