Niu Yang-Ping, Shao Jing-Yu, Jiang Yan-Ling
School of Pharmacy, Zhejiang Univerisity of Technology, 18 Chaowang Road, 310014 Hangzhou, China.
Toxicon. 2007 Sep 1;50(3):322-9. doi: 10.1016/j.toxicon.2007.03.022. Epub 2007 Apr 11.
In this study, the authors report the purification and characterization of antitoxic proteins from the serum of Agkistrodon halys Pallas. Two antitoxic proteins have been successfully isolated by the methods of (NH4)(2)SO(4) fractional precipitation, chromatography and preparative discontinuous polyacrylamide gel electrophoresis (PAGE). We have measured their molecular weights by Sephadex G-150 chromatography and 0.1% SDS-Tris-HCl discontinue PAGE respectively. Antitoxin I was about 138,000+/-40 Da and antitoxin II was about 76,000+/-40 Da, they are all single-chain peptides. We have measured their capacity to neutralize the toxicity of agkistrodotoxin (ATX), and their capacity to inhibit the PLA(2) activity of ATX. The results showed that antitoxin I could increase LD(50) of ATX from 0.25+/-0.05 to 0.445+/-0.13 mg/kg, decrease its PLA(2) activity from 2.36 to 1.72 microm/mg min, and antitoxin II could increase LD(50) of ATX from 0.25+/-0.05 to 0.56+/-0.12 mg/kg, decrease Phospholipase A(2) (PLA(2)) activity from 2.36 to 1.2 microm/mg min. When the natural antitoxins were mixed with different amounts of ATX and inoculated intraperitonially into eight mice, it was found that 0.5 mg antitoxin I could neutralize the toxicity of 0.4 mg ATX and 0.5 mg antitoxin II could neutralize the toxicity of 0.5 mg ATX completely. These antitoxic proteins could neutralize the toxicity of ATX completely and inhibit ATX's PLA(2) activity partially.
在本研究中，作者报道了从蝮蛇血清中分离纯化抗毒蛋白及其特性。通过硫酸铵分级沉淀、层析和制备性不连续聚丙烯酰胺凝胶电泳（PAGE）方法成功分离出两种抗毒蛋白。我们分别用Sephadex G - 150层析和0.1% SDS - Tris - HCl不连续PAGE测定了它们的分子量。抗毒素I约为138,000±40 Da，抗毒素II约为76,000±40 Da，它们均为单链肽。我们测定了它们中和蝮蛇毒素（ATX）毒性的能力以及抑制ATX磷脂酶A2（PLA2）活性的能力。结果表明，抗毒素I可使ATX的半数致死量（LD50）从0.25±0.05提高到0.445±0.13 mg/kg，其PLA2活性从2.36降低到1.72 microm/mg min，抗毒素II可使ATX的LD50从0.25±0.05提高到0.56±0.12 mg/kg，磷脂酶A2（PLA2）活性从2.36降低到1.2 microm/mg min。当将天然抗毒素与不同量的ATX混合并腹腔注射到8只小鼠体内时，发现0.5 mg抗毒素I可完全中和0.4 mg ATX的毒性，0.5 mg抗毒素II可完全中和0.5 mg ATX的毒性。这些抗毒蛋白可完全中和ATX的毒性并部分抑制ATX的PLA2活性。
