Pressure-Induced Denaturation of Bovine Serum Albumin

High hydrostatic pressure induces dissociation in several protein aggregates, and denaturation process in conditions as presence of subdenaturing concentrations of urea. In this work we investigated the pressure effect on bovine serum albumin (BSA), MW = 60,000, in combination with urea. The fluorescence properties of the protein with pressure up to 220 MPa were monitored in order to estimate the degree of denaturation, that was calculated as proportional to the red shift of fluorescence emission spectra. BSA at 0.5 mg/ml was incubated at 20 ^oC in 100 mM Tris HCl buffer pH 7.4, in the presence of urea up to 7.0 M concentration. At atmospheric pressure the denaturation process was observed at urea concentration of 5.0 M or higher. The pressure induced denaturation process even at low urea concentration as 2.5 M. Overall the denaturation degree was higher at higher urea concentration and pressure incubation. Considering the dissociation process as the reaction P + nU ßà PU_n , the stochiometry of urea dissociation is calculated based on the pressure dissociation curves extrapolating the equilibrium constants to atmospheric pressure, and plotting such constants as a function of ln of urea concentration. The understanding of denaturation process depends on determination of thermodynamic parameters that include stoichiometry determinations, as the present proposal.