CN102242098A - Extraction method of prothrombin activator from bothrops atrox venom - Google Patents
Extraction method of prothrombin activator from bothrops atrox venom Download PDFInfo
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Abstract
The invention provided an extraction method of a prothrombin activator from bothrops atrox venom. The prothrombin activator is a protein with the activity of a prothrombin activating enzyme, and is collected by performing SP ion-exchange chromatography, dialysis, cellulose DEAE-Sepharose Fast Flow ion-exchange chromatography and Sephadex-G75 gel filtration chromatography on the bothrops atrox venom. The prothrombin activating enzyme provided by the invention can be used as an active component for preparing medicaments for treating clinical hemorrhage such as surgical hemorrhage, medical hemorrhage, gynecological hemorrhage, facial hemorrhage, pediatric hemorrhage, hemorrhage after tissue biopsy and the like, and treating hemorrhagic disease, so that the prothrombin activating enzyme can play an important role in the field of biological pharmacy and has wide application prospects.
Description
Technical field
The present invention relates to enzyme and extracting method thereof, a kind of extracting method that derives from the former activator of spearhead Agkistrodon halys venom thrombin particularly is provided.
Background technology
Thrombokinase is by activatory factor Xa, Va, Ca
2+And the complex body of phosphatide micelle formation.Factor X is activated as the committed step that Xa is this process.
Factor Va non-enzymatic activity, but can make 350 times of the increased activity of Xa, quicken the generation of zymoplasm.Borrow Ca between phosphatide micelle and enzyme (Xa) and the substrate (thrombogen)
2+Link to each other as bridge.Because of holding, the N of thrombogen peptide chain do not contain 10
γThe carboxyglutamic acid residue.Adjacent carboxyl can with Ca
2+Form complex body.On the other hand, Ca
2+Can combine with phosphate in the phosphatide again, Xa and Va and thrombogen be contacted, so Xa is hydrolyzed to zymoplasm with thrombogen.
Summary of the invention
The object of the present invention is to provide a kind of thrombogen activating enzyme that derives from the spearhead viper venom.
The present invention is for solving the problems of the technologies described above, take following technical scheme: a kind of thrombogen activating enzyme is to have the active albumen of thrombogen activating enzyme with what the spearhead viper venom carried out collecting behind SP ion exchange chromatography, Mierocrystalline cellulose DEAE-SephroseFast Flow ion exchange chromatography and the Sephadex-G75 gel permeation chromatography successively.
The thrombogen activating enzyme that derives from the spearhead viper venom of the present invention has following characteristics: thrombokinase is by activatory factor Xa, Va, Ca
2+And the complex body of phosphatide micelle formation.Factor X is activated as the committed step that Xa is this process.
Thrombokinase extracting method of the present invention may further comprise the steps:
(1) the spearhead viper venom is dissolved in the PBS damping fluid of pH6.0,0.05mol/L, obtaining concentration is the spearhead viper venom liquid of 100-120g/L;
(2) the spearhead viper venom liquid that step (1) is obtained carries out ion exchange chromatography, the solid phase weighting material is SP, elutriant is respectively the PBS damping fluid of pH6.0,0.05mol/L and contains the PBS damping fluid of pH6.0, the 0.05mol/L of 0.25mol/LNaCl, straight line wash-out;
(3) collect elutriant, under the wavelength of 280nm elutriant is carried out the ultraviolet spectrometry range and detect, the collection of illustrative plates that is absorbed is collected the part that contains the thrombogen activating enzyme;
(4) concentrate desalination, with the Tris-HCl damping fluid ultrafiltration and concentration of pH7-8,0.05mol/L and remove NaCl;
(5) ion exchange chromatography once more, the concentrated solution of step (4) carries out ion exchange chromatography, the solid phase weighting material is that Mierocrystalline cellulose solid phase weighting material is DEAE-Sepharose Fast Flow, and elutriant is the Tris-HCl damping fluid straight line gradient elution of the pH7-8, the 0.05mol/L that contain 0-0.2mol/L NaCl;
(6) each peak of elutriant that step (5) is obtained carries out SDS-PAGE gel electrophoresis and determination of activity, collects to contain thrombogen activating enzyme part;
(7) concentrate desalination once more, with the Tris-HCl damping fluid ultrafiltration and concentration of pH7.5,0.01mol/L and remove NaCl;
(8) step (7) concentrated solution is carried out gel permeation chromatography, the solid phase weighting material is Sephadex-G75, and elutriant is the Tris-HCl damping fluid of pH7.5,0.01mol/L, collects elutriant, therefrom collect and contain thrombogen activating enzyme part, obtain spearhead pallas pit viper thrombokinase stoste.
