| Five-paced viper(Deinagkistrodon acutus), is one of the most dangerous snake among ten venomous snake, which subordinate to the Genus Deinagkistrodon, Crotalinae of Viperidae, and mainly distributes in southwest and southeast China. After being bitten by D. acutus, victim may suffer from pain, bleeding, swell, edema, muscle necrosis etc. But some victims may develop neurotoxic symptoms such as blepharoptosis and respiratory failure, which indicates that D. acutus venom could contain neurotoxins. However, the toxicological activities, structure and function relationship, and molecular mechanism of envenomation of D. acutus venom remain to be unclear. So far, it has only been found that the venom of saw-scaled viper(Echis carinatus) could inhibit smooth muscle contraction. In this study, it was firstly confirmed that the venom of D. acutus could inhibit smooth muscle contraction. Then, protein component was isolated and purified from the crude venom. Finally, the biochemical properties, toxicological and pharmacological activities were discussed in detail.A active component, designated as DA-1, that could inhibit mouse ileum contraction, was purified from the venom of D. acutus by three steps(including Sephadex G-50 size-exclusion, DEAE Sepharose Fast Flow ion-exchange liquid chromatography and Hitrap Capto DEAE ion-exchange liquid chromatography). DA-1 was purified into homogeneity via HPLC, and was a monomer with 23 k Da determined by reducing and non-reducing SDS-PAGE. The accurate molecular weight was 22,947.9 Da determined by the MALDI-TOF-TOF mass spectrum. The peptide mass fingerprint(PMF) indicated that DA-1 may be a snake venom metalloproteinase.The results of the biochemical experiment show that DA-1 could significantly cleave the bovine fibrinogen in a dose and time dependent manner. However, DA-1 could only degrade the Aα- and Bβ-chain of fibrinogen, having no effect on its γ-chain, besides DA-1 could cleave fibrin monomers. The fibrino(gen)lytic activities of DA-1 could be inhibited by EGTA or EDTA strongly, affected moderately by β-mercaptoethanol, but not affected by PMSF at all. The hypodermic injection experiment revealed that DA-1 is significantly hemorrhagic activity. All the above results indicated that DA-1 is a kind of hemorrhagic metalloproteinase. Other biochemical experiments showed that DA-1 had phospholipase A2(PLA2) activity and edema activity, but not hemolysis activity.The in vitro pharmacological activities of DA-1 were examined using mouse ileum smooth muscle preparation. DA-1 inhibited the active tension of smooth muscle contraction in a time and concentration manner. The time required to cause 90% blockade was about 22 min at the higher dose 12 μg/ml. The response for all venom dilutions was irreversible as spontaneous contraction could not be restored after washing. However, the protein could not affect the frequency of intestinal contraction even at the higher dose 12 μg/ml. Similarly, the contractile response elicited by exogenous application of Ach and KCl was significantly inhibited by all concentrations of DA-1, and also could not be restored by washing after 60 min. The effect was independent on PLA2 activity of DA-1 as evidenced by the ability of the PLA2 inhibitor 4-bromophenacyl bromide(4-BPB) not affecting this activity.These findings demonstrated that DA-1, which could inhibit ileum smooth muscle contraction, is a hemorrhagic metalloproteinase, and could irreversibly inhibit intestinal contraction in a time and concentration manner. It has fibrino(gen)lytic activity, hemorrhagic activity, edema activity, as well as PLA2 activity, yet the PLA2 activity is not related to the pharmacological activity. This inhibition induced by DA-1 could arise from irreversible damage to muscle tissue or neuro cell. However, the detailed molecular mechanisms of DA-1 still need further study in the future. |
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