CN104593344B - Agkistrodon acutus hemocoagulase atrox C - Google Patents
Agkistrodon acutus hemocoagulase atrox C Download PDFInfo
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- CN104593344B CN104593344B CN201510042881.8A CN201510042881A CN104593344B CN 104593344 B CN104593344 B CN 104593344B CN 201510042881 A CN201510042881 A CN 201510042881A CN 104593344 B CN104593344 B CN 104593344B
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- pbs
- agkistrodon acutus
- nacl
- snake venom
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- 230000002427 irreversible effect Effects 0.000 description 1
- 238000001155 isoelectric focusing Methods 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
- C12N9/6418—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals from snakes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21106—Hepsin (3.4.21.106)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
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- Genetics & Genomics (AREA)
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- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Enzymes And Modification Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention provides a kind of Agkistrodon acutus hemocoagulase atrox C, it is isolated a kind of Hx from Agkistrodon acutus snake venom, molecular weight 29213.4D, isoelectric point 5.7.The enzyme is made up of two chains of α, β, and interchain has the amino acid sequence shown in SEQ ID No.1 by disulfide bond, wherein α chains, and β chains have the amino acid sequence shown in SEQ ID No.2.Agkistrodon acutus hemocoagulase atrox of the present invention is serine protease.Serine protease of the present invention is obtained with following purification process, insoluble matter and foreign protein are removed including being pre-processed by ammonium sulfate precipitation, pass through anion exchange chromatography twice again, collect active eluting peak, through dialyse, be concentrated by ultrafiltration and desalination after produce high purity venom Hx, its Rate activity is not less than 40U/mg albumen, and HPLC purity assays count purifying recovery rate as 0.4%-0.5% up to more than 95%, using snake venom raw material weight.
Description
The present invention is the divisional application of following inventions：
The applying date：On 02 21st, 2013；Application number：201310055128.3；Denomination of invention：Agkistrodon acutus hemocoagulase atrox-
C。
Technical field
The present invention relates to a kind of serine protease, specifically a kind of snake venom blood coagulation enzyme-C, the invention further relates to it
Isolation and purification method.
Background technology
According to the report of domestic and foreign literature, in Crotalinae（Crotalinae）It is more in the presence of a kind of related to blood clotting in snake venom
Protease, normally referred to as " batroxobin "（Thrombin-like enzyme, referred to as：TLC）.Batroxobin and blood coagulation
Enzyme（thrombin）Effect it is similar, can make in blood plasma fibrinogen be converted into fibrin and " solidification ".So far
It was found that containing batroxobin composition in more than 30 kinds of snake venom, and there are more than 20 kinds to be isolated and purified, wherein there are more than 10 species
All or part of amino acid sequence of fibrin ferment is elucidated.The TLC molecular weight having found is more between 29~45kD, most of
For acidoglycoprotein.
In the snake venom TLC having been found that in the past, prlmary structure of protein is mostly single-stranded.Its representative products " vertical root of Dahurian angelica snow "
（Reptilase）It is by Brazilian spearhead pallas pit viper（Bothrops atrox）The fibrin ferment isolated in snake venom, the enzyme precursor is by 255
Individual Amino acid profile, N-terminal have 24 amino acids formed guiding peptides, and organized enzyme contains 231 amino acid, and relative molecular weight 39~
43kD, it is single chain glycoprotein.Zhai Ning etc.（Shanghai Institute of Pharmaceutical Industry ＆ Zhejiang Haizheng Pharmaceutical Co）Report within 2005：From Anhui south five
Walk snake（Agkistrodon acutus）In be separated to a kind of " batroxobin ", there is hemoglutination.SDS-PAGE is shown as single
Chain, reduction and non-reduced electrophoresis molecular weight are respectively 59.25kD and 52.58kD, and display has intrachain disulfide bond, is than work
41.5/u/mg phenylmethylsulfonyl fluoride（PMSF）The irreversible inactivation of the enzyme can be caused.
