Oxidative refolding of reduced, denatured lysozyme in AOT reverse micelles.
The refolding kinetics of the reduced, denatured hen egg white lysozyme in sodium bis(2-ethylhexyl)sulfosuccinate (AOT)-isooctane-water reverse micelles at different water-to-surfactant molar ratios has been investigated by fluorescence spectroscopy and UV spectroscopy. The oxidative refolding of the confined lysozyme is biphasic in AOT reverse micelles. When the water-to-surfactant molar ratio (omega 0) is 12.6, the relative activity of encapsulated lysozyme after refolding for 24 h in AOT reverse micelles increases 46% compared with that in bulk water. Furthermore, aggregation of lysozyme at a higher concentration (0.2 mM) in AOT reverse micelles at omega 0 of 6.3 or 12.6 is not observed; in contrast, the oxidative refolding of lysozyme in bulk water must be at a lower protein concentration (5 microM) in order to avoid a serious aggregation of the protein. For comparison, we have also investigated the effect of AOT on lysozyme activity and found that the residual activity of lysozyme decreases with increasing the concentration of AOT from 1 to 5 mM. When AOT concentration is larger than 2 mM, lysozyme is almost completely inactivated by AOT and most of lysozyme activity is lost. Together, our data demonstrate that AOT reverse micelles with suitable water-to-surfactant molar ratios are favorable to the oxidative refolding of reduced, denatured lysozyme at a higher concentration, compared with bulk water.
Fan
Jun-Bao
JB
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China.
Chen
Jie
J
Liang
Yi
Y
D004143:Dioctyl Sulfosuccinic Acid / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008823:Micelles; D009113:Muramidase / Q000737:chemistry*; D009795:Octanes / Q000737:chemistry*; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D012995:Solubility; D013050:Spectrometry, Fluorescence; D013501:Surface-Active Agents / Q000737:chemistry*; D014867:Water / Q000737:chemistry*
D008823:Micelles; D009795:Octanes; D013501:Surface-Active Agents; D014867:Water; D004143:Dioctyl Sulfosuccinic Acid; D009113:Muramidase; C045798:2,2,4-trimethylpentane