Speciation of mercury in salmon egg cell cytoplasm in relation with metallomics research.
Speciation of mercury in salmon egg cell cytoplasm in relation with metallomics research.
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DOI：
10.1016/j.talanta.2005.09.038
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发表时间：
2005-12
期刊：
影响因子：
6.1
通讯作者：
Takuya Hasegawa;Motoki Asano;Kohei Takatani;Hirotaka Matsuura;T. Umemura;H. Haraguchi
中科院分区：
文献类型：
--
作者：
Takuya Hasegawa;Motoki Asano;Kohei Takatani;Hirotaka Matsuura;T. Umemura;H. Haraguchi
采用表面活性剂介导的高效液相色谱/电感耦合等离子体质谱法（HPLC/ICP-MS）对鲑鱼卵细胞细胞质中汞的形态进行了研究，其中ODS（十八烷基硅）柱包被胆汁酸衍生物CHAPS（3-[（3-胆酰胺丙基）-二甲胺]-1-丙烷磺酸）进行了物种分离。在物种形成分析之前，用ICP-MS在流动注射模式下以m/ z202测定细胞质中的总汞。为了精确测量，将鲑鱼卵细胞细胞质用0.1M Tris（Tris（羟甲基）氨基甲烷）- hno3缓冲液稀释5倍，采用标准添加法。因此，在湿重基础上，细胞质中汞的总浓度估计为12.4ngg−1。然后，用0.1M tris - hno3缓冲液稀释5倍的细胞质，采用表面活性剂介导的高效液相色谱（HPLC）分析，采用紫外吸收检测器和ICP-MS双检测系统。在这些色谱图中，主要观察到与某些蛋白质和小分子对应的两个峰。用0.01M Tris缓冲液或纯水将鲑鱼卵细胞细胞质稀释5倍后，出现一些沉淀物可能是由于细胞质中疏水蛋白的沉淀。经膜过滤去除沉淀后，滤液经表面活性剂介导的HPLC/UV/ICP-MS分析。结果表明，在紫外吸收检测和Hg- s特异性ICP-MS检测的色谱图中，在停留时间接近4.0min时（对应于低分子量区），可以清晰地观察到Hg的小分子种的峰。通过半胱氨酸加标实验，确定与Hg结合的小分子为半胱氨酸。此外，利用chaps包被的ODS柱获得的色谱上的蛋白质部分进一步用SEC（粒径排除层析）进行分析。结果表明，在紫外吸收、汞和硒的所有检测中均出现了分子量为300、50和12kDa的蛋白峰，而在s的检测中出现了两个重合峰。这表明，汞在鲑鱼卵细胞细胞质中与蛋白质中含有硒代半胱氨酸和/或半胱氨酸残基的蛋白质结合。
Speciation of mercury in salmon egg cell cytoplasm was investigated by surfactant-mediated high-performance liquid chromatography/inductively coupled plasma mass spectrometry (HPLC/ICP-MS), where an ODS (octadecylsilica) column coated with a bile acid derivative, CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate), was used for species separation. Prior to the speciation analysis, total Hg in the cell cytoplasm was determined by ICP-MS at m/z 202 in a flow injection mode. For the precise measurement, salmon egg cell cytoplasm was diluted five-fold with 0.1M Tris (Tris(hydroxymethyl)aminomethane)–HNO3buffer solution, and the standard addition method was employed. Thus, the total concentration of Hg in cell cytoplasm was estimated to be 12.4ngg−1on the wet weight basis. Next, the cell cytoplasm diluted five-fold with 0.1M Tris–HNO3buffer solution was analyzed by surfactant-mediated HPLC with the dual detection system of a UV absorption detector and an ICP-MS instrument. Two peaks corresponding to some proteins and small molecules were mainly observed in those chromatograms. When salmon egg cell cytoplasm was diluted five-fold with 0.01M Tris buffer solution or pure water, some precipitates appeared probably because of precipitation of hydrophobic proteins in cytoplasm. After the precipitates were eliminated with a membrane filter, the filtrate was subjected to the analysis by surfactant-mediated HPLC/UV/ICP-MS. As a result, the peaks for small molecular species of Hg were clearly observed at the retention time near 4.0min (corresponding to low-molecular weight zone) in the chromatograms with UV absorption detection as well as with Hg- and S-specific ICP-MS detections. The small molecule bound with Hg was identified as cysteine through the cysteine-spiked experiment. In addition, the protein fraction on the chromatogram obtained by using the CHAPS-coated ODS column was further analyzed by SEC (size exclusion chromatography). Consequently, several protein peaks with molecular weight of 300, 50 and 12kDa were observed in all the detections of UV absorption, Hg and Se, although two peaks among them were coincident in the case of S. These results indicate that Hg in salmon egg cell cytoplasm binds with proteins containing selenocysteine and/or cysteine residues in proteins.