Research closely during the last ten years finds in Crotalinae snake venom TLC that there is also duplex molecule structure interchain is by disulfide bond
Connection.Xin Cheng etc.（Chinese University of Science and Technology）Report within 1999：From long-noded pit viper（Agkistrodon acutus）In be separated to
A kind of " batroxobin ", is named as " Agkisacutacin ".The albumen is made up of two peptide chains, and α-molecular weight subunit 15kD, β-
Molecular weight subunit 14kD.Agkisacutacin can be in hydrolysis of fibrin original α chains.Xiao Changhua（Chinese Academy of Sciences Kunming animal is ground
Study carefully institute）2004 from long-noded pit viper（Agkistrodon acutus）In be separated to two kinds " batroxobins ", be duplex molecule knot
Structure.The A subunits of fibrin ferment I contain 132 amino acid, molecular weight 16kD；B subunits contain 123 amino acid, molecular weight 14kD,
Specific enzyme activity is：160U/mg.The A subunits of fibrin ferment II contain 122 amino acid, molecular weight 15kD；B subunits contain 120 ammonia
Base acid, molecular weight 13kD, specific enzyme activity are：70U/mg.Two kinds of TLC prove through pharmacological evaluation：It is respectively provided with hemostasia effect.Tang
Guia Hill 2004 is from long-noded pit viper（Agkistrodon acutus）In be separated to a kind of " batroxobin ", there is hemoglutination.Should
Enzyme is made up of two subunits of 17kD and 15kD, isoelectric point 5.9.
The present invention illustrate from the Guangxi agkistrodon acutus in China (Agkistrodon acutu) isolated a kind of new in snake venom
Snake venom blood coagulation enzyme-- Agkistrodon acutus hemocoagulase atrox-C.
The content of the invention
It is an object of the invention to provide a kind of snake venom blood coagulation enzyme, and it is isolated one kind from Agkistrodon acutus snake venom
Batroxobin-C.
It is another object of the present invention to provide a kind of method for isolating and purifying above-mentioned Hx.
Hx-C of the present invention is from Chinese agkistrodon acutus（Agkistrodon acutus）Isolated blood in snake venom
Solidifying enzyme.The enzyme has following feature：1. zymoprotein contains 252 amino acid, molecular weight 29213.4D, isoelectric point pI are 5.7.②
It is made up of two chains of α, β, interchain is by disulfide bond.3. α chains contain 129 amino acid, molecular weight 14661.7D, its amino acid
Sequence is as shown in SEQ ID No.1；β chains contain 123 amino acid, molecular weight 14551.7D, its amino acid sequence such as SEQ ID
Shown in No.2.4. enzymatic activity can be by phenylmethylsulfonyl fluoride（PMSF) completely inhibit, it is a kind of serine protease to show it.
The present invention also provides the purification process of above-mentioned Hx, and it comprises the following steps：
1）, snake venom pre-processes through ammonium sulfate precipitation；
2）, by the DEAE-Sephrose FF anion exchange chromatography through pre-equilibration on pretreated snake venom solution,
Post is washed with the PBS of 0.01M pH7.0~7.5, then is divided with the PBS of 0.01M pH7.0~7.5 of the NaCl Han 0.02 and 0.06M
Section elution, collects 0.06M first eluting peak of NaCl；
3）, will above-mentioned eluent suitably concentration after dialysis or through repeatedly dilution ultrafiltration remove NaCl；
4）, by the solution after dialysis again on the DEAE-Sephrose FF chromatographic columns through pre-equilibration, use 0.01M
The PBS of pH7.0~7.5 washes post, then is eluted with the PBS of 0.01M pH7.0~7.5 of the NaCl containing 0.05M, collects 0.05M
Second eluting peak of NaCl elutions；
5）, will above-mentioned eluent suitably concentration after with distilled water dialyse or use Sephadex-G25 post desalinations.
Wherein, step 1）The method of snake venom pretreatment is that snake venom is molten with 0.01M pH7.0~7.5 PBS of appropriate precooling
Solution, supernatant is collected by centrifugation, ammonium sulfate precipitation, collects 70% ammonium sulfate precipitation, it is saturating to precipitate the progress after PBS suspends dissolving
Analyse (bag filter molecular cut off is 7,000D ~ 10,000D), or using cross-flow ultrafiltration（Retaining molecular weight be 5,000D~
10,000D）Method, the repeated multiple times dilution of the lysate that will suspend, desalination is concentrated by ultrafiltration.It can be removed by pretreatment insoluble miscellaneous
Matter and part foreign protein, and reduce solution ion strength.
