Urea potentiates the denaturation of bovine serum albumin induced by high hydrostatic pressure

The combined effect of urea and high pressure on bovine serum albumin (BSA) under denaturing conditions was analyzed using a thermodynamic approach involving a two-state transition and numerical modelling. There was no significant denaturation at pU (-log [Urea]) values lower than -0.7 and pressure up to 100 MPa, with a Gibbs free energy deltaGp,pu > 1116 J/mol. At higher pressure and urea concentration the process of denaturation became more spontaneous, with deltaGp,pu reaching about ‑14000 J/mol at pU = -0.9030 and 250 MPa. Analysis of the volume change of denaturation (deltaV) and the stoichiometric coefficient of urea uptake (miU) revealed the occurrence of heterogeneous protein structures during denaturation with a maximum (deltaV) value of -160 mL/mol and miU  = 16.15 mol U/mol BSA at pU = -0.825 at 248 MPa. Intermediate pressure values and urea concentrations resulted in an apparent structural homogenization of the albumin in solution. These results suggest that the physicochemical treatments used here can provide a useful experimental approach for describing the processes involved in protein folding/unfolding.