Determination of metallothionein by high-performance liquid chromatography with fluorescence detection using an isocratic solvent system.
Anal Biochem
; 258(2): 168-75, 1998 May 01.
Article
em En
| MEDLINE
| ID: mdl-9570826
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.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Cromatografia em Gel
/
Cromatografia Líquida de Alta Pressão
/
Metalotioneína
Tipo de estudo:
Diagnostic_studies
/
Evaluation_studies
Limite:
Animals
/
Humans
/
Male
Idioma:
En
Revista:
Anal Biochem
Ano de publicação:
1998
Tipo de documento:
Article