Andrião-Escarso S H, Soares A M, Rodrigues V M, Angulo Y, Díaz C, Lomonte B, Gutiérrez J M, Giglio J R
Departamento de Bioquímica, Faculdade de Medicina, Universidade de São Paulo, 14049-900, SP, Ribeirão Preto, Brazil.
Biochimie. 2000 Aug;82(8):755-63. doi: 10.1016/s0300-9084(00)01150-0.
Venoms from eight Bothrops spp. were fractionated by ion-exchange chromatography on CM-Sepharose at pH 8.0 for the purification of myotoxins. Chromatographic profiles showed differences regarding myotoxic components among these venoms. B. alternatus, B. atrox and B. jararaca venoms did not show the major basic myotoxic fractions identified in the other venoms. Polyacrylamide gel electrophoresis for basic proteins also showed distinct patterns for these toxins. In vivo, all the isolated myotoxins induced release of creatine kinase due to necrosis of muscle fibers, accompanied by polymorphonuclear cell infiltration, and edema in the mouse paw. In addition, the toxins showed cytotoxic and liposome-disrupting activities in vitro. B. jararacussu bothropstoxins-I (BthTX-I) and II (BthTX-II) were submitted to chemical modifications of: His, by 4-bromophenacyl bromide (BPB) or photooxidation by Rose Bengal (RB); Tyr, by 2-nitrobenzenesulphonyl fluoride (NBSF); and Trp, by o-nitrophenylsulphenyl chloride (NPSC). The myotoxic and cytotoxic activities of BthTX-I, a Lys49 PLA(2) homologue, after modification by BPB, RB, NBSF and NPSC, were reduced to 50%, 20%, 75%, 65% and 13%, 0.5%, 76%, 58%, respectively. However, the edema-inducing and liposome-disrupting activities were not significantly reduced by the above modifications. BPB-treated BthTX-II, an Asp49 PLA(2) homologue, lost most of its catalytic, indirect hemolytic, anticoagulant, myotoxic and cytotoxic activities. The edema-inducing and liposome-disrupting activities were reduced to 50% and 80%, respectively. Lethality caused by BthTX-I and -II was strongly reduced after treatment with BPB or RB, but only partially with NBSF or NPSC. BthTX-I and -II, both native or modified, migrated similarly in a charge-shift electrophoresis. Antibodies raised against BthTX-I or -II, B. asper Basp-II and the C-terminal 115-129 peptide from Basp-II did not show significant differences in their cross-reactivity with the modified toxins, except with RB photooxidized toxins.
为了纯化肌毒素，采用离子交换色谱法在pH 8.0的CM-琼脂糖凝胶上对8种矛头蝮属蛇毒进行了分离。色谱图显示这些蛇毒在肌毒性成分方面存在差异。黑腹矛头蝮、矛头蝮和巴西矛头蝮蛇毒未显示出在其他蛇毒中鉴定出的主要碱性肌毒性组分。碱性蛋白的聚丙烯酰胺凝胶电泳也显示出这些毒素的不同模式。在体内，所有分离出的肌毒素均因肌纤维坏死诱导肌酸激酶释放，伴有多形核细胞浸润和小鼠爪部水肿。此外，这些毒素在体外显示出细胞毒性和脂质体破坏活性。对巴西矛头蝮蛇毒素-I（BthTX-I）和-II（BthTX-II）进行了以下化学修饰：组氨酸用4-溴苯甲酰溴（BPB）修饰或用孟加拉玫瑰红（RB）进行光氧化；酪氨酸用2-硝基苯磺酰氟（NBSF）修饰；色氨酸用邻硝基苯硫氯（NPSC）修饰。作为Lys49磷脂酶A2同源物的BthTX-I经BPB、RB、NBSF和NPSC修饰后，其肌毒性和细胞毒性活性分别降至50%、20%、75%、65%和13%、0.5%、76%、58%。然而，上述修饰并未显著降低其水肿诱导和脂质体破坏活性。经BPB处理的作为Asp49磷脂酶A2同源物的BthTX-II失去了大部分催化、间接溶血、抗凝、肌毒性和细胞毒性活性。其水肿诱导和脂质体破坏活性分别降至50%和80%。用BPB或RB处理后，BthTX-I和-II的致死率大幅降低，但用NBSF或NPSC处理仅部分降低。天然或修饰后的BthTX-I和-II在电荷转移电泳中的迁移情况相似。针对BthTX-I或-II、墨西哥矛头蝮Basp-II以及Basp-II的C末端115-129肽产生的抗体，除了与RB光氧化毒素外，与修饰后的毒素的交叉反应性没有显著差异。
