Accurate determination of the thiol-to-metal ratio in metalloproteins by on-line combination of UV-visspectrophotometry with electrochemistry

Abstract

Determination of the thiol-to-metal ratio in proteins is of great biological relevance. Common methods for the characterization of thiol-containing metalloproteins are applicable to either sulfhydryl groups or metal ions, but not suitable for the simultaneous determination of both. In this study, UV-vis spectrophotometry has been combined in tandem with voltammetry for accurate determination of the thiol-to-metal ratio in metalloproteins. A protein sample and the electrophilic disulphide DTDP (4, 4′-dithiodipyridine) were introduced to the UV-vis detector for spectrophotometric thiol assay and then delivered into the electrochemical flow cell for metal ion determination. Zn₇-metallothionein (Zn₇MT) was used as a model system due to the specific thiol-to-Zn ratio. The feasibility of the method for analysis of formamidopyrimidine-DNA glycosylase (Fpg) containing a zinc-finger consensus sequence was also demonstrated. With this coupled technique, uncertainties that cannot be addressed by the individual techniques are largely circumvented. Such a hyphenated system is amenable to the accurate and convenient determination of both sulfhydryl groups and metal ions in metalloproteins and should be useful for physiological studies.