EVALUATION OF THE CD HEMOGLOBIN AFFINITY ASSAY FOR THE RAPID-DETERMINATION OF METALLOTHIONEIN IN BIOLOGICAL TISSUES
EVALUATION OF THE CD HEMOGLOBIN AFFINITY ASSAY FOR THE RAPID-DETERMINATION OF METALLOTHIONEIN IN BIOLOGICAL TISSUES
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DOI：
10.1016/0041-008x(82)90068-0
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发表时间：
1982-01-01
影响因子：
3.8
通讯作者：
TOAL, BF
中科院分区：
文献类型：
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作者：
EATON, DL;TOAL, BF
由S.Onosaka等人描述的CD/Hb亲和分析的修改。用于定量组织中金属硫蛋白(MT)水平的简便性、敏感性和特异性。五步法包括制备热变性组织匀浆上清液，将上清液与1微克/毫升的109Cd溶液孵育，加入2%的牛Hb溶液，Hb热沉淀，分析上清液中残留的Cd。通过凝胶渗透和离子交换层析纯化MT，并对每个组分进行Cd结合电位分析后，Cd仅在MT特征洗脱的样品中保留在上清液中。该方法可检测到40 ng MT，相当于组织中的浓度为0.8微克MT/g，重复性测定的相对标准偏差为平均值的4%。用氯化镉处理大鼠，证明了该方法能够检测到镉处理后不同组织中MT组织含量的大小变化。虽然认识到了几个潜在的误差来源，但这种分析的简便性和敏感性应该为估计大多数生物组织中的MT含量提供了一种有用的手段，并且对于时间和费用是主要考虑因素的大规模研究尤其有用。
A modification of the Cd/Hb affinity assay described by S. Onosaka, et al. was evaluated for its ease, sensitivity and specificity for quantifying tissue levels of metallothionein (MT). The 5 step method involved preparation of the heat-denatured tissue homogenate supernatant fraction, incubation of the supernatant fraction with 1 .mu.g/ml 109Cd solution, addition of 2% bovine Hb solution, heat precipitation of Hb and analysis of the supernatant fraction for Cd remaining in the fraction. After purification of MT by gel permeation and ion-exchange chromatography and analysis of each fraction for Cd-binding potential, Cd remained in the supernatant fraction only in those samples where MT characteristically eluted. The assay was capable of detecting 40 ng MT, equivalent to a tissue concentration of 0.8 .mu.g MT/g. The relative SD for replicate assays was < 4% of the mean. Treatment of rats with CdCl2 demonstrated the ability of this assay to detect large and small changes in tissue content of MT after Cd treatment in a variety of tissues. Although several potential sources of error were recognized, the ease and sensitivity of this assay should provide a useful means for estimating MT content in most biological tissues and should be particularly useful for large-scale studies where time and expense are major considerations.