In the thrombokinase extracting method of the present invention, step (1) is for obtaining the better implement effect, can be earlier remove impurity in the spearhead viper venom liquid with centrifugation method, centrifugal condition can be selected according to practical situation, as centrifugal 15min under at 4500rpm.
In the thrombokinase extracting method of the present invention, the middle SP ion exchange column in the step (2) before use, earlier with the PBS damping fluid balance of pH6.0,0.05mol/L 5-6 hour, flow velocity was 3.0ml/min; During chromatography, after treating that the spearhead pit viper venom moves to below the glue face fully, above the glue face, add the damping fluid identical with balance liquid, cross the post wash-out with the PBS damping fluid of pH6.0,0.05mol/L then, PBS damping fluid with the pH6.0, the 0.05mol/L that contain 0.25mol/LNaCl carries out the straight line wash-out then, and flow velocity is 3.0ml/min;
In the thrombokinase extracting method of the present invention, the collection method in the step (3) can be: detect the collection of illustrative plates manual collection according to ultraviolet spectrometry range under the 280nm; Detection method with thrombogen activating enzyme part is: SDS-PAGE electrophoresis and blood coagulation activity detect.
In the thrombokinase extracting method of the present invention, step (4) concentrates desalination can select cutoff 10000 molecular weight film cross-flow ultrafiltrations for use, by the dilution of Tris-HCl damping fluid, the ultrafiltration and concentration with pH7-8,0.05mol/L, the redilution mode of ultrafiltration and concentration again reduces solution ion strength to remove micromolecule polypeptide and desalination.
In the thrombokinase extracting method of the present invention, Mierocrystalline cellulose DEAE-Sepharose Fast Flow ion exchange column before use in the step (5), the Tris-HCl damping fluid balance of first usefulness pH7-8,0.05mol/L 5-6 hour, flow velocity is 3.0ml/min; During chromatography, after treating that the spearhead pit viper venom moves to below the glue face fully, above the glue face, add the damping fluid identical with balance liquid, Tris-HCl damping fluid with the pH7-8 that contains 0.2mol/LNaCl, the 0.05mol/L of the Tris-HCl damping fluid of pH7-8,0.05mol/L and equivalent carries out the straight line gradient elution then, and flow velocity is 3.0ml/min; Tris-HCl damping fluid in the described step (5) is preferably the Tris-HCl damping fluid of pH7.5,0.05mol/L..
In the thrombokinase extracting method of the present invention, before the Sephadex-G75 chromatography column uses in the step (8), the Tris-HCl damping fluid balance of first usefulness pH7.5,0.01mol/L 5-6 hour, flow velocity is 3.0ml/min, during chromatography, treat supernatant liquor move to fully the glue face following after, above the glue face, add the buffer solution elution identical with balance liquid, flow velocity is 3.0ml/min, uses the method identical with step (3) to collect the part that contains the thrombogen activating enzyme in the elutriant.
The invention provides a kind of thrombogen activating enzyme that from the spearhead pit viper venom, extracts.Described thrombogen activating enzyme has following characteristics: (1) thrombokinase is by activatory factor Xa, Va, Ca
2+And the complex body of phosphatide micelle formation; (2) extracting method of this activator is based on the chromatographic technique of routine, realize the purpose of clean cut separation by conversion chromatography condition and regulation technology parameter, and can operate at normal temperatures, and have that condition is simple amplifies easily, cost low (the chromatography glue of selection can be recycled), the high and high advantage of product purity of yield.Based on These characteristics, can utilize that thrombogen activating enzyme of the present invention is hemorrhage for activeconstituents preparation treatment surgery, internal medicine is hemorrhage, Obstetric and Gynecologic Department are hemorrhage, department of eye is hemorrhage, paediatrics is hemorrhage and biopsy after the medicine of clinical hemorrhage and hemorrhagic diseases such as hemorrhage, to play a significant role in field of biological pharmacy, have a extensive future.
