SIDE-CHAIN OPTICAL ACTIVITY IN CYSTINE-CONTAINING PROTEINS: CIRCULAR DICHROISM STUDIES*

There have been several reports of optical activity associated with side-chain chromophores in proteins and polypeptides within the last few years.'l- That the sources of optical activity in these cases were side-chain electronic transitions was inferred from the location of actual Cotton effects or irregularities in the optical rotatory dispersion curves in the spectral region 260-300 m,u. In two of the more recent papers6 8 circular dichroism measurements were reported. The occurrence and measurements of side-chain optical activity in proteins is important for several reasons. Estimates of helix content by analysis of optical rotatory dispersion curves', 10 depend on the absence of Cotton effects at wavelengths longer than about 230 mu or on ability to subtract such Cotton effects from the background rotation which is due to peptide bond transitions in the 180-225-mj region. An added complication is that optical activity in the 260-300-m, region heralds the absence of symmetry of one or more side-chain chromnophores and makes it highly probable that Cotton effects due to other transitions in these chromophores will occur at shorter wavelengths where they may go undetected but nonetheless make substantial contributions to optical rotation at longer wavelengths. Finally, because not all residues which absorb light in this spectral region exhibit optical activity, and to the extent that such optical activity is conformation-dependent, the appearance of side-chain Cotton effects is indicative of hindered rotation about the bond(s) connecting the chromophoric group to the asymmetric a-carbon in native proteins. The identification of the responsible chromophores may thus give valuable information about the tertiary structure of proteins and alteration of this structure consequent to interaction with other molecules, change of pH, temperature, and other factors affecting protein structure. In this paper, we report results of circular dichroism measurements of several proteins in the 250-350-m, region of the spectrum. Circular dichroism possesses the inherent advantage of presenting discrete bands whose widths are comparable to the associated spectral bands."l Thus, while the limbs of a Cotton effect are of different sign on each side of the maximum of the absorption band and extend over hundreds of millimicrons, an ellipticity band has a single sign (which may be positive or negative) and a half band width which may be no greater than 15-20 mu. A further and substantial advantage of circular dichroism over optical rotatory dispersion in these measurements is that there is no background signal. Thus, a decision as to the wavelength of a band maximum may be made with considerable confidence from the circular dichroism spectrum. In this paper, we are primarily interested in the optical activity of electronic transitions associated with the disulfide bond at wavelengths longer than 245 m,u. These transitions are only weakly allowed (electrically) in cystine-containing peptides.l2 In proteins, the disulfide spectral bands are difficult to discern be999