Determination of metallothionein by high-performance liquid chromatography with fluorescence detection using an isocratic solvent system.
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.
Miyairi
S
S
Department of Molecular and Biochemical Toxicology, Faculty of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.
Shibata
S
S
Naganuma
A
A
D000818:Animals; D002850:Chromatography, Gel / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005069:Evaluation Studies as Topic; D005456:Fluorescent Dyes; D005464:Fluorobenzenes; D006367:HeLa Cells; D006801:Humans; D008297:Male; D008668:Metallothionein / Q000032:analysis*; D051379:Mice; D008813:Mice, Inbred ICR; D011817:Rabbits; D051381:Rats; D015203:Reproducibility of Results; D012997:Solvents; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D005464:Fluorobenzenes; D012997:Solvents; C037802:7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate; D008668:Metallothionein