Embodiment
Below in conjunction with specific embodiment the present invention is described in further detail
Method therefor is ordinary method if no special instructions among the following embodiment.
Embodiment 1
The extracting method of the described thrombokinase of present embodiment is: get 10g spearhead viper venom dry powder (lot number 080709), PBS damping fluid stirring and dissolving 30min in 4-8 ℃ chromatography cabinet with pH6, the 0.05mol/L of 100ml precooling, the centrifugal 15min of 4500rpm gets supernatant liquor.Before the chromatography earlier with the SP chromatography column with the PBS damping fluid balance of pH6,0.05mol/L 5-6 hour, flow velocity is 3ml/min; During chromatography, after treating that the spearhead pit viper venom moves to below the glue face fully, above the glue face, add the damping fluid identical with balance liquid, cross the post wash-out with the PBS damping fluid of pH6,0.05mol/L then, PBS damping fluid with the pH6, the 0.05mol/L that contain 0.25mol/LNaCl carries out the straight line wash-out then, and flow velocity is 3.0ml/min; Detect the collection of illustrative plates manual collection according to ultraviolet spectrometry range under the 280nm, has thrombogen activating enzyme part through electrophoretic analysis and blood coagulation activity mensuration, merge elutriant, altogether 270ml, with cutoff 10000 molecular weight film cross-flow ultrafiltrations, by the dilution of Tris-HCl damping fluid, the ultrafiltration and concentration with pH7,0.05mol/L, the redilution mode of ultrafiltration and concentration again reduces solution ion strength, concentrating sample 50ml to remove micromolecule polypeptide and desalination; Last sample to cellulose fiber DEAE-Sepharose Fast Flow ion exchange column before use, earlier with the Tris-HCl damping fluid balance of pH7,0.05mol/L 5-6 hour, flow velocity was 3.0ml/min; During chromatography, after treating that the spearhead pit viper venom moves to below the glue face fully, above the glue face, add the damping fluid identical with balance liquid, the Tris-HCl damping fluid straight line gradient elution of the pH7 that contains 0.2mol/LNaCl, the 0.05mol/L of the Tris-HCl damping fluid of pH7,0.05mol/L and equivalent then, flow velocity is 3.0ml/min; Tris-HCl damping fluid with pH7.5,0.01mol/L concentrates desalination once more, concentrating sample 40ml, go up sample Sephadex-G75 chromatography column then, each last sample 10ml, the Tris-HCl damping fluid balance of usefulness pH7.5,0.01mol/L is 5-6 hour earlier, and flow velocity is 3.0ml/min, during chromatography, after treating that supernatant liquor moves to below the glue face fully, add the buffer solution elution identical with balance liquid above the glue face, flow velocity is 3.0ml/min.Collect sample 165ml, results protein 58 mg, the HPLC purity assay is 97.7%, SDS-PAGE is a band.
