CN100584943C - Method for preparing trimeresurus albolabris snake venom 5'-nucleotidase - Google Patents
Method for preparing trimeresurus albolabris snake venom 5'-nucleotidase Download PDFInfo
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Abstract
The invention discloses a method for preparing Trimeresurus albolabris snake venom 5'-nucleotidase. The method comprises the following steps in sequence: (1) pre-treating trimeresurus albolabris snake venom; (2) pre-fractionating through DEAE-SepHadex A-25 anion-exchange chromatography; (3) purifying through SepHadex-G-100 chromatography; and (4) further purifying through CM-SepHadexC -50 ion-exchange chromatography. The method has the advantages that the Trimeresurus albolabris 5'-nucleotidase is extracted from the Trimeresurus albolabris snake venom and purified for the first time, the preparation cost is low, and the purity and the enzyme activity of the prepared Trimeresurus albolabris 5'-nucleotidase is high.
Description
Technical field
The present invention relates to a kind ofly prepare the method for enzyme from animal, specifically, relate to and from the Trimeresurus albolabris (Gray) snake venom, prepare 5 '-method of phosphonuclease.
Background technology
At present, cardiovascular and cerebrovascular diseases such as atherosclerosis, coronary heart disease, cerebral thrombosis have also become the height morbidity of serious harm human health, and a large amount of clinical observation and experiments show, platelet function abnormality, particularly platelet aggregation is unusual, and is in close relations with the generation of these diseases.Therefore, the important step of this class disease of platelet aggregation-against treatment becoming control.The medicine of at present used platelet aggregation-against roughly is divided three classes: (1) suppresses the medicine of platelet economy enzyme.Mainly contain acetylsalicylic acid (Aspirin), prostaglin X (Epoprostenol), Dipyridamole (Dipyridamole); (2) specificity suppresses the medicine of ADP activated blood platelet, as ticlopidine (Ticlopidine); (3) thrombocyte GP IIb/IIIa receptor blocking agent is as A Baixima (Abciximab).Though these medicines have good inhibitory effect to platelet aggregation, all can produce some untoward reactions, as blood pressure drops, heart rate acceleration, headache, dizzy, diarrhoea, hyporrhea, fash and gastrointestinal reaction etc.
Along with thrombocyte physiology, progressively the illustrating of biochemical function, many novel anticoagulants also constantly are being found.People have found to have the active ingredient of anticoagulant from multiple snake venom, in these components wherein a class be 5 in the snake venom '-phosphonuclease, it has the obvious suppression effect to platelet aggregation.[Yu Xiaodong such as Yu Xiaodong, Huang Linong, Xiong Yuliang. green bamboo snake (Trimeresurus Stejnegeri) snake venom 5 '-phosphonuclease is to the inhibition Mechanism Study of platelet aggregation, Chongqing Normal University's journal (natural science edition), 1997,14 (2): 61-68.] from green bamboo snake Yunnan subspecies (T.stejnegeri yunnanensis) venom, isolate 5 '-phosphonuclease.This preparation method's the first step with the DEAE-SepHadexA-50 chromatography column from venom, extract 5 '-the phosphonuclease primary extract, second step was used CM-SepHarose Cl-6B chromatography column purifying, and then be further purified with the method chromatography column of QAE-SepHadexA-50 chromatography column and SepHadexG-100, obtain at last to platelet aggregation inhibited 5 '-phosphonuclease.But this preparation method is the prepared in laboratory method, 5 of gained '-phosphonuclease purity is low, and, in the preparation process to 5 '-the Nucleotide loss of activity is big.
The contriver finds in the research of Trimeresurus albolabris (Gray) (Trimeresurus albolabris) snake venom first, Trimeresurus albolabris (Gray) (Trimeresurus albolabris) snake venom 5 '-phosphonuclease has restraining effect to hematoblastic gathering, but from Trimeresurus albolabris (Gray), prepare 5 at present '-phosphonuclease do not appear in the newspapers.
Summary of the invention
The object of the present invention is to provide a kind of Trimeresurus albolabris (Gray) snake venom 5 '-preparation method of phosphonuclease, with the Trimeresurus albolabris (Gray) 5 that from the Trimeresurus albolabris (Gray) snake venom, prepares high purity, enzymatic activity high '-phosphonuclease.
