Optical Activity of Insulin. II. Effect of Nonaqueous Solvents

A detailed study has been made of the effects of acidic methanol and 2-chloroethanol on the optical activity of insulin. Changes in circular dichroism spectra have been correlated with changes in the optical rotatory dispersion a₀ and b₀ parameters by a mathematical analysis developed in these studies. The maximal effect on the a₀ parameter occurred at 70% 2-chloroethanol and 80% acidic methanol. On the other hand, the -b₀ parameter did not change in either solvent over the concentration range in which - a₀ attained its maximal value, but it did increase to a small extent at higher concentrations. Marked changes in the circular dichroism spectrum of insulin resulted from exposure to these solvents. The effect of either solvent in the near-ultraviolet was a large reduction in intensity without band shifting and an accentuation of fine structure. In the far-ultraviolet region, there was a large increase at 209 nm, a virtual disappearance of a distinct extremum at 222 nm, and an increase in intensity with a blue shift of the 196-nm extremum. Using a mathematical analysis aimed at improved resolution of overlaps of various transitions, the far-ultraviolet spectra of insulin in 0, 70, and 99% methanol were resolved into Gaussian zones, each of which contains one or a small number of actual transitions. The contributions of the various zones to the a₀ and b₀ parameters were evaluated. It was found that experimental a₀ and b₀ values represent the sum of larger contributions which partially cancel each other and that interpretations based on changes observed at individual wavelengths in the circular dichroism spectra would be hazardous. Far-ultraviolet circular dichroism spectral changes between 0 and 80% methanol were attributed primarily to solvent, nonconformational effects on α-helix and β-structure transitions, and to a small increase in unordered polypeptide conformation. Higher concentrations resulted in a small increase in α-helix content. Use of model compounds showed that the changes recorded in the near-ultraviolet circular dichroism spectra were related primarily to changes in side-chain conformation rather than to solvent nonconformational effects. The changes in self-association were correlated with changes recorded in both the near- and far-ultraviolet circular dichroism spectra of insulin in methanol.