The effect of chemical modification on the conformational stability of [beta]-lactoglobulin B

Abstract

To probe the relationship between chemical structure and conformational stability, the urea denaturation of bovine (beta)-lactoglobulin B and four chemically modified derivatives of this protein was investigated. The modifying groups were a series of mercaptans: mercaptopropionic acid, propanethiol, mercaptoethanol, and mercaptoethylamine; they were attached to the single sulfhydryl group of (beta)-lactoglobulin B through a disulfide bond. The reduced specific optical rotation, {(alpha)'}(,N), is -90.1(DEGREES) for (beta)-lactoglobulin B and the mercaptopropionic, propanethiol, and mercaptoethanol derivatives at 365.4 nm, the wavelength used to follow denaturation. In addition, the ORD spectra are similar in the near and far-UV wavelength range. This suggests that the conformation of these derivatives is similar to that of the unmodified protein. In contrast, for the mercaptoethylamine derivative, {(alpha)'}(,N) = -118.7(DEGREES) and the near and far-UV ORD spectra differ substantially. Thus, the conformation of this derivative differs substantially from that of the unmodified protein. In all cases, denaturation was shown to be reversible, and the derivatives were less stable than unmodified (beta)-lactoglobulin B. The midpoints of the isothermal urea denaturation curves at pH 2.83 and 25(DEGREES)C occur at 4.97 M urea for (beta)-lactoglobulin B, and 4.46 M, 4.23 M, 4.19 M, and 1.68 M urea for the mercaptopropionic acid, propanethiol, mercaptoethanol, and mercaptoethylamine derivatives, respectively. An analysis of this data shows that the corresponding decreases in the conformational stability are approximately 1.1, 1.6, 1.7, and 7.3 kcal/mole for the modified proteins.