To achieve these goals, the present invention by following steps from the Trimeresurus albolabris (Gray) snake, prepare 5 '-phosphonuclease:
(1) pre-treatment of Trimeresurus albolabris (Gray) snake venom: get the Trimeresurus albolabris (Gray) snake venom, added concentration in 1: 3 by volume～1: 6 to and be 0.01～0.05mol/L, pH and be in 8.0～9.0 the Tris-HCl damping fluid, in temperature is that 4～20 ℃, rotating speed are centrifugal 10～20min under the condition of 3000～5000r/min, and it is standby to get supernatant liquor;
(2) DEAE-Sephadex A-25 chromatography column initial gross separation: the Tris-HCl damping fluid balance pillar of using concentration 0.01～0.05mol/L, pH8.0～9.0 earlier, supernatant liquor with step (1) gained adds on the DEAE-Sephadex A-25 chromatography column then, use the Tris-HCl buffer solution elution of concentration 0.01～0.05mol/L, pH8.0～9.0 again, elution flow rate is 18～36ml/h, collect elutriant, cryoconcentration, obtain crude product a;
DEAE-Sephadex A-50 is a weak base anion exchanger.After changing the CL-type into the OH-type with NaOH, adsorbable acidic protein.In the venom 5 '-phosphonuclease is basic protein (iso-electric point is pH8.2), all the other all belong to acidic protein.In alkaline environment, acidic protein is all adsorbed by DEAE-SepHadex A-50, have only 5 '-phosphonuclease is not adsorbed.Therefore, by column chromatography 5 '-phosphonuclease just can be in wash-out flows out earlier, other albumen then are attracted on the post, thus just separable 5 '-phosphonuclease.
(3) Sephadex-G-100 chromatography column purifying: the Tris-HCl damping fluid balance pillar of using concentration 0.01～0.05mol/L, pH7.0～8.0 earlier, then crude product a is dissolved in again concentration and is 0.01～0.05mol/L, pH and be in 7.0～8.0 the Tris-HCl damping fluid, add on the SepHadex-G-100 chromatography column, with concentration is that 0.01～0.05mol/L, pH are 7.0～8.0 Tris-HCl buffer solution elution, elution flow rate is 3～12ml/h, collect elutriant with test tube, with elutriant merging, cryoconcentration, obtain crude product b;
Trimeresurus albolabris (Gray) snake venom 5 '-molecular weight of phosphonuclease is about 48.03kDa, select for use the SepHadex-G-100 chromatography column with Trimeresurus albolabris (Gray) snake venom 5 '-phosphonuclease distinguishes.
(4) CM-Sephadex C-50 is further purified: the NaAc damping fluid balance pillar of using concentration 0.01～0.05mol/L, pH 5.5～6.6 earlier, then crude product b is dissolved in concentration 0.01～0.05mol/L, pH and is in 5.5～6.6 the NaAc damping fluid, adding on the CM-Sephadex C-50 chromatography column, is 5.5～6.6 NaAc buffer solution elution again with concentration 0.01～0.05mol/L, pH; Be 5.5～6.6NaAc damping fluid linear gradient elution with concentration 0.01～0.05mol/L, pH then, the concentration range of NaCl is 0.02 to 1mol/L in the elutriant, and elution flow rate is 18～24ml/h, collects freeze-drying behind the elution peak, desalination of this moment, the acquisition product.
CM-Sephadex C-50 is a weakly acidic cation exchanger, in sour environment, 5 '-phosphonuclease is adsorbed.Therefore, earlier all the other impurity are flowed out earlier by column chromatography, 5 '-phosphonuclease then is attracted on the post, then by the NaCl linear gradient elution, thereby just separable 5 '-phosphonuclease.
Products obtained therefrom is detected purification effect through electrophoretic method and HPLC column chromatography.
Electrophoretic method detects: product is detected purification effect (resolving gel concentration 15% concentrates gum concentration 5%) with the SDS-polyacrylamide gel electrophoresis, and the intact back of electrophoresis is with coomassie brilliant blue R250 dyeing, with the mixed solution decolouring that contains 25% ethanol and 10% Glacial acetic acid.As shown in Figure 1, electrophoretogram is rendered as single band.
