Reversible unfolding of the major fraction of ovalbumin by guanidine hydrochloride

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Abstract
The guanidine hydrochloride [Gdn.cntdot.HCl] induced unfolding of the major fraction of ovalbumin (i.e., A1 which contains 2 phosphate groups and constitutes about 77% of the total protein) was investigated systematically by difference spectral and viscosity measurements. As judged by the intrinsic viscosity (3.9 ml/g), the native protein conformation is compact and globular. Difference spectral results showed extensive disruption of the native structure by Gdn.cntdot.HCl. The intrinsic viscosities of ovalbumin A1 in 6 M Gdn.cntdot.HCl with and without 0.1 M .beta.-mercaptoethanol were 31.1 and 27.0 ml/g. These and optical rotation results indicated that the denatured protein existed in a cross-linked random coil conformation in 6 M Gdn.cntdot.HCl alone. In contrast to whole ovalbumin, the denaturation of its A1 fraction by Gdn.cntdot.HCl was fully reversible and obeyed first-order kinetics under different experimental conditions of pH, temperature and the denaturant concentration. The monotonic variation of .DELTA.H [enthalpy] for the unfolding of ovalbumin A1 by Gdn.cntdot.HCl with temperature, the coincidence of the 2 transition curves obtained by measuring 2 independent properties (i.e., reduced viscosity and difference in light absorption at 288 nm (or 293 nm)) as a function of the denaturant concentration and the adherence of the unfolding as well as refolding reactions to first-order kinetics suggested that the transition of ovalbumin A1 can reasonably be approximated by a 2-state model. Analysis of the equilibrium data obtained at pH 7.0 and 25.degree. C according to Aune and Tanford showed that 12 additional binding sites for the denaturant with an association constant of 1.12 were freshly exposed by the unfolding process and that the native protein was marginally more stable (.apprx. 6 kcal/mol) than its unfolded form even under native condition. The temperature dependence of the equilibrium constant for the unfolding of ovalbumin A1 by Gdn.cntdot.HCl which was studied in the range 10-60.degree. C at pH 7.0 can be described by assigning the following values of the thermodynamic parameters for the unfolding process: .DELTA.H = 52 kcal/mol at 25.degree. C; .DELTA.S [entropy] = 153 cal deg-1 mol-1 at 25.degree. C; and .DELTA.Cp [heat capacity] = 2700 .+-. 400 cal deg-1 mol-1.