Sánchez E F, Magalhës A, Mandelbaum F R, Diniz C R
Centro de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias, Belo Horizonte, Brazil.
Biochim Biophys Acta. 1991 Aug 6;1074(3):347-56. doi: 10.1016/0304-4165(91)90084-t.
Hemorrhagic factor II (LHF-II) was isolated from Lachesis muta muta (Bushmaster snake) venom using column chromatographies on Sephadex G-100, CM-Sepharose CL-6B and two cycles on Sephadex G-50. This preparation was devoid of phospholipase A2 as well as of the enzymes active on arginine synthetic substrates (TAME and BAPNA) which are present in the crude venom. LHF-II was homogeneous by SDS-polyacrylamide gel electrophoresis, immunodiffusion and immunoelectrophoresis. Also, a single symmetrical boundary with a value of 2.59 S was obtained by ultracentrifugation. LHF-II contains 180 amino acid residues, has a molecular weight of 22,300, and an isoelectric point of 6.6. It contains one gatom zinc and two gatoms calcium per mol protein. The hemorrhagic factor possesses proteolytic activity toward various substrates such as, casein, dimethylcasein, hide powder azure, fibrinogen and fibrin. It hydrolyzes selectively the A alpha-chain of fibrinogen, leaving the B beta- and gamma-chains unaffected. LHF-II is activated by Ca2+ and inhibited by Zn2+. The hemorrhagic as well as the proteinase activity is inhibited by cysteine and by metal chelators such as EDTA, EGTA and 1,10-phenanthroline. Inhibitors of serine proteinases such as phenylmethanesulfonyl fluoride (PMSF) and soybean trypsin inhibitor (SBTI) have no effect on the hemorrhagic factor.
通过在葡聚糖凝胶G - 100、CM - 琼脂糖CL - 6B上进行柱色谱以及在葡聚糖凝胶G - 50上进行两个循环，从矛头蝮蛇（巴西矛头蝮蛇）毒液中分离出出血因子II（LHF - II）。该制剂不含磷脂酶A2以及粗毒液中存在的对精氨酸合成底物（TAME和BAPNA）有活性的酶。通过SDS - 聚丙烯酰胺凝胶电泳、免疫扩散和免疫电泳，LHF - II是均一的。此外，通过超速离心获得了一个单一的对称边界，沉降系数为2.59 S。LHF - II含有180个氨基酸残基，分子量为22,300，等电点为6.6。每摩尔蛋白质含有1克原子锌和2克原子钙。该出血因子对各种底物如酪蛋白、二甲基酪蛋白、皮粉天青、纤维蛋白原和纤维蛋白具有蛋白水解活性。它选择性地水解纤维蛋白原的Aα链，而Bβ链和γ链不受影响。LHF - II被Ca2 +激活，被Zn2 +抑制。出血活性以及蛋白酶活性被半胱氨酸和金属螯合剂如EDTA、EGTA和1,10 - 菲咯啉抑制。丝氨酸蛋白酶抑制剂如苯甲基磺酰氟（PMSF）和大豆胰蛋白酶抑制剂（SBTI）对该出血因子没有影响。