Specifically, the pretreatment of snake venom can be carried out as follows：Some grams of snake venom is weighed, with snake venom weight 5 ~ 10
The PBS of 0.01M pH7.0~7.5 of times volume precooling stirring and dissolving 30 ~ 60 minutes in 4 ~ 8 DEG C of chromatography cabinet, to lysate
In be slowly added ammonium sulfate to 50% saturation degree, stand 2-4 hours, after 4 ~ 8 DEG C, 5,000 ~ 10,000g centrifugations 10 ~ 30
Minute.Centrifuged supernatant is collected, then ammonium sulfate is slowly added to 70% saturation degree into centrifuged supernatant, is stood overnight, next day
Centrifuged 10 ~ 30 minutes in 4 ~ 8 DEG C, 5,000 ~ 10,000g.Centrifugation is taken to add the PBS of appropriate 0.01M pH7.0~7.5 to suspend
Dissolve to obtain lysate.Lysate is poured into bag filter (molecular cut off 7,000D ~ 10,000D), in cabinet is chromatographed 4 ~ 8 DEG C it is right
The PBS of 0.01M pH7.0~7.5 12 ~ 24 hours, during which changes solution 2 ~ 4 times；Or by lysate molecular weight cutoff
For 5,000 ~ 10,000D milipore filter ultrafiltration concentration desalination is diluted through repeated multiple times addition PBS.
As described above, dialysis step（Depending on liquor capacity）Cross-flow ultrafiltration can be used（Retaining molecular weight is 5,000
~10000D）Method replaces, and is diluted, is concentrated by ultrafiltration by PBS, then dilute, the mode that is concentrated by ultrafiltration again to be to remove micromolecule polypeptide
Solution ion strength is reduced with desalination.
Wherein, step 2）With step 4）The PBS pre-equilibrations DEAE-Sephrose of 0.01M pH7.0~7.5 can be used
FF chromatographic columns, then loading.
Wherein, step 3）With step 5）Purpose be remove solution present in NaCl.Small size eluent can be direct
Dialysed, large volume eluent can be diluted by PBS, is concentrated by ultrafiltration, then dilute, the mode that is concentrated by ultrafiltration again is with protein concentrate
And slough NaCl.The enzyme concentrate being finally purified can directly use Sephadex-G25 post desalinations.
Solution directly freezed after desalination is dried, or adds freeze drying protectant freeze-drying.The freeze drying protectant can be with
It is D-40, mannitol, sucrose, glycerine, gelatin etc..The respective addition of different cryoprotectors is 0.1%-2%
(w/v)。
The blood clotting specific activity of enzyme purified through the inventive method is not less than 40U/mg albumen, polyacrylamide gel electrophoresis
（PAGE）One band, reduce SDS- polyacrylamide gel electrophoresises（Reduce SDS-PAGE）Two bands；HPLC purity assays 95% with
On.In terms of snake venom raw material weight, this law purifying recovery rate is 0.4%-0.5%.
Hx of the present invention has agglutination activity, can be made into various haemostatic medicaments, such as through being suitably diluted to regulation enzyme activity
Unit, then add freeze drying protectant（Gelatin or human serum albumin etc.）, through viral membrane filtration, medical injection jelly is made in freeze-drying
Dry powder pin, for stopping blooding in surgical operation, and various clinical bleedings.It may be made as wound external application hemostatic plaster, pulvis
Or liquid spray.The present invention provides a kind of new Hx, expands Hx species, improves snake venom utilization rate.
Brief description of the drawings
Fig. 1 shows Hx after purification through polyacrylamide gel electrophoresis（PAGE）For a band（Shown in arrow）.
Fig. 2 shows Hx-C after purification through reducing SDS- polyacrylamide gel electrophoresises（RD-SDS-PAGE）For two
Band（Shown in arrow）, wherein M is Protein Marker, and 1 is Hx-C after purification.
Fig. 3 shows the HPLC analysis results of Hx-C after purification.
