Purification And Anti-angiogenesis Activity Of The L-amino Acid Oxidase From Naja Atra Venom

L-amino acid oxidase (EC 1.4.3.2) is a flavoenzyme that catalyzes the oxidative deamination of L-amino acid substrate into a a-Keto acid with production of ammonia and hydrogen peroxidase. L-amino acid oxidase was widely distributed in snake venoms. LAO was one of the important active components,who had many biological effect, including induction of the apoptosis in vascular endothelial cells, inhibition or induction of platelet aggregation , cytotoxicity, antibacterial activity,and so on. Although LAO was not the main toxic components in snake venom, but there is a close relationship between snakebite and symptoms such as bleeding and swelling, tissue necrosis, wound fester.Some recent studies had shown that LAO purified from the venom can induce apoptosis,affect on platelets.So we judged LAO could induce apoptosis of vascular endothelial cells, which suggested us it could affect on vascular and have a certain impact on the role of its anti-tumor blood vessels. Thus the transfer of tumor blood vessels research may have the further development of Value. This research optimized determination of the lao activityand purification, and then observed angiogenesis in vitro and in vivo.These could provide experimental basis for clinical applications.1. Optimization NAV-LAO activity assay methodCompared the sensitivity and reproducibility of methods in assaying activity by three hydrogen Donor (ODA, OPD and TMB). OPD method was sensitive at concentrations of 1.0-0.0025mg·ml-1 .It was significantly higher than the other two methods (p<0.05 or p<0.01), and its RSD was2.1%which could show a good reproducibility.2. Purification and Determination of Physical and Chemical Properties on NAV-LAOThe L-amino acid oxidase was purified from the venom of Naja atra by Crude venom from Naja atra venom was first subjected to a SP-Sepharose FF column that separated it into thirteen major fractions. Activity of LAO was determined by the OPD-based microplate assay. LAO activity was found in the first fraction, which was collected and stored . FractionⅠapplied onto an Heparin SepharoseCL-6B column, then a Pure NAV-LAO was obtained.NA-LAO appeared to be homogeneous protein on 12% SDS-PAGE electrophoresis, under reducing or non-reducing conditions. It suggested that NA-LAO be a single protein chain, with an apparent molecular mass of 58 kDa.3.The effect of NAV-LAO on the proliferation of human umbilical vein endothelial cells (HUVEC)The effects of NAV-LAO on cultured HUVEC cells was observed by MTT method.The results showed that NAV-LAO inhibited proliferation of HUVEC cells and the inhibited effect was dose dependent, and the IC50(24h) value was 21.42μg/ml.4. The effects of NAV-LAO on migration of HUVEC cellsThe migration of HUVEC was observed by a wounding assay.The results showed that cells in the experimental group decreased migration capacity compared with no treatment group at the basis of the Original injury district. there is significant difference compared with the control group(P <0.01).5. The effects of NAV-LAO on tube formation in vitroTube formation assay was applied to measure the effect of anti-angiogenesis. The results showed that NAV-LAO could inhibite HUVEC cells by comparing the number of lumen formation, area,and perimeter, and this effect was dose-dependent manner.6. The effects of NAV-LAO on HUVEC cells apoptosis by Hoechst33342 stainingThe results showed that NAV-LAO could impact on HUVEC cells apoptosis after Hoechst33342 nuclear fluorescence staining. It found that apoptotic cells after the drug significantly more than the control group, and the apoptosis cell size reduced, nuclear condensation , step aside, and apoptotic bodies can be seen.7.Western Blotting Analysis the expression of signaling molecules of caspase3, caspase8Western Blotting analysed the change of expression of caspase3, caspase8 after different concentrations of NAV-LAO treated with HUVEC 24h.The results showed that caspase3, caspase8 expression increased than the control group, and showed a certain degree of dose-dependent.8. The effect of NAV-LAO on angiogenesis of chick chorioallantoic membrane (CAM)The antiangiogenesis effect in vivo of NAV-LAO on CAM assay was observed and the the ratio of area of blood vessel and area of CAM in measure area (VA/CAM) was used to asses the inhibition.The result showed that NAV-LAO inhibited the angiogenesis in vivo: the morphology of blood vessels on CAM were basically normal,but the blood vessels distributed scarcely .The VA/CAM was less than control group significantly.Conclusion: The use of OPD detected L-amino acid oxidase with high sensitivity and repeatability. Through the two-step chromatography from the Naja atra venom , a Pure NAV-LAO was obtained and the molecular weight of 58KD. Through in vitro and in vivo experiments with the NAV-LAO found that had anti-angiogenesis effect.