Effect of ethanol on folding of hen egg-white lysozyme under acidic condition.
The equilibrium and kinetics of folding of hen egg-white lysozyme were studied by means of CD spectroscopy in the presence of varying concentrations of ethanol under acidic condition. The equilibrium transition curves of guanidine hydrochloride-induced unfolding in 13 and 26% (v/v) ethanol have shown that the unfolding significantly deviates from a two-state mechanism. The kinetics of denaturant-induced refolding and unfolding of hen egg-white lysozyme were investigated by stopped-flow CD at three ethanol concentrations: 0, 13, and 26% (v/v). Immediately after dilution of the denaturant, the refolding curves showed a biphasic time course in the far-UV region, with a burst phase with a significant secondary structure and a slower observable phase. However, when monitored by the near-UV CD, the burst phase was not observed and all refolding kinetics were monophasic. To clarify the effect of nonnative secondary structure induced by the addition of ethanol on the folding/unfolding kinetics, the kinetic m values were estimated from the chevron plots obtained for the three ethanol concentrations. The data indicated that the folding/unfolding kinetics of hen lysozyme in the presence of varying concentrations of ethanol under acidic condition is explained by a model with both on-pathway and off-pathway intermediates of protein folding.
Sasahara
Kenji
K
Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, Hokkaido, Japan. sasahara@protein.osaka-u.ac.jp
Nitta
Katsutoshi
K
D000818:Animals; D001703:Biophysics / Q000379:methods*; D002645:Chickens; D002942:Circular Dichroism; D019295:Computational Biology / Q000379:methods; D004529:Egg White; D000431:Ethanol / Q000737:chemistry / Q000494:pharmacology*; D019791:Guanidine / Q000737:chemistry; D006851:Hydrochloric Acid / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009113:Muramidase / Q000737:chemistry*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D040901:Proteomics / Q000379:methods*; D013816:Thermodynamics; D014466:Ultraviolet Rays
D000431:Ethanol; C421864:hen egg lysozyme; D009113:Muramidase; D019791:Guanidine; D006851:Hydrochloric Acid