Embodiment
Following examples further illustrate present disclosure, but should not be construed as limiting the invention.Without departing substantially from
In the case of of the invention spirit and essence, the modifications or substitutions made to the inventive method, step or condition belong to the present invention
Scope.
Unless otherwise specified, the conventional meanses that technological means used in embodiment is well known to those skilled in the art.
The percentage sign " % " being related in the present invention, if not specified, refers to mass percent；But the percentage of solution,
Unless otherwise specified, refer to contain some grams of solute in solution 100ml；Percentage between liquid, refer to the capacity at 20 DEG C
Ratio." with the 10 times of volume precoolings of snake venom weight " statement similar in the present invention, the unit difference of weight and volume therein
It is g and ml.
Snake venom blood coagulation enzyme-the C of embodiment 1 purifying
Take 20g Agkistrodon acutus snake venom dry powder（Lot number：20061001, purchased from Snake Venoms From Guangxi research institute）, with snake venom weight 10
The 0.01M pH7.4 of times volume precooling sodium phosphate buffer（PBS）The stirring and dissolving 60 minutes in 4 DEG C of chromatography cabinet, in
4 DEG C, 10000g centrifuge 10 minutes, collect supernatant.Centrifugation adds the 0.01M of 5 times of volume precoolings of snake venom weight again
PH7.4 PBS agitator treatings, are centrifuged again.Merge centrifuged supernatant twice.Delay under agitation into centrifuged supernatant
The slow ammonium sulfate that adds stands 4 hours at persistently being stirred 20 minutes, 4 DEG C again after ammonium sulfate is completely dissolved, made to 50% saturation degree
Albumen precipitation is complete.The solution is centrifuged 20 minutes in 4 DEG C, 10000g afterwards, collects supernatant.Under agitation on this
Ammonium sulfate is slowly added in clear liquid, ammonium sulfate saturation degree is risen to 70%.Treating ammonium sulfate, thoroughly dissolving is complete, continues 20 points of stirring
Clock, 4 DEG C stand overnight.Next day centrifuges 20 minutes under 4 DEG C, 10000g.Abandoning supernatant, collecting precipitation protein.Protein precipitation
Load bag filter after being dissolved with 80ml 0.01M pH7.4 PBS（Molecular weight is 10000D by value）In, use 0.01M
PH7.4 PBS carries out being slowly stirred dialysis, changes liquid once within during which every 6 hours, changes liquid altogether 3 times.Dialysis is taken out after the completion of dialysis
Liquid（105ml）.
DEAE-Sephrose FF fillers are filled in Φ 3.5cm × 30cm posts, balanced with 0.01M PBS (pH7.4)
Post, it is stand-by.
Dialyzate is loaded on post, post is washed with 0.01M pH7.4 PBS.With the 0.01M of the NaCl containing 0.02M
PH7.4 PBS elutions, then eluted with the 0.01M pH7.4 of the NaCl containing 0.06M PBS, collect first peak of elution.With
The 0.01M pH7.4 of the NaCl containing 1M PBS washes column regeneration.It is stand-by after balance with 0.01M pH7.4 PBS balance columns.
Through enzyme activity determination（With reference to annexOrMethod）And electrophoretic analysis, purpose thing appear in
In the eluting peak of 0.06M NaCl solutions（176ml）.The eluent is poured into bag filter, with 0.01M pH7.4
PBS in 4 DEG C dialysis, during which every 6 hours change solution once, change liquid altogether 3 times.
Dialyzate is loaded on post, post is rinsed with 0.01M pH7.4 PBS.With the 0.01M containing 0.05MNaCl
PH7.4 PBS elutions, collect second peak of elution（145ml）.Post is washed with the PBS of the 0.01M pH7.4 containing 1MNaCl again
It is raw.It is stand-by after balance with 0.01M pH7.4 PBS balance columns.
Through enzyme activity determination（With reference to annexOrMethod）And electrophoretic analysis, purpose thing appear in
In second eluting peak of 0.05M NaCl solutions.Measure concentration of protein in solution is 0.64mg/ml.The solution is used
Deionized water carries out dialysis 16 hours, during which changes liquid 3 times.
