Ketelhut D F J, de Mello M Homem, Veronese E L G, Esmeraldino L E, Murakami M T, Arni R K, Giglio J R, Cintra A C O, Sampaio S V
Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, Avenida do Café, s/n, Bairro Monte Alegre, 14040-903, SP Ribeirão Preto, Brazil.
Biochimie. 2003 Oct;85(10):983-91. doi: 10.1016/j.biochi.2003.09.011.
Acidic phospholipase A(2) (PLA(2)) isoforms in snake venoms, particularly those from Bothrops jararacussu, have not been characterized. This article reports the isolation and partial biochemical, functional and structural characterization of four acidic PLA(2)s (designated SIIISPIIA, SIIISPIIB, SIIISPIIIA and SIIISPIIIB) from this venom. The single chain purified proteins contained 122 amino acid residues and seven disulfide bonds with approximate molecular masses of 15 kDa and isoelectric points of 5.3. The respective N-terminal sequences were: SIIISPIIA-SLWQFGKMIDYVMGEEGAKS; SIIISPIIB-SLWQFGKMIFYTGKNEPVLS; SIIISPIIIA-SLWQFGKMILYVMGGEGVKQ and SIIISPIIIB-SLWQFGKMIFYEMTGEGVL. Crystals of the acidic protein SIIISPIIB diffracted beyond 1.8 A resolution. These crystals are monoclinic with unit cell dimensions of a = 40.1 A, b = 54.2 A and c = 90.7 A. The crystal structure has been refined to a crystallographic residual of 16.1% (R(free) = 22.9%). Specific catalytic activity (U/mg) of the isolated acidic PLA(2)s were SIIISPIIA = 290.3 U/mg; SIIISPIIB = 279.0 U/mg; SIIISPIIIA = 270.7 U/mg and SIIISPIIIB = 96.5 U/mg. Although their myotoxic activity was low, SIIISPIIA, SIIISPIIB and SIIISPIIIA showed significant anticoagulant activity. However, there was no indirect hemolytic activity. SIIISPIIIB revealed no anticoagulant, but presented indirect hemolytic activity. With the exception of SIIISPIIB, which inhibited platelet aggregation, all the others were capable of inducing time-independent edema. Chemical modification with 4-bromophenacyl bromide did not inhibit the induction of edema, but did suppress other activities.
蛇毒中的酸性磷脂酶A(2)（PLA(2)）同工型，尤其是矛头蝮蛇（Bothrops jararacussu）毒液中的此类同工型，尚未得到表征。本文报道了从该毒液中分离出的四种酸性PLA(2)（命名为SIIISPIIA、SIIISPIIB、SIIISPIIIA和SIIISPIIIB）及其部分生化、功能和结构特征。纯化的单链蛋白质含有122个氨基酸残基和七个二硫键，近似分子量为15 kDa，等电点为5.3。各自的N端序列如下：SIIISPIIA - SLWQFGKMIDYVMGEEGAKS；SIIISPIIB - SLWQFGKMIFYTGKNEPVLS；SIIISPIIIA - SLWQFGKMILYVMGGEGVKQ；SIIISPIIIB - SLWQFGKMIFYEMTGEGVL。酸性蛋白SIIISPIIB的晶体衍射分辨率超过1.8 Å。这些晶体为单斜晶系，晶胞参数为a = 40.1 Å，b = 54.2 Å，c = 90.7 Å。晶体结构已精修至晶体学残余因子为16.1%（R(free) = 22.9%）。分离出的酸性PLA(2)的比催化活性（U/mg）分别为：SIIISPIIA = 290.3 U/mg；SIIISPIIB = 279.0 U/mg；SIIISPIIIA = 270.7 U/mg；SIIISPIIIB = 96.5 U/mg。尽管它们的肌毒性活性较低，但SIIISPIIA、SIIISPIIB和SIIISPIIIA表现出显著的抗凝活性。然而，它们没有间接溶血活性。SIIISPIIIB没有抗凝活性，但具有间接溶血活性。除SIIISPIIB抑制血小板聚集外，其他所有蛋白都能诱导与时间无关的水肿。用4-溴苯甲酰溴进行化学修饰并未抑制水肿的诱导，但确实抑制了其他活性。
