| Dilution |
| denaturing-reducing buffer (100 mM Tris-HCl pH 8.5, 6 M GdnHCl, 1 mM EDTA, 100 mM DTT |
| 50 mM acetic acid-NaOH pH 4.5, 4 M GdnHCl, 1 mM EDTA |
| 100 mM Tris-HCl pH 8.2, 3 mM GSH, 0.3 mM GSSG, 1 mM EDTA) including N'-substituted N-methylimidazolium chlorides at various concentrations. |
| None |
| no tag |
| 8.2 |
| 25.0 |
| n/a |
| 24 h |
| GSH/GSSG |
| 3 mM-0.3 mM |
| Denaturation/Reduction and Refolding. Native lysozyme was denatured and reduced for at least for 2 h at room temperature in a denaturing-reducing buffer (100 mM Tris-HCl pH 8.5, 6 M GdnHCl, 1 mM EDTA, 100 mM DTT). The solution was acidified to pH 4.0 with 1 M HCl to prevent disulfide formation and then dialyzed against a stock buffer (50 mM acetic acid-NaOH pH 4.5, 4 M GdnHCl, 1 mM EDTA) according to a reported procedure (21). We checked that the tertiary structure of the denatured and reduced lysozyme was different from that of the native tertiary structure by measuring the intrinsic fluorescence spectrum data (22). The unfolding transition to the random coil state was confirmed by measuring far-UV CD spectra (23). The complete reduction of intrinsic disulfide bonds was checked by Ellman's assay (24).
A solution of denatured and reduced lysozyme (30.0 mg/mL) was then diluted by 30-fold in a refolding buffer (100 mM Tris-HCl pH 8.2, 3 mM GSH, 0.3 mM GSSG, 1 mM EDTA) including N'-substituted N-methylimidazolium chlorides at various concentrations. These diluted solutions are hereafter referred to as refolding solutions. Refolding solutions were incubated at 25 C for 24 h before being subjected to enzymatic activity assays. For kinetic analyses, aliquots of the refolding solutions were withdrawn at the desired times. |
| Enzyme activity |
| None |
| N-substitued N-methylimidazolium cations |
| Various concentrations |
| n/a |
| n/a |
| In this paper they studied effects of various N-substituted N-methylimidazolium Chlorides and conventional additives on aggregation and reactivation of lysozyme |