Biochemistry 1994, 33, 11225-1 1236
11225
Kinetics of Folding of Guanidine-Denatured Hen Egg White Lysozyme and Carboxymethyl(Cy~~,Cys’~~)-Lysozyme: A Stopped-Flow Absorbance and Fluorescence Study? Mary E. Denton,? David M. Rothwarf, and Harold A. Scheraga’ Baker Laboratory of Chemistry, Cornell University, Ithaca, New York 14853- 1301 Received April 7 , 1994; Revised Manuscript Received July 1 1 , 1994”
ABSTRACT: The folding kinetics of hen egg white lysozyme and of a three-disulfide derivative of lysozyme
[carboxymethyl(Cys6,Cys’*7)-henegg white lysozyme] have been studied by absorbance- and fluorescencedetected stopped-flow techniques. A -very-fast” phase with a time constant in the millisecond range has been observed by both absorbance and fluorescence when unfolded lysozyme in 4 M guanidine hydrochloride, 100 m M phosphate buffer, and p H 2.0 is refolded a t 0.5 M guanidine hydrochloride, 100 m M phosphate, and p H 6.7. Data obtained from fluorescence-detected refolding studies show that a transient intermediate is formed during the very-fast refolding phase. This intermediate is characterized by substantial quenching of tryptophan fluorescence, In addition, analysis of the fluorescence data indicates the presence of an additional “burst” phase that occurs within the dead time of the instrument,
