菲律宾蛤仔金属硫蛋白的提取方法优化

Extraction Optimization of Metallothionein from Ruditapes philippinarum

为了利用双壳类动物金属硫蛋白(MTs)指示海洋重金属污染,有必要确定有效提取这种生物标志物的方法。本研究以暴露于含镉海水的菲律宾蛤仔内脏为试验材料,依次采用正交设计和单因素设计对内脏MT(以粗提液中的巯基含量表示)的提取工艺条件进行优化。所得到的最佳匀浆条件为:Tris-HCl缓冲液浓度为0.02 mol/L,pH值7.8,内脏与缓冲液的质量体积比(m/v)为1/5,其中,缓冲液的浓度对提取效果影响较大。最佳除杂条件为:匀浆液首先在4℃、20 000 g离心30 min,再于80℃水浴加热10 min,然后于4℃15 000 g离心10 min。这些结果有助于重金属生物监测中蛤仔MT的经济高效提取和共存杂蛋白干扰的有效去除。

In order to use metallothioneins（MTs）from bivalve to indicate marine heavy metals pollution, it is necessary to ascertain an effective extraction method of this biomarker. In this study, viscus in Ruditapes philippinarum exposed to seawater containing cadmium was used as a material, and extraction conditions for maximum MT recovery from viscus, expressed as content of sulfhydryl group （-SH） in the crude extract, were optimized by orthogonal design and single-factor design. The optimized homogenization process was as follows： the buffer concentration was 0. 02 mol/ L, pH7. 8 and m/v was 1/5. MT yield was primarily affected by Tris-HCl buffer concentration. The optimal process obtained for removing impurity were that the homogenate was centrifuged at 20 000 g at 4 ℃for 30 min, and then the collected supernatant was heated at 80 ℃ for 10 min, followed by a second centrifugation at 15 000 g at 4 ℃ for 10 min. These results help the rapid and economic extraction of MT for monitoring heavy metals and the effective elimination of co-proteins interference.