Spectroscopic and volumetric investigation of cytochrome c unfolding at alkaline pH: characterization of the base-induced unfolded state at 25 degrees C.

We have measured at 25 degrees C the relative specific sound velocity increment, [u], and the partial specific volume, v degrees, of cytochrome c as a function of pH. Our data reveal that the base-induced native to unfolded transition of the protein is accompanied by a volume decrease of 0.014 cm3 g-1 and a compressibility decrease of 3.8 x 10(-6) cm3 g-1 bar-1. These results allow us to conclude that, relative to a fully unfolded conformation, the base-denatured state of cytochrome c has only 70 to 80% of its surface area exposed to the solvent. Recently, we reported a similar result for the acid-denatured state of cytochrome c. Thus, insofar as solvent exposure is concerned, both the base- and the acid-induced unfolded states of cytochrome c retain some order, with 20 to 30% of their surface areas remaining solvent-inaccessible. We discuss the implications of this result in terms of defining potential intermediate states in protein folding pathways.