Conformation of Proteins and Polypeptides. Ii. Optical Rotatory Dispersion and Conformation of the Milk Proteins and Other Proteins in Organic Solvents.

The study of the optical rotatory properties of synthetic polypeptides in organic solvents has contributed considerably to our understanding of the structural organization of globular proteins. However, in contrast to the extensive literature which has accumulated in the past decade on the various polyamino acids (l-3), relatively little work has been done on actual proteins in organic solvents. Weber and Tanford (4) have studied the hydrodynamic behavior and the rotatory dispersion properties of ribonuclease in 2chloroethanol and 2-chloroethanolwater mixtures. The conversion of the native structure of the protein to a more helical structure in chloroethanol-rich solutions has been interpreted by these authors in terms of weakening and destruction of hydrophobic regions in the native protein fold. Sage and Singer (5) have found that the structural alterations in ethylene glycol lead to the normalization of the titration behavior of the three abnormally ionizing tyrosyl residues in this enzyme without any significant alterations in the helical content or irreversible loss of catalytic activity. Presumably, the abnormal tyrosyls are buried in nonhelical hydrophobic regions of ribonuclease. The optical rotatory and hydrodynamic properties of P-lactoglobulin in mixed solvents, containing up to 80% nonaqueous component, have been studied by Tanford et al. (6-8). These studies and studies by others in mixed solvents have led to the hypothesis that hydrophobic forces (9-13) play an important stabilizing role in the maintenance of the native structure of proteins as well as nucleic acids (1416) in aqueous media. Recently, Hamaguchi et al. (17-19) have published a detailed study on the solvent denaturation of lysozyme, employing a variety of organic solvents as denaturing agents. The effects of 2-chloroethanol on the rotatory properties of a number of proteins have been reported by Imahori, Klemperer, and Doty (20) (see also References 1 and 2) and very recently by Shechter and Blout (21,22). These are the only further studies which could serve to augment the findings of the accompanying paper (23) which deals with the extension of the solvent perturbation method of difference spectroscopy to the study of proteins and polypeptides in organic solvents. The need for auxiliary information prompted us to examine the optical rotatory properties of the seven proteins employed in the above paper. The denaturing solvents used in the present work are ethylene glycol, acidic methanol, 2-chloroethanol, 8 M urea, and, in some cases, formic acid. For comparison, we also report the rotatory properties of these proteins in aqueous media. EXPERIMENTAL PROCEDURE'