Sanchez Eladio F, Souza Christiane T, Bello Cynthia A, Richardson Michael, Oliveira Eduardo B, Magalhaes Arinos
Centro de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias, 30510-010, Belo Horizonte, MG, Brazil.
Toxicon. 2003 Jun;41(8):1021-31. doi: 10.1016/s0041-0101(03)00076-x.
Mutalysin II, a zinc endopeptidase possessing direct-acting fibrinolytic activity has been previously purified from bushmaster (Lachesis muta muta) snake venom. We now report a method to isolate two isoforms of natural mutalysin II (mut IIa and mut IIb) using chromatographies on Sephacryl S-200, CM Sepharose CL 6B and Sephadex G-50. The two proteins are monomeric non-glycosylated proteinases with similar molecular masses of 23 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Tryptic peptide mapping of the two native enzymes suggested a large degree of structural similarity. Both isoforms showed high similarity in all enzymatic properties using fibrinogen, fibrin and dimethylcasein as substrates. Thus, the specific fibrinolytic activity was estimated as 12+/-1.04 and 11.5+/-1.02 U/microg for mut IIa and mut IIb, respectively. The antigenic cross-reactivity of both isoforms was examined using rabbit hyperimmune serum or immunoglobulin G anti-mut IIa assays on immunodiffusion microscope slides, indirect enzyme-linked immunoabsorbent assay and western blots. From these experiments it was concluded that the two metalloproteinases mut IIa and mut IIb share identical antigenic structures. Since the stability of mutalysin II is dependent upon the presence of zinc, we examined the EDTA sensitivity of the isoforms of mutalysin II. Thus, the IC(50) values (concentration of EDTA to produce 50% inhibition of dimethylcasein hydrolysis) for mut IIa is 180 microM and 165 microM for mut IIb.
互变溶菌素II是一种具有直接纤溶活性的锌内肽酶，此前已从巨蝮蛇（Lachesis muta muta）毒液中纯化得到。我们现在报告一种使用Sephacryl S - 200、CM Sepharose CL 6B和Sephadex G - 50色谱法分离天然互变溶菌素II的两种同工型（mut IIa和mut IIb）的方法。这两种蛋白质是单体非糖基化蛋白酶，在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳上分子量相似，均为23 kDa。两种天然酶的胰蛋白酶肽图显示出高度的结构相似性。以纤维蛋白原、纤维蛋白和二甲基酪蛋白为底物时，两种同工型在所有酶学性质上都表现出高度相似性。因此，mut IIa和mut IIb的比纤溶活性分别估计为12±1.04和11.5±1.02 U/μg。使用兔超免疫血清或抗mut IIaIgG在免疫扩散显微镜载玻片上进行检测、间接酶联免疫吸附测定和蛋白质印迹法，检测了两种同工型的抗原交叉反应性。从这些实验得出结论，两种金属蛋白酶mut IIa和mut IIb具有相同的抗原结构。由于互变溶菌素II的稳定性取决于锌的存在，我们检测了互变溶菌素II同工型对EDTA的敏感性。因此，mut IIa的IC50值（产生50%二甲基酪蛋白水解抑制的EDTA浓度）为180 μM，mut IIb为165 μM。
