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A comparative study of lysozyme conformation in various reverse micellar systems
Author(s) -
Steinmann Bettina,
Jäckle Hans,
Luisi Pier Luigi
Publication year - 1986
Publication title -
biopolymers
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/bip.360250612
Subject(s) - lysozyme , micelle , chemistry , aqueous solution , substrate (aquarium) , solubilization , micellar solutions , enzyme , micellar liquid chromatography , crystallography , chromatography , organic chemistry , biochemistry , oceanography , geology
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/H 2 O, cetyltrioctylammoniumbromide (CTAB)/CHCl 3 , isooctane/H 2 O; tetraethyleneglycoldodecylether (EO 4 C 12 )/isooctane/H 2 O, and bulk water. CD spectra of lysozyme in reverse micellar solutions were investigated as a function of w 0 (= [H 2 O]/[AOT]) and were compared to the spectra in aqueous solutions. No marked changes were found in the EO 4 C 12 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 NAG 3 , and by inference, when the substrate is bound, e.g., during enzyme turnover. However, in the absence of added NAG, NAG 3 , 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 w 0 using the chromophoric substrate 3,4‐dinitrophenyl‐tetra‐N‐acetyl‐β‐ D ‐chitotetraoside (3,4‐DNP‐NAG 4 ). 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 EO 4 C 12 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.