Optical rotatory dispersion and spectrophotometric studies on fetuin in solutions containing detergent and denaturing reagents

Abstract

The effects of the denaturing agents, urea and guanidine·HCl, and detergents, sodium dodecyl sulphate and hexadecyltrimethylammonium bromide (HTAB), on the optical rotatory dispersion of fetuin have been investigated. It was concluded that fetuin has a high affinity for the cationic detergent HTAB which seems to bind, at one type of binding site only, as demonstrated by both an optical rotatory and Optical density study. The same detergent also increased by the parameters λ_c and $\text{b}{}_{\mathrm{<mtext>o</mtext>}}$, determined from Yang-Doty plots and the Moffitt equation, respectively. The conformation-dependent Cotton trough also shows an increase in amplitude and in addition undergoes a shift to higher wavelengths upon addition of HTAB. Although these observations suggest an increase in helical structure, caution was exercised in interpreting the results in this way since optical artifacts (e.g., higher refractive indexforprotein-micelle complex) may contribute. Modification of the fetuin molecule by removal of sialic acid or cleavage of the disulfide bonds results in a reduction in helical content of the molecule but does not alter the affinity of the protein for HTAB. The effect of HTAB on the various fetuin preparations was opposed by guanidine·HCl but not by urea; this was attributed to a charge effect by the former ions. The red shifts of the Cotton curve and the absorption spectra, upon the addition of HTAB to fetuin solutions are gradual and seem to be related, and it is tempting to attribute both phenomena to possible micelle formation in which tyrosine chromophores are also included. Such protein-micellar complexes would have a higher refractive index than the bulk solvent, and this in itself would cause a red shift of the $\text{π → π}{}^{\mathrm{*}}$ absorption band of the helix.

On the basis of solvent perturbation and spectrophotometric studies, it was concluded that fetuin contains no buried tyrosine groups. A structural similarity of both bovine albumin and fetuin was proposed as a result of the appearance in both cases of a second Cotton trough in the far-ultraviolet region after cleaving the disulfide bonds, indicative in both cases of a more random conformation.