毛蚶血红蛋白的分离纯化及其类酚氧化酶活性研究.docVIP

  毛蚶血红蛋白的分离纯化及其类酚氧化酶 活性研究# 5 10 15 20 25 30 35 40  摘要：为了建立毛蚶血红蛋白（Hb）的分离纯化方法并研究其类酚氧化酶（phenoloxidase， PO）活性，本文利用凝胶过滤柱层析和离心超滤技术对毛蚶 Hb 进行了纯化，通过 SDS、基质辅助激光解析串联飞行时间质谱等方法对其进行了鉴定，再以 L-DOPA 为 特异性底物对其类 PO 活性以及常用激活剂的激活作用进行了研究。实验结果显示，利用上 述方法能够得到纯度很高的两种毛蚶 Hb 样品——HbI 和 HbII，HbII 分子由分子量为 28.90 kDa 和 30.28 kDa 的两个亚基组成，HbI 分子仅为分子量 28.97 kDa 的单一亚基；HbI 和 HbII 两种分子对底物 L-DOPA、儿茶酚和对苯二酚均具有类 PO 活性，且其类 PO 活性均可为异 丙醇所激活。 关键词：毛蚶；血红蛋白；纯化；类酚氧化酶活性；L-DOPA 中图分类号：Q55 Studies on purification of hemoglobin from blood clam Scapharca kagoshimensis and its phenoloxidase-like activity characterization XU Bin1, JING Zhao1,2, ZHANG Yanan1,3, FAN Tingjun1 (1. Department of Marine Biology, College of Marine Life Sciences, Ocean University of China, ShanDong QingDao 266003; 2. Department of Histology and Embryology, Qingdao University Medical College, ShanDong QingDao 266021; 3. Department of Biology, Qingdao University Medical College, ShanDong QingDao 266021) Abstract: To identify the phenolxoidase (PO)-like activity of blood clam hemoglobin (Hb), Hb were prepared and purified from the haemolymph of Scapharca kagoshimensis by sephacryl S-100 gel-filtration chromatography and centrifugal ultra-filtrating techniques. After characterized by SDSand MALDI-TOF mass sectrometry, the PO-like activity and its activation of the purified Hb was investigated by using L-DOPA as a specific substrate. The results showed the Scapharca Hb, including molecules of HbI and HbII, was efficiently purified. HbII consists of two subunits with molecular weight of 28.90 kDa and 30.28 kDa respectively, while HbI consists of only one subunit with a molecular weight of 28.97 kDa. Both HbI and HbII had PO-like activity on L-DOPA, catechol and hydroquinone, and the PO-like activity could be further activated by isopropanol. Therefore, it can be concluded that Scapharca Hb have PO-like activity which might play important roles in non-specific immunity. This is the first report of the PO-like activity of Scapharca Hb which is of great