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  • Determination of metallothionein by high-performance liquid chromatography with fluorescence detection using an isocratic solvent system.

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

Analytical biochemistry (1998-05-08)
S Miyairi, S Shibata, A Naganuma
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Supelco
7-Fluorobenzofurazan-4-sulfonic acid ammonium salt, for HPLC derivatization, LiChropur, ≥98.5% (HPLC)
Sigma-Aldrich
7-Fluorobenzofurazan-4-sulfonic acid ammonium salt, ≥98%