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.(ABSTRACT TRUNCATED AT 250 WORDS)
Chaffotte
A F
AF
Unité de Biochimie Cellulaire, CNRS URA 1129, Institut Pasteur, Paris, France.
Guillou
Y
Y
Goldberg
M E
ME
D000818:Animals; D002645:Chickens; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry; D004529:Egg White; D019791:Guanidine; D006146:Guanidines; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009113:Muramidase / Q000737:chemistry*; D017510:Protein Folding; D013056:Spectrophotometry, Ultraviolet
D004220:Disulfides; D006146:Guanidines; D009113:Muramidase; D019791:Guanidine