Biochemical properties and conformation in liquid on two hemorrhagic toxins from the venom of deinagkistrodon acutus from human

Two hemorrhagic toxins(HaHT-1 and HaHT-2) from the venom of Deinagkistrodon acutus from Hunan have been purified using Sephadex G-75 gel filtration, QAE-Sephadex A-50 and CM-Sephadex C-25 ion exchange chromatography. They were homogeneous judged by polyacrylamide gel electrophoresis and SDS-polyacrylamide electrophoresis. The both consist of single polypeptide chain with similar molecular weight of 23.5kDa. HaHT-1 is composed of 213 amino acid residues and HaHT-2 consists of 207 amino acid residues, respectively. As determined by polyacrylamide gel isoelectric focusing, their isoelectric points are 5.6 for HaHT-1 and 5.2 for HaHT-2, respectively. They were strong hemorrhagic toxins with proteolytic activity. EDTA and cysteine could inhibit both hemorrhagic and proteolytic activity but trypsin had no effect, suggesting that the toxins are metalloproteins and disulfide bridges which had contribution to the consormation of activities. Analyses for metals indicated that zinc and calcium were present in both toxins with roughly 0.5 mol zinc and 1 mol calcium per 1 mol protein.Circular dichroism (CD) was used to examine the conformation of HaHT-1 and HaHT-2. In the far CD spectrum, it was showed a wide negative peak at 217 nm for HAHT-1 and a negative peak at 2.8 nm with a shoulder peak at 217 nm for HaHT-2. Although there is some indication of a high β-sheet content, it is possible that the aromatic region contributed to this complex spectrum for HaHT-2. The secondary structure contents calculated by the method of Chen and Yang showed that HaHT-1 was found to be 36.9%α-helix, 35.5%β-sheet% and 27.6% randon corl and HaHT-2 to be 23.4%α-helix, 31.3%β-sheet% and 45.3% randon coil. The effects of pH, temperature and EDTA on CD spectra of the hemorrhagins could be found that the conformation of HaHT-1 and HaHT-2 have some changes. As they were calculated by the method of Chen and Yang, the contents of α-helix were decreased while that randon coil were increased.