Reversible fast-dimerization of bovine serum albumin detected by fluorescence resonance energy transfer.
Self-association of bovine serum albumin (BSA) was explored using fluorescence resonance energy transfer (FRET) between two populations of the protein labeled separately with either fluorescein-5'-isothiocyanate (FITC) or eosin-5'-isothiocyanate (EITC). The energy transfer reached the steady state after 5 s at 25 degrees C, indicating a fast exchange between oligomer subunits. The dependence of the energy transfer efficiency on the protein concentration and its reversion by unlabeled BSA demonstrate that association between BSA monomers occurs through a reversible path that involves specific interactions between the protein molecules. Because energy transfer took place even after blocking Cys 34 with iodoacetamide, this residue might not be involved in the reversible self-association process. The number of subunits forming the oligomer and its dissociation constant were determined from measurements of energy transfer as a function of the donor-acceptor ratio and of the total protein concentration. Analysis of these data indicated that BSA is in a monomer-dimer equilibrium with a dissociation constant of 10 +/- 2 microM at 25 degrees C in 10 mM MOPS-K (pH 5.8).
Levi
Valeria
V
Departamento de Química Biológica, Instituto de Química y Fisicoquímica Biológicas, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junin 956, 1113, Buenos Aires, Argentina.
González Flecha
F Luis
FL
D000818:Animals; D002417:Cattle; D019281:Dimerization; D004735:Energy Transfer; D004801:Eosine Yellowish-(YS) / Q000031:analogs & derivatives*; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D012710:Serum Albumin, Bovine / Q000737:chemistry*
C039217:fluorescein isothiocyanate bovine serum albumin; D012710:Serum Albumin, Bovine; C012957:eosine-5-isothiocyanate; D016650:Fluorescein-5-isothiocyanate; D004801:Eosine Yellowish-(YS)