Alhama José, Romero-Ruiz Antonio, López-Barea Juan
Department of Biochemistry and Molecular Biology, University of Córdoba, Severo Ochoa Building, 2nd floor, Campus de Rabanales, Highway A-4, Km 396a, 14071 Córdoba, Spain.
J Chromatogr A. 2006 Feb 24;1107(1-2):52-8. doi: 10.1016/j.chroma.2005.11.057. Epub 2005 Dec 15.
In this paper, we describe a highly specific, sensitive and reliable method for total metallothionein (MT) quantification by RP-HPLC coupled to fluorescence detection following reaction with monobromobimane of thiols from metal-depleted MT after heat-denaturation of extracts in the presence of sodium dodecyl sulphate (SDS). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the identity of the peak resolved (t(R)=16.44) with MT: a highly fluorescent protein of approximately 8.3 kDa, in agreement with the high thiol content and low MT size. Other heat-resistant and Cys-containing proteins of 35 kDa were efficiently separated. The new method was successfully used to quantify MT content in digestive gland of clams from southern Spanish coastal sites with different metal levels, and is proposed as a tool for using MTs as biomarker in monitoring programmes.
在本文中，我们描述了一种高度特异、灵敏且可靠的方法，用于通过反相高效液相色谱（RP-HPLC）结合荧光检测对总金属硫蛋白（MT）进行定量。该方法是在十二烷基硫酸钠（SDS）存在下对提取物进行热变性后，使金属耗尽的MT中的硫醇与单溴联苯胺反应。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）证实了保留时间为16.44的洗脱峰与MT的一致性：MT是一种约8.3 kDa的高荧光蛋白，这与高硫醇含量和较小的MT尺寸相符。其他35 kDa的耐热且含半胱氨酸的蛋白质也被有效分离。该新方法成功用于对西班牙南部沿海不同金属水平地区蛤类消化腺中的MT含量进行定量，并被提议作为一种工具，用于将MT用作监测计划中的生物标志物。
