Isolation,purification And Characterization Of Two Key Components ?Aminopeptidase And Phospholipase A₂? From Snake Venom

Snakebite envenoming has been defined by WHO as a neglected tropical disease that took place in tropical or subtropical poor rural areas.Snakebite envenoming usually causes physical impairment,economical burden,and psychological panic sequelae to snakebite victims.The first prerequisite of snakebite is to understand the composition of snake venom and the mechanism of its pathogenesis.Previous research showed that,the snake venom was a complex mixture,containing a variety of enzymes,proteins,bioactive peptides and inorganic compounds.Because of the different species of venomous snakes,the relative contents of these compounds may vary and they mainly target at the nervous system,blood system,and muscle systems of the victims,causing injuries or disorders in the cardiovascular system,nervous system,or muscle systems.Therefore,it is very important to understand the components and functions of snake venom for saving the snakebitten victims.Chinese Cobra?Naja atra?is one of the species of venomous snakes in China,and its venom contain neurotoxins and haemotoxins,which produced the neurotoxicity and haemotoxicity.Although many studies on the main components of the snake venom have been reported,there is still a lack of fully systematic research on the composition of the snake venom,especially,the research on aminopeptidase of the snake venom is not yet reported.Therefore,this study,for the first time,mainly aims at isolating and identifying aminopeptidase from the snake venom since no report has been made on the aminopeptidase identified from the N.atra venom,and also PLA₂ was isolated,purified and identified from the N.atra venom because there are several PLA₂ subtypes in the snake venom.These studies provide the basis for understanding the molecular mechanisms of the toxic pathogenesis of snakebite envenoming.After a series of experiments,we obtained the main results,which will be summarized as follows:1.By the bite-dish method to collect the fresh venom,the venom was dried under the low-temperature into the powder,the N.atra venom carried on the Hitrap-sepharos Fast flow cation exchange column,then the peak with the aminopeptidase activity was loaded on the Amastatin-ah Sepharose 4b affinitycolumn.Finally,the components with aminopeptidase activity were obtained.The component was identified on the reductive 10%SDS-PAGE,and the results showed a single band and its molecular weight was about 150 kD;also HPLC analysis showed that the obtained component presented a single peak.These results indicated that the component obtained with aminopeptidase activity may be almost a single component.The final concentration of purified target protein was 1.4mg/ml examined by BCA.2.The experiment results about the activity and properties of aminopeptidase component obtained indicated that,five kinds of synthetic substrates?Asp-pNA,Leu-pNA,Glu-pNA,Arg-p NA and Ala-pNA?were used to test the hydrolysis activity of the component from snake venom,and only Glu-pNA among them was the most suitable substrate;the optimum temperature of the hydrolysis was 35?;the experiments about the effect of metal ions on the activity of aminopeptidase component showed that some metal ions(such as Ba²⁺,Ca²⁺,Sr²⁺etc.)could significantly increase the activity of the enzyme,but also metal ions(such as Ni²⁺,Cu²⁺,Zn²⁺,Hg²⁺and Cd²⁺)significantly inhibited the activity of the enzyme.In particular,when the concentrations of these metal ions reached up to 1 mm,the ions completely inhibited the activity of the enzyme;the experiments about the effect of pH values on the activity of aminopeptidase component showed that the enzyme can completely hydrolyze the substrate when the pH of the reaction solution was 8.5.The aminopepidase isolated from the snake venom were mixed with the IgY antibody,the results showed that there was no neutralizing effect between aminopepidase and the IgY antibody.The result indicated that the amount of aminopepidase component was very low in the snake venom,and it did not cause the production of anti-aminopepidase component antibody;3.By two steps,Hitrap-sepharos Fast flow cation exchange chromatography and HPLC chromatography,PLA₂ was isolated from the venom,which was identified by SDS-PAGE as a single band,and its molecular weight was approximately 17 kD;The component was analyzed by HPLC and showed a single peak.These results suggested that the components obtained with PLA₂ activity as a single component.4.The PLA₂ activity assay was used to test the enzyme activity,and the influence effect of temperature,pH,and metal ions against on the enzyme activity.The experiment showed that with increasing pH,the egg yolk hydrolysis activity would be increased,and when the pH was about 10,the hydrolysis activity was the strongest;the optimum temperature for the enzyme to hydrolyze egg yolk was 37?.Some ions like K⁺,Na⁺,and Ca²⁺increased the enzyme activity of PLA₂,while Zn²⁺and Fe²⁺inhibited the activity of the enzyme.5.We immunized chickens with N.atra venom,and obtained the anti-snake venom Ig Y antibody form egg yolk.The PLA₂ isolated from the snake venom were mixed with the IgY antibody,the result showed that the PLA₂ and the IgY showed a dose-effect relationship of neutralization,and its ED₅₀0 was 4.568?g.The result indicated that the amount of PLA₂ was rather high in the sanke venom,so it may cause the production of anti-PLA₂ component antibody.In summary,we successfully obtained two key components,aminopeptidase and PLA₂,from N.atra venom,and their enzyme activities and some properties were preliminarily measured and analyzed,and the effect of anti-snake venom IgY neutralizing the activities of these two components was preliminarily evaluated.These results provided a basis for the preparation of anti-snake venom antibody and for further understanding the molecular mechanism of the snakebite envenoming.However,the amino acid sequence and structure of the two components will be need to be studied in the future.