Characterization Of A New L-amino Acid Oxidase From Agkistrodon Blomhoffii Ussurensis Snake Venom

Snake venom comprise mixtures of various bioactive proteins, polypeptides, metal ions, carbohydrates, amines and trace amounts of lipids. L-amino acid oxidases (LAAOs) are widely distributed in snake venoms and are thought to contribute to the toxicity upon envenomation via the oxidative stress arising from the production of H₂O₂. So far, various LAAOs have been purified from different snake venoms and identified to exhibit induction or inhibition of platelet aggregation, anticoagulant activity, stimulation of oedema formation, hemorrhage, and antimicrobial, antiviral, cytotoxic and antiproliferative effects on tumor cells, leishmanicidal functions, and anti-HIV activity. SV-LAAOs are very interesting bioactive substances showing a wide range of pharmacological activites.An L-amino acid oxidase (Akbu-LAAO) was isolated from the venom of Agkistrodon blomhoffii ussurensis snake using DEAE Sephadex A-50 ion-exchange, Sephadex G-75 gel filtration and high performance liquid chromatographies. It showed a single band on sodium-dodecyl sulfate–polyarylamide gel electrophoresis (SDS-PAGE). Combined result from MALDI-TOF masss spectroemtery analysis indicated that Akbu-LAAO was a homogenous molecule composed by two identical subunits with the molecular weight of ¹24.4 kDa. Tryptic in-gel digested peptides from was further analyzed by high performance liquid chromatography-electro-spray ionization tandem mass spectrometry. Protein identification was performed by TurboSequest Biowroks, and ten peptides from Akbu-LAAO, VVEELKR, YPVKPSEEGK, KFWEDEGIHGGK, VTVVYQTPAK, HDDIFAYEK, IKFEPPLPPK, SAGQLYEESLGK, FDEIVGGMDK, RFDEIVDGMDK, and FDEIVDGMDKLPTAM#YR were sequenced by mass spectroetry analysis. Although protein sequence alignment indicated that the Akbu-LAAO shares homologies with peptides of LAAOs from the venoms of Calloselasma rhodost, Agkistrodon halys, Daboia russellii siamensis, and Trimeresurus stejnegeri, Akbu-LAAO is a novel snake venom LAAO. Horseradish peroxide coupling method anlysis indicated that Akbu-LAAO reacts optimally with its enzymatic substrate, L-Leu, at pH 4.7 with a Km of 2.1 mM. Inductively coupled plasma-atomic emission spectroscopy (ICP-AES) measurements showed that four Zn²⁺ ions are contained per Akbu-LAAO. Zn²⁺ is unessential for the hydrolytic activity of Akbu-LAAO. However, the maximum emission fluorescence intensity of Akbu-LAAO decreases by 61% on removal of Zn²⁺ by indicating that zinc probably helps maintain structural integrity of Akbu- LAAO. The additions of exogenous metal ions, including Mg²⁺, Mn²⁺, Ca²⁺, Ce³⁺, Nd³⁺, Co²⁺ and Tb³⁺, increase the L-Leu hydrolytic activity of the enzyme, on the other hand, Ni²⁺, La³⁺ and Gd³⁺ slightly decrease the hydrolytic activity of Akbu-LAAO, Fe²⁺, Cu²⁺and Eu²⁺/Eu³⁺ have little effect on its hydrolytic activity. Akbu-LAAO shows apparent anti-aggregation and de-aggregation effects on human and rabbit platelets with different effects, this difference in degree of the inhibition of platelet aggregation for different animals suggests that animal blood platelets may not be a good model for human blood platelets, the result showed that Akbu-LAAO will play an important role in treating cardiovascular disease. It also exhibits a strong bacteriostasis effect on Staphylococcus aureus, 18 fold that of cephalosporin C under the same conditions. Akbu-LAAO shows promise as a potential antibacterial agent.Taken together, a novel LAAO is obtained from Agkistrodon blomhoffii ussurensis snake venom by a four-step purification. It shows promise for drug development value and clinical application. The resusts from current work on the characterizations of the biochemical, enzymatic, structural and biological propertises of Akbu-LAAO provide useful clues for its further delevopment and application.