Embodiment 2
The extracting method of the described thrombokinase of present embodiment is: get 10g spearhead viper venom dry powder (lot number 080709), PBS damping fluid stirring and dissolving 30min in 4-8 ℃ chromatography cabinet with pH6, the 0.05mol/L of 100ml precooling, the centrifugal 15min of 4500rpm gets supernatant liquor.Before the chromatography earlier with the SP chromatography column with the PBS damping fluid balance of pH6,0.05mol/L 5-6 hour, flow velocity is 3ml/min; During chromatography, after treating that the spearhead pit viper venom moves to below the glue face fully, above the glue face, add the damping fluid identical with balance liquid, cross the post wash-out with the PBS damping fluid of pH6,0.05mol/L then, PBS damping fluid with the pH6, the 0.05mol/L that contain 0.25mol/LNaCl carries out the straight line wash-out then, and flow velocity is 3.0ml/min; Detect the collection of illustrative plates manual collection according to ultraviolet spectrometry range under the 280nm, has thrombogen activating enzyme part through electrophoretic analysis and blood coagulation activity mensuration, merge elutriant, altogether 270ml, with cutoff 10000 molecular weight film cross-flow ultrafiltrations, by the dilution of Tris-HCl damping fluid, the ultrafiltration and concentration with pH7,0.05mol/L, the redilution mode of ultrafiltration and concentration again reduces solution ion strength, concentrating sample 50ml to remove micromolecule polypeptide and desalination; Last sample to cellulose fiber DEAE-Sepharose Fast Flow ion exchange column before use, earlier with the Tris-HCl damping fluid balance of pH7.5,0.05mol/L 5-6 hour, flow velocity was 3.0ml/min; During chromatography, after treating that the spearhead pit viper venom moves to below the glue face fully, above the glue face, add the damping fluid identical with balance liquid, the Tris-HCl damping fluid straight line gradient elution of the pH7.5 that contains 0.2mol/LNaCl, the 0.05mol/L of the Tris-HCl damping fluid of pH7.5,0.05mol/L and equivalent then, flow velocity is 3.0ml/min; Tris-HCl damping fluid with pH7.5,0.01mol/L concentrates desalination once more, concentrating sample 40ml, go up sample Sephadex-G75 chromatography column then, each last sample 10ml, the Tris-HCl damping fluid balance of usefulness pH7.5,0.01mol/L is 5-6 hour earlier, and flow velocity is 3.0ml/min, during chromatography, after treating that supernatant liquor moves to below the glue face fully, add the buffer solution elution identical with balance liquid above the glue face, flow velocity is 3.0ml/min.Collect sample 202ml, results protein 65 mg, the HPLC purity assay is 98.6%, SDS-PAGE is a band.
Embodiment 3
The extracting method of the described thrombokinase of present embodiment is: get 10g spearhead viper venom dry powder (lot number 080709), PBS damping fluid stirring and dissolving 30min in 4-8 ℃ chromatography cabinet with pH6, the 0.05mol/L of 100ml precooling, the centrifugal 15min of 4500rpm gets supernatant liquor.Before the chromatography earlier with the SP chromatography column with the PBS damping fluid balance of pH6,0.05mol/L 5-6 hour, flow velocity is 3ml/min; During chromatography, after treating that the spearhead pit viper venom moves to below the glue face fully, above the glue face, add the damping fluid identical with balance liquid, cross the post wash-out with the PBS damping fluid of pH6,0.05mol/L then, PBS damping fluid with the pH6, the 0.05mol/L that contain 0.25mol/LNaCl carries out the straight line wash-out then, and flow velocity is 3.0ml/min; Detect the collection of illustrative plates manual collection according to ultraviolet spectrometry range under the 280nm, has thrombogen activating enzyme part through electrophoretic analysis and blood coagulation activity mensuration, merge elutriant, altogether 270ml, with cutoff 10000 molecular weight film cross-flow ultrafiltrations, by the dilution of Tris-HCl damping fluid, the ultrafiltration and concentration with pH7,0.05mol/L, the redilution mode of ultrafiltration and concentration again reduces solution ion strength, concentrating sample 50ml to remove micromolecule polypeptide and desalination; Last sample to cellulose fiber DEAE-Sepharose Fast Flow ion exchange column before use, earlier with the Tris-HCl damping fluid balance of pH8,0.05mol/L 5-6 hour, flow velocity was 3.0ml/min; During chromatography, after treating that the spearhead pit viper venom moves to below the glue face fully, above the glue face, add the damping fluid identical with balance liquid, the Tris-HCl damping fluid straight line gradient elution of the pH8 that contains 0.2mol/LNaCl, the 0.05mol/L of the Tris-HCl damping fluid of pH8,0.05mol/L and equivalent then, flow velocity is 3.0ml/min; Tris-HCl damping fluid with pH7.5,0.01mol/L concentrates desalination once more, concentrating sample 40ml, go up sample Sephadex-G75 chromatography column then, each last sample 10ml, the Tris-HCl damping fluid balance of usefulness pH7.5,0.01mol/L is 5-6 hour earlier, and flow velocity is 3.0ml/min, during chromatography, after treating that supernatant liquor moves to below the glue face fully, add the buffer solution elution identical with balance liquid above the glue face, flow velocity is 3.0ml/min.Collect sample 182ml, results albumen 55mg, the HPLC purity assay is 98%, SDS-PAGE is a band.
