Miyairi S, Shibata S, Naganuma A
Department of Molecular and Biochemical Toxicology, Faculty of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.
Anal Biochem. 1998 May 1;258(2):168-75. doi: 10.1006/abio.1998.2578.
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.
金属硫蛋白（MT）是一种低分子量蛋白质，在重金属解毒和抗氧化应激保护中发挥作用。为阐明其生理作用，需要一种灵敏且便捷的MT测定方法。因此，利用独特的荧光标记试剂7-氟苯并-2-恶唑-1,3-二氮杂茂-4-磺酸盐（SBD-F）开发了一种用于MT的等度溶剂系统高效液相色谱（HPLC）方法。带有两个串联分离柱的HPLC系统，即昭和电工RSpak RP18 - 413柱（苯乙烯二乙烯基苯聚合物凝胶填充柱）和Puresil C18柱（ODS凝胶填充柱），成功地将SBD标记的MT与生物干扰物分离。SBD标记的MT很稳定，可保存至少1周，荧光强度无任何变化。虽然无法检测到汞结合的MT，但该方法适用于以市售兔MT为标准测定主要的MT，如锌结合的MT、镉结合的MT和铜结合的MT。仅用1×10⁴个培养细胞的等分试样就可以测定细胞中的MT浓度。目前这种使用串联柱HPLC系统和等度洗脱的方法可能有助于监测培养细胞以及动物组织中MT的浓度。
