A comparative study of lysozyme conformation in various reverse micellar systems

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, k_cat tends to be smaller than in aqueous solution (with quite similar K_M), whereas in the EO₄C₁₂ system (at pH 7.0) the turnover number is larger and K_M 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.