On the Structural Stability and Solvent Denaturation of Proteins

Abstract

Abstract The effects of the water-miscible straight chain and branched alcohols and glycols on the native conformation of sperm whale myoglobin, cytochrome c, and α-chymotrypsinogen have been investigated by spectral, difference spectral, and optical rotatory dispersion methods. Based on the midpoints of the denaturation transitions, that is, the amount of alcohol or glycol required to produce 50% denaturation at 25°, it is concluded that the effectiveness of the alcohols as protein denaturants increases with increasing chain length or hydrocarbon content, in conformance of what is expected of the disorganization of the hydrophobic interior of these proteins revealed by their detailed three-dimensional x-ray structure. As a rule, branching of the hydrocarbon portion of the alcohols tends to reduce their effectiveness as protein denaturants. The glycols are found to be less effective than the corresponding alcohols, suggesting that increased polarity or hydrogen-bonding capacity is of secondary importance when compared with the effects of increasing hydrocarbon content. The theories of Peller and Flory, used to account for the effects of denaturants and salts on the temperature transition of proteins and polypeptides, were extended to the analysis of isothermal denaturation data, with appropriate binding and Setschenow parameters based on free energy of transfer data taken from the literature or calculated from N-acetyl-l-tryptophan ethyl ester solubilities. The denaturation midpoints, Sm, based on the assumption of a hydrophobic mechanism of alcohol-protein side chain interactions, are found to be in satisfactory agreement with the experimentally obtained Sm values. Both the increased hydrophobicity with increasing chain length and the somewhat reduced effectiveness of the alcohols on branching are in most cases correctly predicted. For the straight chain alcohols, fairly satisfactory agreement with the experimental data is also obtained with binding constants based on estimates of free energies of hydrophobic bond formation, obtained by Scheraga and associates using the Nemethy and Scheraga theory of hydrophobic bonding.