小球藻经60 μmol/L的锌胁迫培养5天后,离心收集藻泥,经超声波细胞破碎后,离心得到上清液即蛋白质粗提液.粗提液经葡聚糖凝胶层析柱G-75及脱盐柱G-25分离、纯化后,收集的蛋白组分经真空冷冻干燥后获得小球藻锌结合蛋白.实验测定了在不同pH值下小球藻锌结合蛋白的紫外光谱特征,发现该蛋白质的紫外光谱特征与兔肝金属硫蛋白(Zn-MTs)十分相似.经阴离子交换层析柱(DEAE Sephadex A-25)及反相液相色谱分离表明,小球藻锌结合蛋白的色谱行为与兔肝Zn-MTs有一定的相似性.此外,经Tricine-SDS-PAGE法测定该蛋白质的分子量约为8.2 kDa;蛋白质分子中半胱氨酸含量达15%以上.由此可见,小球藻在锌胁迫下可诱导生成与兔肝Zn-MTs相似的锌结合蛋白质,即类金属硫蛋白(Zn-MT-like).每克鲜藻可诱导Zn-MT-like蛋白达0.2 g以上.%C. vulgaris was cultivated for five days under the stress of 60 μmol/L of Zn (ZnCl). After harvest by centrifugation, the algal cells were washed with EDTA and water, and were homogenized by a supersonic cell dis-integrator. After centrifugation, the supernatant, a crude protein-containing extract, was separated and purified with a gel filtration column (Sephadex G-75, 3.5 i.d. ×80 cm) and a desahing gel filtration column (Sephadex G-25, 1.5 i.d. ×30 cm), respectively. The purified Zn-binding protein was characterized by the methods of UV spectrum and the different chromatograms. In addition, the molecular weight of the purified Zn-binding protein measured with Tricine-SDS-PAGE was about 8.2 ku. The content of cysteine in the protein was more than 15%. Results indicated that the Zn-binding proteins from C. vulgaris were the analogues of Zn-binding metallothioneins, and were referred as Zn-MT-like. In addition, 0.2% (w/w) of purified Zn-MT-like could be obtained from fresh alga.
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