Reversible unfolding of the major fraction of ovalbumin by guanidine hydrochloride

Abstract

The guanidine hydrochloride induced unfolding of the major fraction of ovalbumin (i.e. A1 which contains two phosphate groups and constitutes about 77% of the total protein) was investigated systematically by difference spectran 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 guanidine hydrochloride 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 guanidine hydrochloride alone. Strikingly, in contrast to whole ovalbumin, the denaturation of its A1 fraction by guanidine hydrochloride was fully reversible and obeyed first-order kinetic law under different experimental condit ions of pH, temperature, and the denaturant concentration. The monotonic variation of deltaH for the unfolding of ovalbumin A1 by guanidine hydrochloride with temperature, the coincidence of the two transition curves obtained by measuring two independent properties (namely reduced viscosity and difference in light absorption at 288 nm (or 293 nm) as a function of the denaturant concentration, and finally the adherence of the unfolding as well as refolding reactions to first-order kinetic law suggested that the transition of ovalbumin. A1 can reasonably be approximated by a two-state mode. Analysis of the equilibrium data obtained at pH 7.0 and 25 degrees C according to Aune and Tanford (Aune, K.C.,and Tanford, C. (1969), Biochemistry 8, 4586) 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 (approximately 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 guanidine hydrochloride which was studied in the range 10-60 degrees C at pH 7.0 can be described by assigning the following values of the thermodynamic parameters for the unfolding process: deltaH = 52 kcal/mol at 25 degrees C; deltaS = 153 cal deg-1 mol-1 at 25 degrees C; and delta Cp = 2700 +/- 400 cal deg-1 mol-1.

Characterization of a Human α1-Antitrypsin Variant That Is as Stable as Ovalbumin

“…For instance, the equilibrium unfolding transition of inhibitory serpins including ␣ 1 AT (Fig. 2) is very complex (5,(27)(28)(29), whereas the unfolding transition of ovalbumin is more or less fitted to a two-state model when monitored either by far UV CD signal (5), intrinsic fluorescence (30), or by UV absorbency and intrinsic viscosity (31). The unfolding midpoint of C73A mutant ovalbumin, which could not form disulfide bonds, was very close to that of Multi-7 ␣ 1 AT (Fig.…”

Comparison of the effects of inorganic and organic (hydrophobic) cations on the physical states of proteins and phospholipid bilayer membranes

“…Since, to a first approximation, To and AH0 for lysozyme, ribonuclease and ovalbumin are similar (cf. data in [7,14,15]), for a given concentration of solute (i.e., a fixed 6yh), 6 T is proportional to A.4 for these proteins. Now Chothia [ 161 and Teller [ 171 have given expressions for AA for the transition from native to random coil state as a function of molecular weight; from this approach, if all three proteins undergo the same degree of unfolding on denaturation, 6T for ovalbumin should be larger by a factor of -3.5 than the value for lysozyme or ribonuclease.…”

Structural Stability as a Probe for Molecular Evolution of Homologous Albumins Studied by Spectroscopy and Bioinformatics

“…The free energy change of GuHClinduced unfolding of ovalbumin [30] and HSA [3] is 6 kcal mol -1 each and BSA is 4 kcal mol -1 [5]. The free energy changes are 5.6 and 3.5 kcal mol -1 for RSA and PSA, respectively, as observed in urea-induced unfolding of secondary structure.…”