Total protein content is 93mg in dialysis solution, and directly freezed is dried.Freeze-dried powder measure specific activity of enzyme is 48U/mg
Albumen, ultimate yield 0.47%.PAGE is a band（See Fig. 1）, reduction SDS-PAGE is two bands（See Fig. 2）, its molecular weight
Each substantially 15kD and 14.5kD.HPLC purity assays 97.3%（See Fig. 3 and table 1）.Isoelectric focusing electrophoresis determines the enzyme etc.
Electric point pI is 5.7.
The HPLC quantitative results of table 1
Its amino acid sequence is determined using DENOVO methods, determines the amino acid sequence of two bands respectively such as SEQ ID No.1
Shown in SEQ ID No.2.α chains（SEQ ID No.1）Containing 129 amino acid, molecular weight 14661.7Dalton；β chains（SEQ
ID No.2）Containing 123 amino acid, molecular weight 14551.7Dalton.Complete Hx-C molecular weight is 29213.4Da.α chains
With β interchains by disulfide bond.
Snake venom blood coagulation enzyme-the C of embodiment 2 purifying
Take 50g Agkistrodon acutus snake venom dry powder（Lot number：20061101, purchased from Snake Venoms From Guangxi research institute）, with snake venom weight 10
The 0.01M pH7.4 of times volume precooling PBS stirring and dissolving 60 minutes in 4 DEG C of chromatography cabinet, in 4 DEG C, 10000g centrifugations 10
Minute, collect supernatant.The PBS stirrings that centrifugation adds the 0.01M pH7.4 of 5 times of volume precoolings of snake venom weight again are outstanding
It is floating, centrifuge again.Merge centrifuged supernatant twice.Ammonium sulfate is slowly added into centrifuged supernatant under agitation to 50%
Saturation degree, 4 hours are stood at persistently being stirred 20 minutes, 4 DEG C again after ammonium sulfate is completely dissolved, makes albumen precipitation complete.Afterwards
The solution is centrifuged 20 minutes in 4 DEG C, 10000g, collects supernatant.
Ammonium sulfate is slowly added into the supernatant under agitation, ammonium sulfate saturation degree is risen to 70%.Treat ammonium sulfate
Thoroughly dissolving is complete, continues to stand overnight at stirring 20 minutes, 4 DEG C.Next day centrifuges 20 minutes under 4 DEG C, 10000g.Discard
Clear liquid, collecting precipitation protein.Protein precipitation 200ml 0.01M pH7.4 PBS dissolves, and is fitted into bag filter, uses 0.01M
PH7.4 PBS is stirred dialysis, changes liquid once within every 6 hours, changes liquid altogether 3 times.Dialyzate is taken out after the completion of dialysis
（223ml）.
DEAE-Sephrose FF fillers are filled in Φ 5cm × 30cm posts, are buffered with pH7.4 0.01M sodium ascorbyl phosphates
After liquid balance columns.By dialyzate loading.Post is washed with 0.01M pH7.4 PBS.With the 0.01M pH7.4 of the sodium chloride containing 0.02M
PBS elution.Eluted again with the 0.01M pH7.4 of the sodium chloride containing 0.06M PBS, collect first peak of elution（395ml）.
Post is washed with the 0.01M pH7.4 of the sodium chloride containing 1M PBS.It is stand-by after balance with 0.01M pH7.4 PBS balance columns.
Through enzyme activity determination（With reference to annexOrMethod）And electrophoretic analysis, purpose thing appear in 0.06M NaCl solutions
In eluting peak.The eluent is poured into bag filter, with 0.01M pH7.4 PBS in 4 DEG C of dialysis, changes liquid one within during which every 6 hours
It is secondary, liquid is changed altogether 3 times.
By in dialyzate loading to post.Post is washed with 0.01M pH7.4 PBS.With the 0.01M of the NaCl containing 0.05M
PH7.4 PBS takes off post, collects second peak of elution（375ml）.Post is washed with the 0.01M pH7.4 of the sodium chloride containing 1M PBS
Regeneration.With 0.01M pH7.4 PBS balance columns.It is stand-by after balance.
The eluent is poured into bag filter, with 0.01M pH7.4 PBS in 4 DEG C of dialysis, changes liquid one within during which every 6 hours
It is secondary, liquid is changed altogether 3 times.Total protein content is 230mg in dialysis solution, is directly freeze-dried.Freeze-dried powder specific activity of enzyme is
50U/mg albumen, ultimate yield 0.46%.PAGE and reduction SDS-PAGE and HPLC chromatogram are consistent with embodiment 1,
HPLC purity assays 98.1%.
