Tetrodotoxin-binding protein in the haemolymph of shore crab Hemigrapsus sanguineus

本文报道了一种海岸（Hemigraphsus sanguineus）对河豚毒素（Tetrodotoxin，TTX）具有高抗性的血淋巴，其血淋巴中含有分子量大于2000 k和400 k的TTX结合蛋白。通过使用500 k mol.wt.截留滤膜的超滤、在ConA-Sepharose柱上的凝集素亲和层析和在TSKgel G3000柱上的凝胶过滤HPLC来纯化TTX结合蛋白（mol.wt.为400 k）之一，监测对TTX对小鼠的致死活性的中和作用。由此获得的蛋白质在非变性PAGE中仅给出一条带，证实其同质性。SDS-聚丙烯酰胺凝胶电泳测得TTX结合蛋白的分子量在非还原条件下约为82 k，在还原条件下约为72 k和82 k，表明TTX结合蛋白的分子量与TTX结合蛋白的分子量有关。 ...更多信息 TTX结合蛋白至少由两个亚基组成，对TTX无中和作用，为酸性糖蛋白，pI 3.5，富含Asp和Glu，不含Trp，含还原糖6%，氨基糖12%，选择性结合TTX，中和能力为6.7 mouse unit/mg protein。然而，它的中和活性几乎失去了治疗与酶（胰蛋白酶，蛋白酶XIV，淀粉酶和淀粉葡糖苷酶）和化学洗涤剂（1% SDS，1%二硫苏糖醇，8 M尿素和6 M盐酸胍），这表明参与蛋白质和糖部分的结合TTX和TTX结合蛋白的空间构象的重要性。此外，TTX结合蛋白与金属离子（Cu^<2+>、Cr^<6+>和Zn^<2+>）的预孵育显著抑制了中和活性。这是可能的TTX结合蛋白的功能作为一种金属载体蛋白在血淋巴中的无毒海岸蟹血红血。少

A shore crab Hemigrapsus sanguineus is reported to be resistant to tetrodotoxin (TTX) administrated intrahemocoelically, although it contains no detectable amount of TTX.This research project was carried out to clarify the mechanism of high resistance of the crab H.sanguineus to TTX.Analysis by gel filtration on Sepharose 6B revealed that the haemolymph contained TTX-binding proteins with mol.wt.of >2000k and 4OOk. One of the TTX-binding proteins (mol.wt.of 40Ok) was purified, monitoring the neutralizing effect against the lethal activity of TTX to mice, by ultrafiltration using a 500k mol.wt.cut-off filter membrane, lectin affinity chromatography on a ConA-Sepharose column, and gel filtration HPLC on a TSKgel G3000 column. The protein thus obtained gave only one band in native-PAGE, confirming its homogeneity. The mol.wt.of the TTX-binding protein was estimated to be about 82k under non-reducing conditions and about 72k and 82k under reducing conditions by SDS-PAGE, indicating that th … More e TTX-binding protein was composed of at least two subunits which appeared to have no neutralizing effect to TTX.The TTX-binding protein was an acidic gycoprotein with pI 3.5, abundant in Asp and Glu but absent in Trp, and contained 6% reducing sugar and 12% of amino-sugar.The TTX-binding protein selectively bound to TTX, with a neutralizing ability to TTX of 6.7 mouse unit/mg protein. However, its neutralizing activity was almost lost by treatments with enzymes (trypsin, protease XIV, amylase and amyloglucosidase) and chemical detergents (1% SDS, 1% dithiothreitol, 8M urea and 6M guanidine hydrochloride), suggesting the involvement of both proteineous and sugar moieties in the binding to TTX and the importance of the steric conformation of the TTX-binding protein. Moreover, the neutralizing activity was significantly inhibited by pre-incubation of the TTX-binding protein with metal ions (Cu^<2+>, Cr^<6+> and Zn^<2+>). It is possible that the TTX-binding protein functions as a metal carrier protein in the haemolymph of the non-toxic shore crab H.sanguineus. Less