Photoactive hemolytic toxin in Chattonella marina

轮藻属鞭毛虫Chattonella marina(C.marina)是一种赤潮的致病生物，对鱼类有很强的毒性。在本研究中，我们发现从该鞭毛虫制备的无细胞甲醇提取物对兔红细胞具有很强的溶血活性。有趣的是，提取物的溶血活性完全依赖于光，在黑暗中即使在很高的浓度下也没有检测到溶血活性。甲醇提取物在Sephadex LH-20柱上的凝胶过滤层析表明，提取物含有血凝素和溶血剂，负责这些活性的物质被分别洗脱。这些结果表明，其血凝和溶血活性来源于不同的化合物。凝胶过滤后得到的溶血组分(F4)对滨海梭子藻本身和其他种类的浮游植物的生长有明显的抑制作用。此外，F4对包括人肿瘤细胞(HeLa细胞)在内的多种哺乳动物细胞株均表现出较强的细胞毒作用，且呈剂量依赖关系。细胞毒性也是光依赖的，在黑暗中测试的任何细胞系都没有显示出细胞毒性作用。通过制备性薄层层析和随后的高效液相色谱进一步纯化，得到一种主要的溶血剂，其形式为高纯度。由于赤潮异弯藻、黄盾盘藻和日本纤毛虫等鞭毛虫的甲醇提取物对兔红细胞具有光依赖性溶血活性，因此推测这些鞭毛虫中普遍存在光依赖性溶血剂。

Chattonella marina (C.marina), a raphidophycean flagellate, is a causative organism of red tide, and highly toxic to fish. In this study, we found that the cell-free methanol extract prepared from this flagellate exhibited potent hemolytic activity against rabbit erythrocytes. Interestingly, the hemolytic activity of the extract was absolutely light-dependent, and no hemolytic activity was detected in the dark even at very high concentration. Gel filtration chromatography of the methanol extract on a column of Sephadex LH-20 revealed that the extract contained hemagglutinin as well as hemolytic agents, and the substances responsible for these activities were separately eluted. These results suggest that the hemagglutinating and hemolytic activities were derived from distinct compounds. The hemolytic fraction obtained after gel filtration (F4) caused marked inhibition of the growth of C.marina itself and other species of phytoplanktons. Furthermore, F4 showed a potent cytotoxicity toward various mammalian cultured cell lines including human tumor cells (HeLa cells) in a dose-dependent manner. The cytotoxicity was also light-dependent, and no cytotoxic effect was exhibited in any cell lines tested in the dark. After further purification procedures via preparative thin-layer chromatography and subsequent HPLC, a major hemolytic agent was obtained as highly purified form. Since the methanol extracts prepared from other raphidophycean flagellates such as Heterosigma akashiwo, Olisthodiscus luteus, and Fibrocapsa japonica showed light-dependent hemolytic activity toward rabbit erythrocytes, it was suggested that the light-dependent hemolytic agents commonly exist at least in these raphidophycean flagellates.