The HPLC column chromatography detects: get product 20mg, be dissolved in 750 μ l 0.1%TFA solution, the centrifugal 5nim of 4500r/min, get supernatant liquor, be splined on the C18 post of crossing with the 0.1%TFA solution equilibria, with 0-66.5% acetonitrile (being dissolved in 0.1%TFA solution) gradient elution, use 66.5% acetonitrile 0.1%TFA eluant solution at last.Flow velocity: 2ml/min, 1ml/ manages collection.As shown in Figure 2, the next single protein peak of wash-out in the time of 10-12 minute.
This shows, the Trimeresurus albolabris (Gray) snake venom 5 of the present invention preparation '-not containing other impurity in the phosphonuclease, purity is higher.
With finally the preparation 5 '-the pure product of phosphonuclease, with AMP is substrate, measure its active power, 5 '-the active power of Nucleotide is 330.33 μ g Pi/min/mg, when being substrate 5 with ADP '-the active power of Nucleotide is 123.56 μ g Pi/min/mg, therefore, the Trimeresurus albolabris (Gray) snake venom 5 of the present invention preparation '-activity of 5 '-nucleotidase is higher.
Desalination method in the above-mentioned preparation process (4) adopts Sephadex G-25 chromatography column desalination.
Freeze drying process is for being lower than vacuum lyophilization under-50 ℃ of temperature in the above-mentioned preparation process (4).
Beneficial effect of the present invention:
(1) the present invention set up first utilize chromatography from Trimeresurus albolabris (Gray) (Trimeresurusalbolabris) snake venom, separate and purifying 5 '-phosphonuclease and be suitable for the preparation method of industrial applications;
(2) the prepared Trimeresurus albolabris (Gray) 5 of the present invention '-phosphonuclease purity height, enzymic activity is big.
(3) the selected filling cost of material of the present invention is cheap, and the whole process of preparation cost is low;
Description of drawings
Fig. 1 be Trimeresurus albolabris (Gray) snake venom 5 '-the SDS-PAGE collection of illustrative plates of phosphonuclease;
Fig. 2 be Trimeresurus albolabris (Gray) snake venom 5 '-the HPLC column chromatography chromatogram figure of phosphonuclease;
Fig. 3 is DEAE-Sephadex A-25 column chromatography figure;
Fig. 4 is Sephadex G-100 column chromatography figure;
Fig. 5 is CM-Sephadex C-50 column chromatography figure;
Fig. 6 be Trimeresurus albolabris (Gray) snake venom 5 '-phosphonuclease is to the influence of ADP (20uM) inductive rabbit platelet aggregation.
Embodiment
Further the present invention is illustrated below in conjunction with embodiment:
(1) preparation process
The pre-treatment of step (1) Trimeresurus albolabris (Gray) snake venom: at first employing is stung the ware method of gathering venom and is gathered snake venom from the Trimeresurus albolabris (Gray) snake, with the snake venom stored refrigerated of gathering.
Get the Trimeresurus albolabris (Gray) snake venom, added concentration in 1: 3 by volume to and be 0.01mol/L, pH and be in 8.0 the Tris-HCl damping fluid, centrifugal 20min under 4 ℃, the condition of 5000r/min, it is standby to get supernatant liquor.
The initial gross separation of step (2) DEAE-Sephadex A-25 chromatography column:
DEAE-SephadexA-25 ion exchange layer analysis method: chromatographic column: 2.0cm * 60cm glass packed column; Filler: DEAE-Sephadex A-25; Detect wavelength: 280nm; Flow velocity: 18ml/h.
Use the Tris-HCl damping fluid balance pillar of concentration 0.01mol/L, pH8.0 earlier, supernatant liquor with step (1) gained adds on the DEAE-SephadexA-25 chromatography column then, use the Tris-HCl buffer solution elution of concentration 0.01mol/L, pH8.0 again, collect elutriant with the 4/mL test tube, reduce to below 0.05 up to the A280 absorption value, merge all elutriants, low temperature reflux concentrates, and obtains crude product a.
Separating spectrum as shown in Figure 3, wash-out goes out 4 peaks, carry out 5 to collecting all peaks '-activity of 5 '-nucleotidase detects, and finds that the strongest active peak is the 1st peak.
