Osmotic pressure method to measure salt induced folding/unfolding of bovine serum albumin

Abstract

A new approach has been developed to monitor protein folding by utilizing osmotic pressure and a range of salt concentrations in a well characterized protein, bovine serum albumin (BSA). It is hypothesized that both the ‘effective’ osmotic molecular weight, A e , and the solute/solvent interaction parameter, I, in the empirical relation M solvent M solute = ( RTϱ A e ) 1 gp + I [1] can be used as measures of protein folding. I is a measure of solvent perturbed by the solute and is thought to depend directly upon the solvent accessible surface area (ASA). It is reasoned that larger solvent accessible surface area of an unfolded or denatured protein should perturb more water and produce larger I-values. Thus I-values allow calculation of a unfolded protein fraction, f u a , due to changes in relative solvent accessible surface area. It has been observed that A e decreases for filamentous, denatured proteins due to segmental motion of the molecule [2]. This allows calculation of unfolded protein fraction from the effective molecular weight, f u m . Colloid osmotic pressure of BSA was measured in a range of salt concentrations at 25°C, and pH = 7 (above the isoelectric point of BSA at pH = 5.4). Both S and I were used to monitor protein folding as the salt concentration was varied. In general, larger and variable I-values and smaller A e were observed at salt concentrations less than 50 mmolal NaCl ( I max = 8.9), while constant I = 4.1 and A e = 66,500 were observed above 50 mmolal NaCl. The two expressions for fractional unfolding ( f u a and f u m ) are in general agreement. Small differences in the parameters below 50 mmolal salt concentration are explained with well known shifts in the relative amounts of α-helix, β-sheet and random coil in denatured BSA. The relative amounts of these shifts agree with predictions in the literature attributed to continuous BSA expansion rather than an ‘all-or-none’ conversion.