Determination of metallothionein by high-performance liquid chromatography with fluorescence detection using an...

Metallothionein (MT) is a low-molecular-weight protein which plays a role in detoxification of heavy metals and protection against oxidative stress. A sensitive and convenient determination method for MT is necessary to clarify its physiological roles. High-performance liquid chromatography (HPLC) with an isocratic solvent system for MT was, therefore, developed utilizing an unique fluorescence labeling reagent, ammonium 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonate (SBD-F). The HPLC system with two separation columns, Shodex RSpak RP18-413 column (a styrene divinylbenzene polymer gel-packed column) and Puresil C18 column (an ODS gel-packed column), connected in tandem successfully separated SBD-labeled MT from biological interference. The SBD-labeled MT was stable and could be stored for at least 1 week without any changes in fluorescence intensity. Although Hg-MT was not detectable, this method is applicable to determination of major MTs such as Zn-MT, Cd-MT, and Cu-MT using commercially available rabbit MT as a standard. Determination of the MT concentration in cells was possible in aliquots of only 1 x 10(4) cultured cells. The present method using a tandem column HPLC system with isocratic elution might be useful for monitoring the concentration of MT in cultured cells as well as in animal tissues.