Step (3) Sephadex-G-100 chromatography column purifying:
Sephadex-G-100 chromatography method: chromatographic column: 1.0cm * 100cm glass packed column; Filler: Sephadex-G-100; Detect wavelength: 280nm; Flow velocity: 3ml/h.
Use the Tris-HCl damping fluid balance Sephadex-G-100 chromatography column of concentration 0.01mol/L, pH7.0 earlier, then crude product a is dissolved in again concentration and is 0.01mol/L, pH and be in 7.0 the Tris-HCl damping fluid, add on the SepHadex-G-100 chromatography column, add on the SepHadex-G-100 chromatography column, with concentration is that 0.01mol/L, pH are 7.0 Tris-HCl buffer solution elution, collect elutriant with test tube, reduce to below 0.05 up to the A280 absorption value, elutriant merging, low temperature reflux are concentrated, obtain crude product b;
Separating spectrum as shown in Figure 4, wash-out goes out 3 peaks, carry out 5 to collecting all peaks '-activity of 5 '-nucleotidase detects, and finds that the strongest active peak is the 2nd peak.
Step (4) CM-Sephadex C-50 is further purified:
CM-Sephadex C-50 ion exchange layer analysis method: chromatographic column: 2.0cm * 30cm glass packed column; Filler: DEAE-SephadexA-25; Detect wavelength: 280nm; Flow velocity: 18ml/h.
Use the NaAc damping fluid balance CM-Sephadex C-50 chromatography column of concentration 0.01mol/L, pH 5.5 earlier, then crude product b is dissolved in concentration 0.01mol/L, pH and is in 5.5 the NaAc damping fluid, adding on the CM-Sephadex C-50 chromatography column, is 5.5 NaAc buffer solution elution again with concentration 0.01mol/L, pH; Then with concentration 0.01mol/L, pH 5.5NaAc damping fluid linear gradient elution, the concentration range of NaCl is 0.02 to 1mol/L in the elutriant, and elution flow rate is 18ml/h, collect the elution peak of this moment, separating spectrum washes 3 peaks as shown in Figure 5, wherein peak 3 tools 5 '-activity of 5 '-nucleotidase.Peak 3 is merged collection,, obtain product with being lower than vacuum lyophilization under-50 ℃ of temperature behind the Sephadex G-25 chromatography column desalination.
(2) purity qualification test:
With the preparation 5 '-the pure product of phosphonuclease, detect purification effect (resolving gel concentration 15% concentrates gum concentration 5%) with the SDS-polyacrylamide gel electrophoresis, the intact back of electrophoresis is with coomassie brilliant blue R250 dyeing, with the mixed solution decolouring that contains 25% ethanol and 10% Glacial acetic acid.As shown in Figure 1, electrophoretogram is rendered as single band.
With the preparation 5 '-the pure product of phosphonuclease, detect purification effect with the HPLC column chromatography: get the sample 20mg that obtains at last, be dissolved in 750 μ l 0.1%TFA solution, the centrifugal 5nim of 4500r/min, get supernatant liquor, be splined on the C18 post of crossing with the 0.1%TFA solution equilibria,, use 66.5% acetonitrile 0.1%TFA eluant solution at last with 0-66.5% acetonitrile (being dissolved in 0.1%TFA solution) gradient elution.Flow velocity: 2ml/min, 1ml/ manages collection.As shown in Figure 2, the next single protein peak of wash-out in the time of 10-12 minute.
(3) target compound qualification test:
For whether the crude product a that detects gained is the target product that needs preparation, crude product a carried out the platelet aggregation-against test and with AMD be substrate measure 5 '-the activity of 5 '-nucleotidase test.
