一种无毒的抗凝血金属蛋白酶：从印度眼镜蛇（眼镜蛇指名亚种）毒液中纯化及特性鉴定

A non-toxic anticoagulant metalloprotease: purification and characterization from Indian cobra (Naja naja naja) venom.

作者信息

Jagadeesha D K, Shashidhara murthy R, Girish K S, Kemparaju K

机构信息

Department of Studies in Biochemistry, University of Mysore, Manasa Gangothri, Mysore -570 006, India.

出版信息

Toxicon. 2002 Jun;40(6):667-75. doi: 10.1016/s0041-0101(01)00216-1.

DOI:10.1016/s0041-0101(01)00216-1

PMID:12175602

Abstract

A non-toxic potent anticoagulant metalloprotease NN-PF(3) has been purified to homogeneity from the Indian cobra (Naja naja naja) venom through a combination of column chromatography and electrophoresis. NN-PF(3) is a single chain protein with a molecular weight of 68 kDa by SDS-PAGE. It hydrolysed casein, gelatin, haemoglobin and bovine fibrinogen, but did not hydrolyse bovine serum albumin or synthetic substrates such as TAME, BAEE and BAPNA. EDTA, EGTA and cyanide inhibited the enzymatic activity while 1,10-phenanthroline, PMSF, leupetin and pepstatin did not show any effect. NN-PF(3) is a metalloprotease containing Ca(2+) and Zn(2+) at a molar ratio of 1:1.2 and 1:0.4, respectively, as revealed by atomic absorption spectroscopy. NN-PF(3) was non-lethal up to an i.p. dose of 15 mg/kg body weight of mice and is devoid of myotoxicity, cytotoxicity and haemorrhagic activity. It is weakly oedematic, but strongly anticoagulant in property and the effect observed was both dose and time dependent.

摘要

一种无毒的强效抗凝金属蛋白酶NN-PF(3)已通过柱色谱和电泳相结合的方法从印度眼镜蛇（Naja naja naja）毒液中纯化至同质。通过SDS-PAGE分析，NN-PF(3)是一种分子量为68 kDa的单链蛋白质。它能水解酪蛋白、明胶、血红蛋白和牛纤维蛋白原，但不能水解牛血清白蛋白或诸如TAME、BAEE和BAPNA等合成底物。EDTA、EGTA和氰化物抑制其酶活性，而1,10-菲咯啉、PMSF、亮抑酶肽和胃蛋白酶抑制剂则无任何作用。原子吸收光谱显示，NN-PF(3)是一种金属蛋白酶，分别含有摩尔比为1:1.2和1:0.4的Ca(2+)和Zn(2+)。NN-PF(腹腔注射剂量高达15 mg/kg体重的小鼠时无致死性，且无肌毒性、细胞毒性和出血活性。它有轻微的水肿作用，但抗凝作用很强，并呈剂量和时间依赖性。