Activity and fluorescence changes of lactate dehydrogenase induced by guanidine hydrochloride in reverse micelles

Denaturants activate several multimeric enzymes in reverse micelles [Garza-Ramos, G., Darszon, A., Tuena de Gómez-Puyou, M. & Gómez-Puyou, A. (1992) Eur. J. Biochem. 205, 509-517]. Here, the effect on activity and intrinsic fluorescence of pig heart lactate dehydrogenase (LDH) in reverse micelles [formed with 0.2 M cetyltrimethylammonium bromide in octane/hexanol (8.6:1, by vol.)] was explored at various water and guanidine hydrochloride (Gdn/HCl) concentrations. Emission fluorescence spectra of LDH in aqueous media and in micelles were similar. As in all aqueous media, 1.0 M Gdn/HCl in the water phase of reverse micelles produced fluorescence quenching and a blue shift of the maximal emission. In 5.0 M Gdn/HCl, instead of the red shift and significant quenching seen in water, the maximum emission further shifted to the blue and was only slightly quenched. Gdn/HCl titrations of activity and fluorescence changes of LDH in micelles with different water contents showed that at Wo ([H2O]/[surfactant]) of 6.6, 8.3, or 12.5, increasing concentrations of Gdn/HCl up to 0.6 M produced small changes in fluorescence, whereas activity increased several-fold. At higher denaturant concentrations, activity decreased with significant fluorescence changes. In reverse micelles with 1 M Gdn/HCl, Vmax but not Km of LDH decreased with time. Under these conditions, there was progressive quenching of LDH fluorescence. The results show that in reverse micelles different Gdn/HCl concentrations induce variations in activity with or without alterations of the intrinsic fluorescence of LDH. The results also indicate that in reverse micelles, concentrations of Gdn/HCl below 1.0 M cause an enhancement of protein flexibility; this is accompanied by a marked increase in activity without important changes in intrinsic fluorescence. 1.0 M Gdn/HCl produces perturbations of inter-subunit contacts that lead to fluorescence quenching and loss of catalytic activity, probably as consequence of dimerization of tetrameric LDH.