The platelet aggregation-against test method is: fresh rabbit blood is collected in the plastics tubing of silanization, and with (V/V) anti-freezing in 9: 1 of 3.8% Trisodium Citrate, the centrifugal 10min of room temperature (1000r/min) gets upper strata liquid and is platelet rich plasma (PRP).Residue blood continues centrifugal 15min (3000r/min), isolates platelet poor plasma (PPP), as the platelet count of surveying among periodic contrast or the adjusting PRP.Press BornShi turbidimetry principle, adopt the LBY-NJ4 platelet aggregation instrument to measure.Test-results is shown in Figure 6.(A is contrast, the curve of platelet aggregation when not containing 5 '-phosphonuclease among Fig. 6; B, C, D are for adding 25 μ g/ml respectively, the curve of platelet aggregation when 50 μ g/ml and 100 μ g/ml)
5 '-the activity of 5 '-nucleotidase test method is as follows:
Reaction system is formed: (10-1000mg/ml fits over and contains 1.2 * 10-2M MgCl snake venom solution
20.05M pH 8.8 glycine buffers in) 0.2ml, 3.6 * 10
-2M AMP (, transferring pH to 8.8) 1.0ml with above-mentioned damping fluid configuration, reaction cumulative volume 1.2ml.Behind 37 ℃ of insulation 30min, add 1ml 10% trichoroacetic acid(TCA) stopped reaction, place 20min, filter, get the generation of filtrate 1ml, go out to measure the change of absorbancy at 660nm by Fiske-Subbarrow method mensuration inorganic phosphorus.Measurement result is: Trimeresurus albolabris (Gray) snake venom 5 '-the active power of Nucleotide is 330.33 μ g Pi/min/mg, Trimeresurus albolabris (Gray) snake venom 5 when being substrate with ADP '-the active power of Nucleotide is 123.56 μ g Pi/min/mg.
The pre-treatment of step (1) Trimeresurus albolabris (Gray) snake venom: with embodiment 1, the pH of different is Tris-HCl damping fluid is 8.3.
The initial gross separation of step (2) DEAE-Sephadex A-25 chromatography column:
The pH of Tris-HCl damping fluid is 8.3, and with 25ml/h flow velocity wash-out DEAE-Sephadex A-25 chromatography column, all the other steps are identical with embodiment 1.
Step (3) Sephadex-G-100 chromatography column purifying:
The pH of Tris-HCl damping fluid is 7.6, and with 8ml/h flow velocity wash-out Sephadex-G-100 chromatography column chromatography column, all the other steps are identical with embodiment 1.
Step (4) CM-Sephadex C-50 is further purified:
The pH value of NaAc damping fluid is 5.8, and the flow velocity during the liquidus gradient elution is 20ml/h, and all the other steps are identical with embodiment 1.。
The pre-treatment of step (1) Trimeresurus albolabris (Gray) snake venom:
Get the Trimeresurus albolabris (Gray) snake venom, added concentration in 1: 6 by volume to and be 0.05mol/L, pH and be in 9.0 the Tris-HCl damping fluid, in temperature is that 20 ℃, rotating speed are centrifugal 10min under the condition of 3000r/min, and it is standby to get supernatant liquor, and all the other steps are identical with embodiment 1.
(2) DEAE-Sephadex A-25 chromatography column initial gross separation: the Tris-HCl damping fluid balance pillar of using concentration 0.05mol/L, pH9.0 earlier, supernatant liquor with step (1) gained adds on the DEAE-SephadexA-25 chromatography column then, use the Tris-HCl buffer solution elution of concentration 0.05mol/L, pH9.0 again, elution flow rate is 36ml/h, collect elutriant, cryoconcentration, obtain crude product a; All the other steps are identical with embodiment 1.
(3) Sephadex-G-100 chromatography column purifying:
Use the Tris-HCl damping fluid balance pillar of concentration 0.05mol/L, pH8.0 earlier, then crude product a is dissolved in again concentration and is 0.05mol/L, pH and be in 8.0 the Tris-HCl damping fluid, add on the SepHadex-G-100 chromatography column, with 12ml/h flow velocity wash-out, collect elutriant with test tube, with elutriant merging, cryoconcentration, obtain crude product b; All the other steps are identical with embodiment 1.
(4) CM-Sephadex C-50 is further purified:
Use the NaAc damping fluid balance pillar of concentration 0.05mol/L, pH 6.6 earlier, then crude product b is dissolved in concentration 0.05mol/L, pH and is in 6.6 the NaAc damping fluid, adding on the CM-Sephadex C-50 chromatography column, is 6.6 NaAc buffer solution elution again with concentration 0.05mol/L, pH; Be 6.6NaAc damping fluid linear gradient elution then with concentration 0.05mol/L, pH, the concentration range of NaCl is 0.02 to 1mol/L in the elutriant, elution flow rate is 24ml/h, collect elution peak, with being lower than vacuum lyophilization under-50 ℃ of temperature behind the Sephadex G-25 chromatography column desalination, the acquisition product.All the other steps are identical with embodiment 1.
