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Equilibrium and kinetic folding of hen egg‐white lysozyme under acidic conditions

The equilibrium and kinetic folding of hen egg‐white lysozyme was studied by means of circular dichroism spectra in the far‐ and near‐ultraviolet (UV) regions at 25°C under the acidic pH conditions. In equilibrium condition at pH 2.2, hen lysozyme shows a single cooperative transition in the GdnCl‐induced unfolding experiment. However, in the GdnCl‐induced unfolding process at lower pH 0.9, a distinct intermediate state with molten globule characteristics was observed. The time‐dependent unfolding and refolding of the protein were induced by concentration jumps of the denaturant and measured by using stopped‐flow circular dichroism at pH 2.2. Immediately after the dilution of denaturant, the kinetics of refolding shows evidence of a major unresolved far‐UV CD change during the dead time (<10 ms) of the stopped‐flow experiment (burst phase). The observed refolding and unfolding curves were both fitted well to a single‐exponential function, and the rate constants obtained in the far‐ and near‐UV regions coincided with each other. The dependence on denaturant concentration of amplitudes of burst phase and both rate constants was modeled quantitatively by a sequential three‐state mechanism, U↔I↔N, in which the burst‐phase intermediate (I) in rapid equilibrium with the unfolded state (U) precedes the rate‐determining formation of the native state (N). The role of folding intermediate state of hen lysozyme was discussed. Proteins 2002;49:472–482. © 2002 Wiley‐Liss, Inc.