Behaviour in different denaturant agents and structural characterization by fluorescence techniques of Haloferax mediterranei D-2-hydroxyacid dehydrogenase

Abstract The halophilic enzyme D-2-hydroxyacid dehydrogenase (D2-HDH) from Haloferax mediterranei is found to be an oligomeric enzyme composed of two identical subunits. Fluorescence spectra of native and denatured protein and effect of denaturants such as urea and guanidine hydrochloride on enzyme activity of halophilic D-2-hydroxyacid dehydrogenase have been analysed. Native D2-HDH shows the maximum emission at 340 nm. The denaturation process caused an exposure to the solvent of the tryptophan residues, as manifested by the red shift of the emission maximum from 340 to 350 nm. When urea was used as denaturant agent the enzyme required long incubation times, higher to 24 h, to unfold. Fluorescence quenching by KI and acrylamide was also carried out; showing that the tryptophan residues are mainly located near the enzyme surface.