Voltammetric Investigation of Zinc Release from Metallothioneins Modulated by the Glutathione Redox Couple and Separated with a Porous Membrane

Glutathione (GSH), in addition to serving as a redox buffer in cellular environment, has been suggested as a modulator in metal regulation and homeostasis by metallothioneins (MTs). The interactions of MTs with both GSH and its oxidized form GSSG have been shown to govern the direction of metal transfer. Common methods for the determination of zinc release from MTs modulated by GSH/GSSG either involve radioactive species or enzymes or are labor‐intensive. In this study, upon separation of Zn²⁺ from the reaction mixture of MTs and GSH with a centrifugal filter membrane, differential pulse voltammetry (DPV) was used for the Zn²⁺ quantification. The same approach is extended to the studies of metal transfer between Zn₇MT with a GSH/GSSG mixture and that between Zn₇MT with GSSG. The concomitant conversion between the free thiol and disulfide bonds was confirmed with UV‐vis spectrophotometry. The results demonstrate that GSSG, GSH, and the GSH/GSSG mixture all modulate zinc release from Zn₇MT. The percentage of zinc release increases in the order of GSH, GSSG, and the GSH/GSSG mixture. The new approach is demonstrated to be well suited for investigation of redox regulation of MT and its reaction with zinc‐containing enzymes.