Claims (3)
- A Trimeresurus albolabris (Gray) snake venom 5 '-preparation method of phosphonuclease, it is characterized in that may further comprise the steps:(1) pre-treatment of Trimeresurus albolabris (Gray) snake venom: get the Trimeresurus albolabris (Gray) snake venom, added concentration in 1: 3 by volume～1: 6 to and be 0.01～0.05mol/L, pH and be in 8.0～9.0 the Tris-HCl damping fluid, in temperature is that 4～20 ℃, rotating speed are centrifugal 10～20min under the condition of 3000～5000r/min, and it is standby to get supernatant liquor;(2) DEAE-Sephadex A-25 chromatography column initial gross separation: the Tris-HCl damping fluid balance pillar of using concentration 0.01～0.05mol/L, pH8.0～9.0 earlier, supernatant liquor with step (1) gained adds on the DEAE-Sephadex A-25 chromatography column then, use the Tris-HCl buffer solution elution of concentration 0.01～0.05mol/L, pH8.0～9.0 again, elution flow rate is 18～36ml/h, collect elutriant, cryoconcentration, obtain crude product a;(3) Sephadex-G-100 chromatography column purifying: the Tris-HCl damping fluid balance pillar of using concentration 0.01～0.05mol/L, pH7.0～8.0 earlier, then crude product a is dissolved in again concentration and is 0.01～0.05mol/L, pH and be in 7.0～8.0 the Tris-HCl damping fluid, add on the Sephadex-G-100 chromatography column, with concentration is that 0.01～0.05mol/L, pH are 7.0～8.0 Tris-HCl buffer solution elution, elution flow rate is 3～12ml/h, collect elutriant with test tube, with elutriant merging, cryoconcentration, obtain crude product b;(4) CM-Sephadex C-50 is further purified: the NaAc damping fluid balance pillar of using concentration 0.01～0.05mol/L, pH 5.5～6.6 earlier, then crude product b is dissolved in concentration 0.01～0.05mol/L, pH and is in 5.5～6.6 the NaAc damping fluid, adding on the CM-Sephadex C-50 chromatography column, is 5.5～6.6 NaAc buffer solution elution again with concentration 0.01～0.05mol/L, pH; Be 5.5～6.6NaAc damping fluid linear gradient elution with concentration 0.01～0.05mol/L, pH then, the concentration range of NaCl is 0.02 to 1mol/L in the elutriant, and elution flow rate is 18～24ml/h, collects freeze-drying behind the elution peak, desalination of this moment, the acquisition product.
- According to the described Trimeresurus albolabris (Gray) snake venom 5 of claim 1 '-preparation method of phosphonuclease, it is characterized in that: the desalination method in the described preparation process (4) adopts Sephadex G-25 chromatography column desalination.
- According to the described Trimeresurus albolabris (Gray) snake venom 5 of claim 1 '-preparation method of phosphonuclease, it is characterized in that: freeze drying process is for being lower than vacuum lyophilization under-50 ℃ of temperature in the described preparation process (4).
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| 竹叶青（Trimeresurus stejnegeri）蛇毒5’-核苷酸酶对血小板聚集功能的抑制机制研究. 余晓东等.重庆师范学院学报（自然科学版），第14卷第2期. 1997 * |
| 竹叶青（Trimeresurus stejnegeri）蛇毒5'-核苷酸酶对血小板聚集功能的抑制机制研究. 余晓东等.重庆师范学院学报（自然科学版），第14卷第2期. 1997 |
| 竹叶青（Trimeresurus stejnegeri）蛇毒－对5’-核苷酸酶的分离纯化及理化和酶学性质研究. 余晓东等.重庆师范学院学报（自然科学版），第13卷第4期. 1996 * |
| 竹叶青（Trimeresurus stejnegeri）蛇毒－对5'-核苷酸酶的分离纯化及理化和酶学性质研究. 余晓东等.重庆师范学院学报（自然科学版），第13卷第4期. 1996 |
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