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Guanidinium chloride-induced inactivation of firefly luciferase and its reactivation Ekaterina I. Dementieva*; LG Maloshenok; NN Ugarova Chemistry Department, Lomonosov Moscow State University, Moscow, 119899, Russia      *(eid@enzyme.chem.msu.ru) Inactivation of recombinant Luciola mingrelica firefly luciferase under the action of guanidinium chloride and reactivation of the enzyme after the removal of the denaturing agent were studied by measuring the enzyme activity and spectra of tryptophan fluorescence. Monomer-dimer interactions were shown to play a major role in the inactivation. The proposed kinetic scheme for the inactivation involves: (1) lag-period without loss of luciferase activity due to small conformational changes in the active dimers; (2) inactivation of the enzyme due to reversible dissociation of dimers into inactive monomers; (3) irreversible inactivation of monomers. The steps (2) and (3) are similar to the kinetic steps of thermoinactivation of the luciferase at elevated temperatures. The kinetic constants and equilibrium constants of the dissociation of dimers into monomers at different concentrations of guanidinium chloride were calculated. The removal of guanidinium chloride resulted in total or partial restoration of the luciferase activity. The reactivation follows a second-order reaction kinetics and is proposed to involve association of inactive monomers to the active dimers. [Poster: dementie.ekater.22143]

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