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

Abstract

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.

The Alkali Molten Globule State of Horse Ferricytochrome c: Observation of Cold Denaturation

“…54 Major structural perturbations occur at above pH 12. 31,55 The tertiary structure is fully lost at pH 13 (Figure 1(a)), but a fraction of destabilized helical structure is retained. This is the gross structural identity of the U B state, which has also been likened to an equilibrium intermediate.…”

Compressibility as a means to detect and characterize globular protein states.

“…tCharacterized as a N-to-PU transition based on CD measurements (22) Biochemistry: Chalikian and Breslauer 1014 Biochemistry: Chalikian and Breslauer of (3-7) x 10-6 cm3.g-lbar-for the N-to-PU transitions listed in We propose that this relationship can be understood in terms of the foregoing discussion. Significantly, however, the existence of the empirical relationship and its utility do not depend on the details of our interpretation.…”

Compressibility changes accompanying conformational transitions of apomyoglobin

“…This contribution results from proton transfer reactions accompanying protonation/ deprotonation of titrable amino acids, which also cause a relaxation increase in ultrasonic absorption. 26,[53][54][55][56] The relaxation contribution to relative molar sound velocity increment, D[u] rel ¼ ÀD() rel / (2!c), can be calculated from the relaxation increase in ultrasonic absorption expressed per wavelength, D() rel ( is the coefficient of ultrasonic absorption; is the ultrasonic wavelength; ! is the angular frequency of ultrasound; and is the relaxation time of the proton-transfer reaction).…”

“…To reduce the complexity, we use a previously described approach that, in particular, has been used for treating pH-dependent data on sperm whale apomyoglobin. 56,58 Recall that a pH-induced transition of a protein occurs as a result of a small number of ionizable amino acid residues which exhibit ''abnormal'' dissociation constants. 57 Consequently, the effect of acidic pH on protein stability can be considered in terms of the binding of protons to a relatively small number of ionizable residues.…”