| Agkistrodon blomhoffii Ussurensis belongs to Viperidae Agkistrodon subfamily, which is one of the most poisonous snakes in our country, and it widely distributes in the northwest, northeast, central, southwest regions, and Taiwan province, as well as Korean Peninsula. Agkistrodon blomhoffii Ussurensis venom is the main resource for producing antithrombotic drug "thrombin-like enzyme (TLE, defibrase)". However, except for TLE, other components with bioactivities have not been comprehensively studied yet.5'-nucleotidase is a kind of specific phosphatase, which catalyzes the hydrolysis of the 5'-phosphate bond in the nucleotide molecules, and yields corresponding nucleoside and inorganic phosphate.5'-nucleotide is a common reagent widely used in molecular biology research. In recent years, it was noticed that the activity of 5'-nucleotide was closely related with many diseases, and can be used as positive control for the diagnosis of these diseases, e.g. liver and gallbladder disease, coronary heart disease, lung cancer, gastrointestinal tumors, hyperthyroidism, bone disease, throat cancer, etc. Except for bacteria and plant, snake venom is also a good resource for isolating 5'-nucleotidase. Although 5'-nucleotidase could be isolated from almost all kinds of snake venoms, venom of rattle snake and viperine snake showed higher concentration. Study on the purification and characterization of 5'-nucleotidase from the venom of Agkistrodon blomhoffii Ussurensis will be valuable for utilization of the venom, which has been commercially used for isolating TLE.In this dissertation, DEAE-SephadexA-25 ion exchange chromatography, followed by Sephadex-G-75 gel filtration chromatography, were applied to isolate and purify 5'-nucleotidase from the venom of Agkistrodon blomhoffii Ussurensis. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was then used to determinate the molecular weight (MW) and purity of the enzyme protein. Optimal temperature and pH, effects of metal ions on the activity, and other characteristics of the enzyme were studied. Furthermore, study on the protective effect of simple sugars on the enzyme protein, as well as biological effects on the platelet aggregation induced by ADP, were carried out.MW of 5'-nucleotidases is 70 kDa, and it is identified as a glycoprotein through periodic acid silver nitrate Alcian blue staining. The specific activities of the purified 5'-nucleotidase was 562.44 U/mg when using AMP as substrate. The optimal temperature is 55℃, and the optimal pH is pH 9.0. The enzyme is thermostable, and has high activity in basic environment. The enzymatic activity could be inhibited by Cu2+ and Fe3+, especially by Fe3+; while Mg2+, Ca2+ and K+, are agonists, among them Ca2+ showed highest effects.P-mercaptoethanol also showed some effects to activate the enzymatic in a relative low concentration (0.05mmol/L-0.25mmol/L). In a relative low concentration of EDTA (0.05 mmol/L-0.25mmol/L), it usually activated the enzyme, however, inhibition effects would be observed when the EDTA concentration was higher than 0.3 mmol/L. This phenomena maybe due to chelating capability of EDTA:in a lower concentration, it would capture Cu2+and Fe3+, which are inhibitors to the enzyme; while in a higher concentration, it will even bind and remove Mg2+, Ca2+ and K+, which are needed to activate the enzyme.In this dissertation, protective effects to the enzyme protein of simple sugars were also investigated. Four carbohydrates, i.e. glucose, fructose, trehalose and sucrose, were tested in the experiments. Results suggested that glucose, fructose, trehalose have obviously protective effect to the 5'-nucleotidase. These studies have not been reported yet, and the specific mechanisms need to be further studied.The 5'-nucleotidase has facilitating effect on platelet aggregation, in the procedure induced by ADP. This result, interestingly, is on the contrary to reported properties of 5'-nucleotidases from other snake venoms. More experiments are scheduled to investigate the mechanism.
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