杂色鲍血蓝蛋白的分离纯化及其酚氧化酶活性研究

Purification and phenoloxidase (PO) activity of hemocyanin in Haliotis diversicolor Reeve

利用超速离心和CsCl梯度离心的方法,从杂色鲍(Haliotis diversicolor Reeve)血淋巴中分离纯化血蓝蛋白(Hcs),SDS-PAGE检测提取的血蓝蛋白由1种亚基构成,分子量为400kD;Native-PAGE电泳检测2个亚基(Isoform)分别为HdH1、HdH2;透射电镜下其四级结构为圆柱体,以多十聚体、二十聚体和少量的十聚体形式存在于血淋巴中。杂色鲍血蓝蛋白在以邻苯二酚为底物的条件下表现出微弱的酚氧化酶活性(Km=25.66mmol/L),胰蛋白酶和SDS能使其酚氧化酶活性增强;以左旋多巴(L-DOPA)为底物则不表现出酚氧化酶活性。

Hemocyanins （Hcs） from haemolymph of Haliotis diversicolor Reeve were purified by ultracentrifugation and CsCl gradient centrifugation. SDS-PAGE shows that Hc was composed of uniform subunits with the molecular weight about 400 kD, and the native Hcs contains two isforms of HdH1 and HdH2, which was identified by Native-PAGE. Structure of Hc was cylinder under electron microscope by negative staining, in various forms of multidecamers, didecamers and a few decamers dissolved in haemolymph. Hcs of Haliotis diversicolor Reeve showed weak phenoloxidase activity with catechol as substrate （Km=25.66）. Trypsinase and SDS could increase this activity. However, Hcs didn＇ t show phenoloxidase activity with L-DOPA as substrate.