Optical Activity of Insulin. I. On the Nature of the Circular Dichroism Bands

Detailed studies of the optical rotatory dispersion and circular dichroism spectra of insulin were carried out to define the difficulties encountered at present in the analysis of protein optical activity in terms of specific conformations. Spectra were recorded at 27°, at elevated temperatures, and subsequent to reaction with N-acetylimidazole. Tyrosine acetylation resulted in a marked diminution and blue shifting of the near-ultraviolet circular dichroism spectrum; the 274-nm extremum was broadened into two distinct extrema, indicating a major contribution from tyrosine moieties to the near-ultraviolet circular dichroism spectrum. The contribution of unordered polypeptide segments to the far-ultraviolet circular dichroism spectrum of insulin was assessed from circular dichroism spectra of thermally denatured insulin. At 70° the far-ultraviolet circular dichroism spectrum of insulin is quite different from that usually obtained with unordered polypeptides in solution, suggesting the maintenance of appreciable amounts of residual order. Lack of a blue shift of the 209-nm extremum at 70° suggested that the negative band of the unordered conformation is located above 200 nm, as is found in polypeptide films. Heating to 70° resulted in a marked reduction of intensity in the near-ultraviolet region with no blue shift. The far-ultraviolet circular dichroism spectrum of native insulin contains two anomalous qualitative features: the position of the positive extremum at 196 nm and the high magnitude of the 209-nm extremum relative to that at 222 nm. These were analyzed in terms of shifts in position and intensity of α-helical and β-structure transitions. The principal limitations to interpreting these spectra exactly are the inadequacy of polypeptide models for protein polypeptide optical activity and the inability to assess quantitatively the contributions made by side chains to far-ultraviolet circular dichroism.