海藻中类金属硫蛋白的分离表征及对Zn,Cu的诱导响应

Separation and Characterization of Metallothionein-like Protein in Algae and Its Response to Zn and Cu Induced

以海带为试验载体,利用葡聚糖凝胶柱,建立了海藻中类金属硫蛋白(MT-like)的分离、提取纯化技术;采用紫外分光光度法比对类金属硫蛋白与兔肝Zn-MTs的紫外吸收,并用凝胶电泳法对其分子量范围进行表征,同时利用质谱法精确测定其分子量;并在ZnCl2,CuCl2,ZnCl2+CuCl2不同条件下胁迫培养藻体,考察了海带对重金属的富集能力,探索藻体中MT-like对Zn及Cu元素的诱导响应。结果显示,MT-like与兔肝Zn-MTs在294 nm处有相似的紫外吸收峰,在SDS-PAGE凝胶电泳图上显像类似,分子量范围相近,经电喷雾离子源质谱测定该蛋白质的分子量约为6.5 ku。另外,海带对重金属的富集能力较强,对Zn元素的富集能力明显高于Cu元素;Zn2+对藻体MT-like的产生有促进作用,而MT-like随着Cu2+诱导时间的增长先增加后降低,且Cu2+对Zn2+诱导存在拮抗效应。

With the kelp as a test vector,a new method of seperation and purification of metallothionein-like protein（ MT-like） in algae was developed by using sephadex column. Then the MT-like protein and the Zn-MTs standard were measured by UV spectrophotometry. The molecular weight of MT-like protein were characterized by gel electrophoresis and accurately analysed by mass spectrometry. The enrichment ability of heavy metals was studied,and the response of MT-like to Zn and Cu was investigated after the kelp was cultured with ZnCl2,CuCl2,ZnCl2＋ CuCl2. The results showed that the MT-like and Zn-MTs had a similar UV absorption peak at 294 nm and displayed the similar range in SDS-PAGE gel electrophoresis. The molecular weight of the MT-like determined by electrospray ionization mass spectrometry was about 6. 5 ku. The kelp showed a strong enrichment ability for metals,in which the enrichment ability for Zn was stronger than that for Cu. The Zn2 ＋could promote the MT-like production,while the growth of MT-like induced with Cu2 ＋increased at first,and then decreased. The Cu2 ＋had an antagonistic effect on Zn2 ＋inducing process.