Monteiro R Q, Yamanouye N, Carlini C R, Guimarães J A, Bon C, Zingali R B
Departamento de Bioquímica Médica, Cidade Universitária, Rio de Janeiro, Brazil.
Toxicon. 1998 Jan;36(1):153-63. doi: 10.1016/s0041-0101(97)00061-5.
Bothrojaracin (BJC) is a potent thrombin inhibitor isolated from the venom of Bothrops jararaca. Venoms from individual snakes have been shown to vary in BJC content, and more than one molecular variant (isoform) has been identified in the same venom. In order to determine whether the production of this protein and its isoforms varies under seasonally invariant conditions, an analysis was made of BJC isolated from venoms collected individually once a month for 10 months from two female B. jararaca snakes kept under conditions of constant temperature and photoperiod. The crude venom from each individual snake exhibited a characteristic pattern of protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with no noticeable variation throughout the collecting period. BJC from individual venoms was purified by gel filtration on Sephacryl S-200 followed by an affinity column (PPACK-thrombin Sepharose). BJC content and other activities such as phospholipase A2, azocaseinolytic activity and inhibition of thrombin-induced platelet aggregation varied considerably among the samples. Purified BJC from both snakes inhibited fibrin coagulation and migrated as a single band of 27,000 mol. wt on SDS-PAGE. However, the BJC pattern on non-denaturing PAGE differed between the two snakes, with four to six bands per sample each month, which were all recognized by polyclonal anti-BJC antibodies. The isoelectric focusing pattern of BJC was also characteristic for each snake, with only minor differences throughout the collecting period. These results indicate that under seasonally invariant conditions: (1) there was a considerable variation over the 10-month period in the production of BJC and other important venom activities such as phospholipase A2 and proteinases; (2) individual B. jararaca snakes produced a distinctive array of BJC isoforms; and (3) despite quantitative differences, there were essentially no qualitative differences in the production of BJC isoforms by individual snakes during the 10-month period.
矛头蝮蛇毒素（BJC）是一种从巴西矛头蝮蛇毒液中分离出的强效凝血酶抑制剂。已证明不同个体蛇的毒液中BJC含量存在差异，并且在同一毒液中已鉴定出不止一种分子变体（同工型）。为了确定这种蛋白质及其同工型的产生在季节性不变的条件下是否会发生变化，对从两条饲养在恒温恒光周期条件下的雌性巴西矛头蝮蛇每月单独采集一次毒液，持续10个月所分离出的BJC进行了分析。每条蛇的粗毒液在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）上呈现出特征性的蛋白条带模式，在整个采集期间没有明显变化。通过在Sephacryl S-200上进行凝胶过滤，随后通过亲和柱（PPACK-凝血酶琼脂糖）对单个毒液中的BJC进行纯化。样品之间的BJC含量以及其他活性，如磷脂酶A2、酪蛋白分解活性和对凝血酶诱导的血小板聚集的抑制作用差异很大。两条蛇纯化后的BJC均能抑制纤维蛋白凝固，并且在SDS-PAGE上迁移为一条27,000分子量的单带。然而，两条蛇在非变性PAGE上的BJC模式不同，每个样品每月有四到六条带，这些带均能被多克隆抗BJC抗体识别。BJC的等电聚焦模式对每条蛇来说也是特征性的，在整个采集期间只有微小差异。这些结果表明，在季节性不变的条件下：（1）在10个月的时间里，BJC以及其他重要的毒液活性，如磷脂酶A2和蛋白酶的产生有相当大的变化；（2）单个巴西矛头蝮蛇产生了独特的一系列BJC同工型；（3）尽管存在数量差异，但在10个月期间，单个蛇产生BJC同工型的过程中基本上没有质量差异。
