Spectroscopic characterization of thioredoxin covalently modified with monofunctional organoarsenical reagents

Abstract

Thioredoxin upon reduction with mercaptoethylamine was subjected to covalent modification by the monofunctional organoarsenical reagents H2NPhAsO and HO(CH2)4AsCl2. The degree of modification was monitored by the percentage loss in free thiol content as measured by the reaction with the thiol reagent 5,5'-dithiobis(2-nitrobenzoic acid). The modification results in the formation of a stable 15-membered cyclic dithioarsenite ring that readily extrudes the arsenic moiety upon the addition of 2,3-dimercaptopropanol. The conformational effects of this modification were monitored by steady-state fluorescence and circular dichroism. On the basis of circular dichroic spectra, it appeared that the protein experiences no significant backbone conformational change from this modification. The degree of conformational change was found to be within the range observed upon reduction of the oxidized thioredoxin. Steady-state fluorescence revealed that the arsenicals caused strong quenching of the tryptophan fluorescence. Stern-Volmer titrations revealed that the quenching was a function of both the nature of the organic group and its covalent attachment to the "spatially close" thiols. The analysis of the spectroscopic results obtained with the arsenical reagents provides further insight into the nature of the conformational change that has been observed upon reduction of thioredoxin.