Claims (10)
1. extracting method that derives from the former activator of spearhead Agkistrodon halys venom thrombin, it is characterized in that: the former activator of described spearhead Agkistrodon halys venom thrombin is to have the active albumen of thrombogen activating enzyme with what the spearhead viper venom carried out collecting behind SP ion exchange chromatography, Mierocrystalline cellulose DEAE-Sephrose Fast Flow ion exchange chromatography and the Sephadex-G75 gel permeation chromatography successively; Described first time, the SP ion exchange chromatography carried out the straight line wash-out with the PBS damping fluid of pH6.0,0.05mol/L and the PBS damping fluid that contains pH6.0, the 0.05mol/L of 0.25mol/LNaCl respectively; Described Mierocrystalline cellulose DEAE-Sephrose FastFlow ion exchange chromatography carries out the straight line gradient elution with the Tris-HCl damping fluid of the pH7-8 that contains 0.2mol/LNaCl, the 0.05mol/L of the Tris-HCl damping fluid of pH7-8,0.05mol/L and equivalent; The described Sephadex-G75 gel permeation chromatography Tris-HCl buffer solution elution of pH7.5,0.01mol/L.
2. it is characterized in that according to the described extracting method that derives from the former activator of spearhead Agkistrodon halys venom thrombin of claim 1: described thrombokinase is by activatory factor Xa, Va, Ca
2+And the complex body of phosphatide micelle formation; Factor X is activated as the committed step that Xa is this process.
3. it is characterized in that according to the described extracting method that derives from the former activator of spearhead Agkistrodon halys venom thrombin of claim 1: the step of the preparation of the former activator of described spearhead Agkistrodon halys venom thrombin is:
(1) snake venom pre-treatment;
(2) the pre-treatment snake venom is carried out ion exchange chromatography, the solid phase weighting material is SP, carries out the straight line wash-out with the PBS damping fluid of pH6.0,0.05mol/L and the PBS damping fluid that contains pH6.0, the 0.05mol/L of 0.25mol/LNaCl respectively;
(3) collect elutriant, under the wavelength of 280nm elutriant is carried out the ultraviolet spectrometry range and detect, the collection of illustrative plates that is absorbed is collected the part that contains the thrombogen activating enzyme;
(4) above-mentioned elutriant is carried out ultrafiltration and concentration and remove NaCl;
(5) ion exchange chromatography once more, the concentrated solution of step 4) carries out ion exchange chromatography, the solid phase weighting material is DEAE-Sephrose Fast Flow, carries out the straight line gradient elution with the Tris-HCl damping fluid of the pH7-8 that contains 0.2mol/LNaCl, the 0.05mol/L of the Tris-HCl damping fluid of pH7-8,0.05mol/L and equivalent;
(6) collect elutriant, under the wavelength of 280nm elutriant is carried out the ultraviolet spectrometry range and detect, the collection of illustrative plates that is absorbed is collected the part that contains the thrombogen activating enzyme;
(7) ultrafiltration and concentration is removed NaCl;
(8) step (7) concentrated solution is carried out gel permeation chromatography, the solid phase weighting material is Sephadex-G75, and elutriant is the Tris-HCl damping fluid of pH7.5,0.01mol/L.
4. according to the preparation process of the former activator of the described spearhead Agkistrodon halys venom thrombin of claim 3, it is characterized in that: the pretreated method of described step (1) snake venom is with the PBS damping fluid dissolving of snake venom with pH6.0, the 0.05mol/L of an amount of precooling; Step (1) also comprises the step of removing the impurity in the spearhead viper venom liquid with centrifugation method.
