Oliveira F, Rodrigues V M, Borges M H, Soares A M, Hamaguchi A, Giglio J R, Homsi-Brandeburgo M I
Departamento de Ciências, Faculdade de Filosofia, Ciências e Letras, FAFI, Araguari-MG, Brazil.
Biochem Mol Biol Int. 1999 Jun;47(6):1069-77. doi: 10.1080/15216549900202193.
A basic serine protease which is active on casein and fibrinogen was purified from Bothrops moojeni venom using a single step chromatography on a CM-Sepharose fast flow column. The enzyme, MOO3, was not hemorrhagic and presented only a trace of blood-clotting activity. Synthetic chromogenic substrates (azoacasein and azoalbumin) where not hydrolyzed by MOO3. Using polyacrylamide gel electrophoresis at pH 4.3, MOO3 showed as a single protein band. Using sodium dodecyl sulfate-polyacrylamide electrophoresis, MOO3 behaved as a single-chain protein with an approximate mol. weight of 27,000, both in the presence and absence of beta-mercaptoethanol. Its pI was 7.8 by electrofocusing. The enzyme did not contain neutral carbohydrates and its N-terminal amino acid was alanine. The amino acid composition showed 249 residues/mole, a high content of hydrophilic amino acids and 14 half-cystine residues, which should account for 7 disulfide bonds. The protease cleaved the A-alpha chain faster than the B-beta of bovine fibrinogen and showed no effect on the delta-chain. Specific esterolytic activity of MOO3 on alpha-N-tosyl-l-arginine methyl ester was 29.64 mumol min-1 x mg-1. MOO3 represented 1.42% (w/w) of the initial desiccated venom. Its proteolytic activity was inhibited by beta-mercaptoethanol, leupeptin, phenylmethylsulphonyl fluoride and ethylenediamine tetraacetate.
通过在CM-琼脂糖快速流动柱上进行一步色谱法，从矛头蝮蛇毒液中纯化出一种对酪蛋白和纤维蛋白原有活性的碱性丝氨酸蛋白酶。这种酶，即MOO3，没有出血活性，仅呈现出微量的凝血活性。合成生色底物（偶氮酪蛋白和偶氮白蛋白）未被MOO3水解。在pH 4.3条件下使用聚丙烯酰胺凝胶电泳，MOO3显示为单一蛋白条带。使用十二烷基硫酸钠-聚丙烯酰胺电泳，无论有无β-巯基乙醇，MOO3均表现为单链蛋白，其分子量约为27,000。通过等电聚焦法测定其等电点为7.8。该酶不含中性碳水化合物，其N端氨基酸为丙氨酸。氨基酸组成显示每摩尔含有249个残基，亲水性氨基酸含量高，有14个半胱氨酸残基，这应形成7个二硫键。该蛋白酶切割牛纤维蛋白原的A-α链比B-β链快，对δ链无作用。MOO3对α-N-甲苯磺酰-L-精氨酸甲酯的比酯解活性为29.64 μmol min⁻¹·mg⁻¹。MOO3占初始干燥毒液的1.42%（w/w）。其蛋白水解活性受到β-巯基乙醇、亮抑酶肽、苯甲基磺酰氟和乙二胺四乙酸的抑制。
