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The compactness of ribonuclease A with intact disulfide bonds and reduced ribonuclease A was investigated by synchrotron small-angle X-ray scattering. The R g values and the Kratky plots showed that non-reduced ribonuclease A maintain a compact shape with a R g value of about 17.3 A in 8 M urea. The reduced ribonuclease A is more expanded, its R g value is about 20 A in 50 mM Tris-HCl buffer at pH 8.1 containing 20 mM DTT. Further expansions of reduced ribonuclease A were observed in the presence of high concentrations of denaturants, indicating that reduced ribonuclease A is more expanded and is in neither a random coil [A. Noppert et al., FEBS Lett. 380 (1996) 179–182] nor a compact denatured state [T.R. Sosnick and J. Trewhella, Biochemistry 31 (1992) 8329–8335]. The four disulfide bonds keep ribonuclease A in a compact state in the presence of high concentrations of urea.

The denaturation of dimeric creatine kinase (CK) induced by urea and guanidine hydrochloride (GuHCl) has been studied by small angle X-ray scattering (SAXS), which is a direct way to measure the changes in the overall dimensions of a protein molecule. The radii of gyration (Rg) of CK are 29+/-0.4 angstroms in the native state and 46+/-1.5 angstroms in the unfolded state in either 8 M urea or 3 M GuHCl. The transition curves of urea denaturation derived from the Rg values and the zero angle intensity (I(0)) are similar to that from intrinsic fluorescence, indicating that the changes in the molecular shape, the tertiary structure and the dissociation of the subunits proceed simultaneously. In the case of GuHCl-induced denaturation, the dramatic increases both in Rg and in I(0) in 0.3-0.5 M GuHCl suggest clearly that soluble aggregates form at low GuHCl concentrations. The aggregates dissociate and the molecule unfolds at higher GuHCl concentrations. The results suggest that the mechanisms of CK denaturation in urea and in GuHCl are somewhat different and the intermediate in GuHCl denaturation can much more easily form soluble aggregates.