The unfolding of beta-lactoglobulin at pH 3 by urea, formamide, and other organic substances.

We have reported in a previous paper (1) that optical rotatory dispersion measurements indicate that the native conformation of P-lactoglobulin, in aqueous solution, contains few, if any, regions with an a-helical structure. It appears likely, from the many studies of Jirgensons (2) that this conclusion is applicable to a large number of globular proteins, and we have suggested that this indicates the most important factor in the determination of protein structure in aqueous solutions to be the hydrophobic force which has its origin in the hydrogen-bonded structure of the solvent, water. We have also shown previously (1) that the addition of a variety of organic substances to aqueous P-lactoglobulin at pH 3 leads eventually to a conformation which, again on the basis of optical rotatory dispersion, has a high content of cY-helices. In other words, when the concentration of water is sufficiently depleted, the hydrophobic forces vanish and the tendency to form intramolecular hydrogen bonds becomes the predominant factor in the determination of structure. Our previous study (1) also showed that the change in conformation which results from the addition of organic substances to aqueous P-lactoglobulin is not a single reaction, but that it occurs in two stages: first an unfolding of the native structure and then a refolding to the predominantly helical structure. This finding was taken as further proof that the conformation in aqueous solution differs from the helical conformation in a fundamental way. If the difference were simply due to the shift of a helix-coil equilibrium, with helices favored in the water-poor solvents but progressively disrupted as the water content is increased, then a smooth transition from one conformation to the other would have occurred. The chief purpose of the present paper is to compare the organic substances used in our earlier study with two substances not used previously, urea and formamide. These substances are expected to interfere with the formation of CO. . .HN hydrogen bonds, and should thus prevent the refolding reaction leading to a helical conformation. On the other hand, urea and formamide should have no special effect on the unfolding reaction if the native conformation owes its stability to the hydrophobic forces produced