KINETIC RESOLUTION OF PEPTIDE-BOND AND SIDE-CHAIN FAR-UV CIRCULAR-DICHROISM DURING THE FOLDING OF HEN EGG-WHITE LYSOZYME

The kinetics of regain of the native ellipticity in the far-and near-UV spectra have been investigated during the refolding at pH 7.8 and 20-degrees-C of guanidine-unfolded, nonreduced hen egg white lysozyme. Stopped-flow studies showed that the ellipticities at 260 and 289.5 nm exhibit biphasic kinetics with rate constants of about 50 s-1 and 2.5 s-1 for the rapid and slow phase, respectively. The ellipticity in the far-UV obeyed triphasic kinetics. In addition to a rapid and a slow phase with rate constants similar to those observed in the near-UV, a "burst" of ellipticity was shown to occur in the dead time of the experiments. The effects of low pH and of concentrations of guanidine ranging from 0.075 to 1.5 M on the rapid and slow rate constants were studied. Under all conditions investigated, the rate constants observed in the far- and near-UV for a given phase were the same, thus suggesting that the molecular events observed in the two regions of the UV spectrum are either identical or strongly coupled. Continuous-flow experiments at different wavelengths between 214 and 240 nm under conditions where the dead time for the observation was only 4 ms, followed by a detailed analysis of the kinetics of ellipticity change at each wavelength, provided the spectrum of the molecular species formed at the end of the burst phase. This spectrum was found to closely fit that predicted from the secondary structure of native lysozyme. It was shown that the changes in far-UV ellipticity that occur during the rapid and slow phases do not reflect significant changes in the secondary structure of the polypeptide chain. Rather, they reflect essentially changes in the state of side chains, with an important contribution arising from disulfide bonds. Thus, the complete nativelike secondary structure of lysozyme seems to be practically formed in less than 4 ms.