Characterization of the oligomeric states of BSA using light scattering techniques

ERNESTO A. ROMAN; JAVIER SANTOS; F. LUIS GONZÁLEZ FLECHA

Montevideo

Congreso; 6th International Conference on Biological Physics, 5th Southern Cone Biophysics Congress; 2007

Sociedad Argentina de Biofisica

Serum albumin is the most abundant protein in the circulatory system. At neutral pH, the crystal structure of this 66 kD protein reveals a heart-shaped molecule organized into three similar structural domains (1). These are alfa helical and include loops and a large number of disulfide bonds. It was described that aqueous solutions of BSA present a monomer-dimer equilibrium with a dissociation constant of 10 mM at 25 degrees and pH 6 (2). Despite the BSA acid-base transition had been extensively characterized, no previous work had accurately described how these changes are reflected in hydrodynamic properties of the different oligomeric structures of this molecule. In this work, we analyze the effects of pH on the oligomeric states of BSA using static and dynamic light scattering combined with size exclusion chromatography. In these studies, BSA monomer (Sigma (St. Luois, MO)) at 10mg/ml was prepared using phosphate buffer 10 mM with NaCl 100 mM. The same buffer was used to equilibrate the Sepharosa S-200 column at different pHs. All solutions were prepared using bidistilled water filtered through a 20 nm filter and degassed before use. Elution chromatogram of monomeric BSA showed four different oligomeric forms. The molecular masses of these molecules were obtained from static light scattering data using the three angle detector demonstrating that these species are the monomer, dimer, trimer, and small quantities of higher molecular weight oligomer. At lower pHs only dimer and monomer were detected. Hydrodynamic radii and diffusional coefficients of monomer and dimer were calculated from the dynamic light scattering data. At lower pHs hydrodynamic radii becomes higher and diffusional coefficient becomes smaller. Dimer and trimer were collected and reinjected. In both cases, we observed the monomer, dimer, and trimer, demonstrating the reversible character of the forces stabilizing these oligomeric forms.With grants from UBA, CONICET, and ANPCyT.Referencias: [1] Carter D., Ho JX. Advances in Protein Chemistry, 1994, (45) 153-203[2] Levi V., Gonzalez Flecha FL. Biochimica et Biophysica Acta, 2002, (1599) 141-148