Purification and characterization of antitoxic proteins from the serum of Agkistrodon halys Pallas

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)2SO4 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 PLA2 activity of ATX. The results showed that antitoxin I could increase LD50 of ATX from 0.25±0.05 to 0.445±0.13 mg/kg, decrease its PLA2 activity from 2.36 to 1.72 μm/mg min, and antitoxin II could increase LD50 of ATX from 0.25±0.05 to 0.56±0.12 mg/kg, decrease Phospholipase A2 (PLA2) activity from 2.36 to 1.2 μm/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 PLA2 activity partially.