Castro H C, Dutra D L, Oliveira-Carvalho A L, Zingali R B
Departamento de Bioquímica Médica, ICB/CCS, Universidade Federal do Rio de Janeiro, Brazil.
Toxicon. 1998 Dec;36(12):1903-12. doi: 10.1016/s0041-0101(98)00111-1.
Bothroalternin (MW 27 kDa), a new member of the family of C-type lectins is a thrombin inhibitor which was purified from pooled B. alternatus venom by affinity chromatography on PPACK-thrombin-Sepharose, followed by size exclusion and reverse-phase on HPLC columns. Material retained on the affinity column contained proteins with apparent molecular weights ranging from 20 to 60 kDa on SDS-PAGE and inhibited aggregation of rabbit platelets induced by alpha-thrombin (IC50 = 28 microg/ml). A single band of approximately 27 kDa was recognized in Western-blot assays using polyclonal antibodies raised against bothrojaracin, a thrombin inhibitor purified from B. jararaca venom (Zingali et al., 1993). The immunological similarity of this fraction to bothrojaracin was confirmed by ELISA and competitive ELISA. Further purification by size exclusion and reverse-phase on HPLC, produced a single homogenous peak called bothroalternin. This protein was highly homologous to bothrojaracin (95% in its N-terminal sequence-for residues 1 to 25) but displaying lower inhibitory effect on thrombin induced platelet aggregation (Ic50 = 0.19 microg/ml) compared to bothrojaracin (IC50 = 0.06). Altogether, bothroalternin is a new thrombin inhibitor isolated from Bothrops alternatus venom and has been characterized as a bothrojaracin-like protein.
Bothroalternin（分子量27 kDa）是C型凝集素家族的新成员，是一种凝血酶抑制剂，通过在PPACK - 凝血酶 - 琼脂糖上进行亲和层析，随后在HPLC柱上进行尺寸排阻和反相层析，从混合的巴西矛头蝮蛇毒中纯化得到。亲和柱上保留的物质在SDS - PAGE上显示表观分子量范围为20至60 kDa的蛋白质，并抑制α - 凝血酶诱导的兔血小板聚集（IC50 = 28微克/毫升）。在使用针对从巴西矛头蝮蛇毒中纯化的凝血酶抑制剂bothrojaracin产生的多克隆抗体进行的蛋白质印迹分析中，识别出一条约27 kDa的单带（Zingali等人，1993年）。通过ELISA和竞争性ELISA证实了该组分与bothrojaracin的免疫相似性。通过在HPLC上进行尺寸排阻和反相进一步纯化，产生了一个单一的均一峰，称为bothroalternin。该蛋白质与bothrojaracin高度同源（其N端序列中1至25位残基的同源性为95%），但与bothrojaracin相比，对凝血酶诱导的血小板聚集的抑制作用较低（Ic50 = 0.19微克/毫升）（bothrojaracin的IC50 = 0.06）。总之，bothroalternin是从巴西矛头蝮蛇毒中分离出的一种新的凝血酶抑制剂，已被鉴定为一种类似bothrojaracin的蛋白质。
