| Snake venom is mixture of proteins, excreted by snake's toxin gland. It has broad range of biological activities, containing many kinds of proteins, polypeptides, enzymes and other small molecules. It is the animal venom which has been studied and used mostly so far. a-neurotoxin, which was isolated from the venom of cobras, has been used as an ideal tool for studying the function and probing the structure of acetylcholine receptor (AChR) for several decades, because it specifically and virtually irreversibly binds to AChR with high affinity, α-neurotoxins, with high stability, rich of disulfide bonds, lots of natural homologues, it is also used as model for studying the folding, structure-function relationship of protein.For the binding mechanism of long neurotoxin to AChR, as well as the structure of AChR, great progresses have been made in recent years. However, much less data are available for the binding of short neurotoxin to AChR. Actually, it was noticed that the model was not completely applicable to short neurotoxins. The debate also implied the model of AChR might have some errors, and deeper understanding on the short neurotoxin to AChR is valuable for understanding the fine structure of ligand binding center of the receptor, as well as open-close mechanism of the ion channel. Furthermore, little information is known on the binding of long and short neurotoxins to δ/γ subunits, which were thought to determine the specificities and selectivity of receptors.To study the binding mechanism of short neurotoxin to the AChR, followed with our previous modeling work, a short-chain neurotoxin cobrotoxin was isolated from Naja atra venom by sequential cation-exchange chromatography, Sehpadex CM-C-25 and Source 15S. Cobrotoxin showed a single band and homogeneity on SDS-PAGE. Gel image analysis and HPLC results showed the purity was above 98%. And a 15 residue high affinity peptide (HAP), which was designed and derived from the sequence of Loop F of the γ subunit of mouse AChR, was synthesized. The binding capability of the short neurotoxin and the HAP were tested with BIAcore biochip and chromatography.Assays done on BIAcore did not showed significant binding of HAP peptide to the neurotoxin. This might imply the binding of HAP to toxin is in to slow progress, since theHAP just follows through the neurotoxin-coated chip in a short time. However, positive results were gotten with liquid chromatography.Binding capability of cobrotoxin with HAP was determined with cation-exchange chromatography. Results indicated that HAP could bind to cobrotoxin with 2:1, and the binding constant was 4.8 × 10~8. These suggested that LoopF and cobrotoxin had strong affinity, achieving the demand of high structure determination with NMR or X-Ray. These also showed evidence that the Loop F of δ/γ subunit had strong affinity to the ligand, and might play essential role in the ligand selectivity, which is accordant to literatures.At the same time, a thrombin-like enzyme was isolated from Agkistrodon blomhoffii ussurensis venom by anion-exchange chromatography and gel filtration, that is DEAE-Sephadex A-25, Sephadex G-75, Source 15Q. The enzyme showed a single band on SDS-PAGE, with MW of 35.5k Da under reduced conditions. The clotting specific activity of the enzyme was 41.5 U/mg on human plasma and the specific activity of arginine esterase was 454.4 U/mg on BAEE. The enzyme's activity to clotting fibrinogen was inhibited by PMSF, but not by EDTA. These above results showed that the enzyme was a serine protease, but not a metalloproteinase. |
PDF Full Text Request