Quantification of oxidized metallothionein in biological material by a Cd saturation method

A rapid and easy to perform method for determining oxidized metallothionein (MT) is described. The main features of the procedure are that oxidized MT is converted into native MT with 2-mercaptoethanol as reducing agent and Zn2+ as metal donor, and MT is subsequently quantified via Cd saturation. The procedure was effective in recovering MT oxidized either by Cu2+ or by neutralization of apothionein at pH 3, independent of the amount of MT, the origin of sample, degree of oxidation, and oxidation method. As demonstrated by HPLC analysis, the method is highly specific for MT. The described procedure provides information on both the total concentration of MT independent of its thiol redox state and the metal binding capacity of the protein; thus, it is of particular interest in studying the role of MT in metal metabolism and toxicity.