5. according to the preparation process of the former activator of the described spearhead Agkistrodon halys venom thrombin of claim 1, it is characterized in that: the SP ion exchange column before use in the step (2), the PBS damping fluid balance of first usefulness pH6.0,0.05mol/L 5-6 hour, flow velocity is 3.0ml/min; During chromatography, after treating that the spearhead pit viper venom moves to below the glue face fully, above the glue face, add the damping fluid identical with balance liquid, carry out the straight line wash-out with the PBS damping fluid of pH6.0,0.05mol/L and the PBS damping fluid that contains pH6.0, the 0.05mol/L of 0.25mol/LNaCl respectively then, flow velocity is 3.0ml/min.
6. according to the preparation process of the former activator of the described spearhead Agkistrodon halys venom thrombin of claim 1, it is characterized in that: the collection method in the described step (3) is for detecting the collection of illustrative plates manual collection according to ultraviolet spectrometry range under the 280nm; Detection method with thrombogen activating enzyme active part is: with equal-volume and be preheated to 37 ℃ collection liquid and Quality Control blood plasma and be blended in 37 ℃ of following water-baths, collection unit that blood plasma solidified in 20 seconds is divided into have the thrombogen activating enzyme active, carries out the SDS-PAGE gel electrophoresis again and confirm.
7. according to the preparation process of the former activator of the described spearhead Agkistrodon halys venom thrombin of claim 1, it is characterized in that: the concentration method in the described step (4) is with the Tris-HCl damping fluid ultrafiltration and concentration of pH7-8,0.05mol/L and removes NaCl.
8. according to the preparation process of the former activator of the described spearhead Agkistrodon halys venom thrombin of claim 1, it is characterized in that: Mierocrystalline cellulose DEAE-Sephrose Fast Flow chromatography column before use in the described step (5), the Tris-HCl damping fluid balance of first usefulness pH7-8,0.05mol/L 5-6 hour, flow velocity is 3.0ml/min; During chromatography, after treating that the spearhead pit viper venom moves to below the glue face fully, above the glue face, add the damping fluid identical, carry out the straight line gradient elution then with balance liquid, flow velocity is 3.0ml/min, uses the method identical with step 3) to collect the part that contains the thrombogen activating enzyme in the elutriant.
9. according to the preparation process of the former activator of the described spearhead Agkistrodon halys venom thrombin of claim 1, it is characterized in that: the concentration method in the described step (7) is with the Tris-HCl damping fluid ultrafiltration and concentration of pH7.5,0.01mol/L and removes NaCl.
10. according to the preparation process of the former activator of the described spearhead Agkistrodon halys venom thrombin of claim 1, it is characterized in that: before the Sephadex-G75 chromatography column uses in the described step (8), the Tris-HCl damping fluid balance of first usefulness pH7.5,0.01mol/L 5-6 hour, flow velocity is 3.0ml/min, during chromatography, after treating that supernatant liquor moves to below the glue face fully, above the glue face, add the buffer solution elution identical with balance liquid, flow velocity is 3.0ml/min, uses the method identical with step (3) to collect the part that contains the thrombogen activating enzyme in the elutriant.
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| CN2010101667368A CN102242098A (en) | 2010-05-10 | 2010-05-10 | Extraction method of prothrombin activator from bothrops atrox venom |
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| CN2010101667368A CN102242098A (en) | 2010-05-10 | 2010-05-10 | Extraction method of prothrombin activator from bothrops atrox venom |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104531631A (en) * | 2014-12-23 | 2015-04-22 | 青岛康原药业有限公司 | Method for preparing venin antithrombotic enzyme |
| CN109943554A (en) * | 2017-12-21 | 2019-06-28 | 舒泰神（北京）生物制药股份有限公司 | A method of extracting factor X activator from snake venom |
-
2010
- 2010-05-10 CN CN2010101667368A patent/CN102242098A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104531631A (en) * | 2014-12-23 | 2015-04-22 | 青岛康原药业有限公司 | Method for preparing venin antithrombotic enzyme |
| CN109943554A (en) * | 2017-12-21 | 2019-06-28 | 舒泰神（北京）生物制药股份有限公司 | A method of extracting factor X activator from snake venom |
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Application publication date: 20111116 |