Preparation of separated alpha and beta subunits of electron-transferring flavoprotein in unfolded forms and their restoration to the native holoprotein form.
Electron-transferring flavoprotein (ETF) isolated from pig kidney is a heterodimer containing one FAD and one AMP [Sato, K. et al. (1993) J. Biochem. 114, 215-222]. This paper presents a method for separation of the alpha and beta subunits on a preparative scale. The subunits were separated by cation-exchange chromatography in the presence of 6 M urea at pH 7.6. The subunits were obtained in unfolded forms with denaturant. The unfolded subunits were restored to the heterodimeric form having FAD and AMP with high yield after incubation with FAD and AMP under non-denaturing conditions. The far-UV CD spectrum, the fluorescence and absorption spectra of the bound FAD, and the enzyme activity of the reconstituted ETF were all identical to those of the ETF isolated from pig kidney. Thus, an experimental system was established for the reconstitution of ETF from the four components, alpha, beta, FAD, and AMP. Analyses of the absorption spectra of the separated subunits suggested that the contents of aromatic residues were one tryptophan and six tyrosines in alpha and one tryptophan and one tyrosine in beta. The possibility that one subunit binds with FAD or AMP in the absence of the other subunit was examined by measuring the flavin and protein fluorescence spectra, but no spectral changes reflecting such binding were detected. This result suggests that the coexistence of both the subunits is necessary for the binding of FAD and AMP with the protein.
Sato
K
K
Department of Physiology, Kumamoto University School of Medicine.
Nishina
Y
Y
Shiga
K
K
D000249:Adenosine Monophosphate / Q000737:chemistry; D000818:Animals; D044927:Electron-Transferring Flavoproteins; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry; D005420:Flavoproteins / Q000737:chemistry*; D007668:Kidney / Q000737:chemistry; D010446:Peptide Fragments / Q000302:isolation & purification*; D011485:Protein Binding; D017510:Protein Folding; D013552:Swine
D044927:Electron-Transferring Flavoproteins; D005420:Flavoproteins; D010446:Peptide Fragments; D005182:Flavin-Adenine Dinucleotide; D000249:Adenosine Monophosphate