A comparative study of lysozyme conformation in various reverse micellar systems

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Abstract
The activity and conformation of lysozyme solubilized in apolar solvents via reverse micelles was investigated. The systems used were sodium di‐2‐ethylhexylsulfosuccinate (AOT)/isooctane/H2O, cetyltrioctylammoniumbromide (CTAB)/CHCl3, isooctane/H2O; tetraethyleneglycoldodecylether (EO4C12)/isooctane/H2O, and bulk water. CD spectra of lysozyme in reverse micellar solutions were investigated as a function of w0 (= [H2O]/[AOT]) and were compared to the spectra in aqueous solutions. No marked changes were found in the EO4C12 or in the CTAB systems with respect to water, which indicates that no sizeable conformational changes of the enzyme occurred upon solubilization in the reverse micellar systems. In agreement with previous studies [C. Grandi, R. E. Smith, and P. L. Luisi (1981) J. Biol. Chem. 256, 837–843] dramatic conformational changes can be inferred in the AOT system on the basis of CD studies. This is taken as an indication that the enzyme denatures in this micellar system. This is particularly striking because the enzyme is fully active in AOT reverse micelles. The apparent paradox is solved by the observation that the native CD spectrum (and by inference, the native conformation) is maintained when lysozyme is bound to NAG or NAG3, and by inference, when the substrate is bound, e.g., during enzyme turnover. However, in the absence of added NAG, NAG3, or substrate, the enzyme in the AOT reverse micellar system rapidly denatures. Together with CD studies, fluorescence and nmr data confirm the hypothesis of an irreversible denaturation of lysozyme in the AOT system, the denaturation being slowed down when the substrate is present. The activity of the enzyme has been studied as a function of pH and w0 using the chromophoric substrate 3,4‐dinitrophenyl‐tetra‐N‐acetyl‐β‐D‐chitotetraoside (3,4‐DNP‐NAG4). Generally speaking, the kinetic parameters are comparable to those found in bulk water solution. More detailed, in the CTAB system, kcat tends to be smaller than in aqueous solution (with quite similar KM), whereas in the EO4C12 system (at pH 7.0) the turnover number is larger and KM is smaller than in water. In the AOT system, the kinetic parameters at pH 7.0 are also quite comparable to those found in water.