OSMOTIC-PRESSURE METHOD TO MEASURE SALT-INDUCED FOLDING UNFOLDING OF BOVINE SERUM-ALBUMIN

A new approach has been developed to monitor protein folding by utiliz ing osmotic pressure and a range of salt concentrations in a well char acterized protein, bovine serum albumin (BSA). It is hypothesized that both the 'effective' osmotic molecular weight, A(e), and the solute/s olvent interaction parameter, I, in the empirical relation M(solvent)/ M(solute) = (RT rho/A(e))1/pi+I [1] can be used as measures of protein folding. I is a measure of solvent perturbed by the solute and is tho ught to depend directly upon the solvent accessible surface area (ASA) . It is reasoned that larger solvent accessible surface area of an unf olded or denatured protein should perturb more water and produce large r I-values. Thus I-values allow calculation of a unfolded protein frac tion, f(u)(a), due to changes in relative solvent accessible surface a rea. It has been observed that A(e) decreases for filamentous, denatur ed proteins due to segmental motion of the molecule [2]. This allows c alculation of unfolded protein fraction from the effective molecular w eight, f(u)(m). Colloid osmotic pressure of BSA was measured in a rang e of salt concentrations at 25 degrees C, and pH = 7 (above the isoele ctric point of BSA at pH = 5.4). Both S and I were used to monitor pro tein folding as the salt concentration was varied. In general, larger and variable I-values and smaller A(e) were observed at salt concentra tions less than 50 mmolal NaCl (I-max = 8.9), while constant I = 4.1 a nd A(e) = 65,500 were observed above 50 mmolal NaCl. The two expressio ns for fractional unfolding (f(u)(a) and f(u)(m)) are in general agree ment. Small differences in the parameters below 50 mmolal salt concent ration are explained with well known shifts in the relative amounts of alpha-helix, beta-sheet and random coil in denatured BSA. The relativ e amounts of these shifts agree with predictions in the literature att ributed to continuous BSA expansion rather than an 'all-or-none' conve rsion.