The binding distance of benzidine to hemoglobin (Hb) and the hydrodynamic radii of Hb in glycerol-water mixtures were determined by a fluorescence quenching technique and dynamic light scattering measurements, respectively, and utilized to investigate the interaction between glycerol and the protein and its influence on the conformation of the protein in aqueous solution, together with the analysis of the fluorescence spectra and absorption spectra of Hb. The results suggested that glycerol promote a more compact conformation of protein in aqueous solution through preferential hydration. It was found that the decrease in hydrogen-bonding capacity of the solvent played an important role in stabilizing protein conformation and the breaking down of the hydrogen bond network was attributed to the further folding of protein at a high glycerol concentration. Our studies indicated that heme microenvironment was retained in glycerol mixtures, however, a significant conformational change was observed in the peptide segments outside the heme cavity with a consequent perturbation to aggregation of subunits of Hb.

Key words: hemoglobin, glycerol, conformation, fluorescence quenching, dynamic light scattering