Occurrence and characterization of stable intermediate state(s) in the unfolding of ovomucoid by guanidine hydrochloride

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Abstract
Reversible unfolding of ovomucoid by guanidine hydrochloride, as followed by viscosity and difference-spectral measurements at 25.degree. C, pH 6, occurred in 2 distinct steps involving at least 3 major conformational states, i.e., the native, intermediate and completely denatured states, occurring, respectively, in 60 mM-sodium phosphate buffer, 3.5 M-guanidine hydrochloride and 6 M-guanidine hydrochloride. The overall native conformation of ovomucoid, as indicated by its intrinsic viscosity (5.24 ml/g) and gel-filtration behavior, differs significantly from that of a typical globular protein. Exposures of tyrosine residues in native ovomucoid measured by difference spectroscopy following perturbation with glycerol, ethylene glycol and dimethyl sulfoxide were, respectively, 0.42, 0.56 and 0.57. Of the exposed phenolic groups, only 1 titrated normally (pKint, 9.91, electrostatic-interaction factor, w, 0.04). Results on difference spectra, solvent pertubation, phenolic titration and intrinsic viscosity (7.4 ml/g) taken together showed that although ovomucoid in 3.5 M-guanidine hydrochloride was significantly unfolded, it retained a degree of native structure, removable with 6 M-guanidine hydrochloride. In the latter, all 6 tyrosine residues were available for titration, and the intrinsic viscosity of ovomucoid increased to 9.4 ml/g. The characteristic fine structures in circular-dichroism spectra of ovomucoid, associated with the elements of native structure, were abolished in 6 M-guanidine hydrochloride, suggesting that the completely denatured state is structureless and presumably behaves as a cross-linked random coil. The latter state was shown by analysis of the results on guanidine hydrochloride-dependence on the transition, intermediate .dblarw. denatured, to be less stable than the intermediate state under native conditions by about 46 kJ/mol at 25.degree. C. Attempts were made to interpret the above results in the light of available information on the amino acid sequence of ovomucoid.