Snake venom blood coagulation enzyme-the C of embodiment 3 purifying
Weigh 100 grams of Agkistrodon acutus snake venom dry powder（Lot number 20061101, purchased from Snake Venoms From Guangxi research institute）With 1 liter of precooling
0.01M pH7.4 PBS stirring and dissolving 60 minutes in 4-8 DEG C of chromatography cabinet, 4 DEG C of 10000g are centrifuged 20 minutes, in collection
Clear liquid.Centrifugation adds the 0.01M pH7.4 of 5 times of volume precoolings of snake venom weight PBS stirring suspensions again, centrifuges again.
Merge centrifuged supernatant twice.Ammonium sulfate is slowly added into centrifuged supernatant under agitation to 50% saturation degree, treats sulfuric acid
Ammonium stands 4 hours at persistently being stirred 20 minutes, 4 DEG C again after being completely dissolved, and makes albumen precipitation complete.Afterwards by the solution in 4 DEG C,
10000g is centrifuged 20 minutes, collects supernatant.Ammonium sulfate is slowly added into the supernatant under agitation, ammonium sulfate is satisfied
70% is risen to degree.Treating ammonium sulfate, thoroughly dissolving is complete, stops stirring after continuing stirring 20 minutes, 4 DEG C stand overnight.Next day is in 4
DEG C, 10000g centrifuge 20 minutes.Abandoning supernatant, collecting precipitation protein.Protein precipitation 400ml pH7.4 0.01M phosphoric acid
Sodium salt buffer solution, is fitted into bag filter, is dialysed with 0.01M pH7.4 PBS, changes liquid once within every 6 hours, changes liquid altogether
3 times.Dialyzate is taken out after the completion of dialysis.
DEAE-Sephrose FF fillers are filled in Φ 7cm × 30cm posts, after 0.01M pH7.4 PBS balance columns.
By dialyzate loading.Post is washed with 0.01M pH7.4 PBS.Eluted with the PBS of the 0.01M pH7.4 containing 0.02MNaCl, then
Eluted with the PBS of the 0.01M pH7.4 containing 0.06MNaCl.Collect first peak of elution（876ml）.With containing 1MNaCl's
0.01M pH7.4 PBS washes column regeneration, stand-by after balance with 0.01M pH7.4 PBS balance columns.
Through enzyme activity determination（With reference to annexOrMethod）And electrophoretic analysis, purpose thing appear in 0.06M NaCl solutions
In eluting peak.With the tangential flow ultrafilters of Millipore Pellicon 2（0.1M2Cut off 5k films）Eluent ultrafiltration is dense
200ml is reduced to, adds 1 liter of 0.01M pH7.4 of precooling PBS, then ultrafiltration is to 200ml.Ultrafiltration concentration process circulation 3
It is secondary.
It will be concentrated by ultrafiltration on liquid loading to post, be eluted with the PBS of the 0.01M pH7.4 containing 0.05MNaCl.Collect 0.05M
Second eluting peak of NaCl solution（810ml）.Column regeneration is washed with the 0.01M pH7.4 of the NaCl containing 1M PBS, uses 0.01M
PH7.4 PBS balance columns, it is stand-by after balance.
By the eluent tangential flow ultrafilters of Millipore Pellicon 2（0.1M2Cut off 5k films）Ultrafiltration is dense
It is reduced to 200ml.By in the concentrate loading to Sephadex-G25 posts, post desalination is washed with deionized water, collects eluting peak
230ml.It is 503mg to determine total protein in the solution, and Rate activity is 47U/mg albumen, ultimate yield 0.5%.
Desalination collection liquid PAGE and reduction SDS-PAGE and HPLC chromatogram are consistent with embodiment 1.HPLC purity reaches
97.5%.
1% mannitol, 0.5% gelatin are added as freeze drying protectant by desalination collection liquid volume.After filtrate packing cillin bottle
It is freeze-dried.
Snake venom blood coagulation enzyme-the C of embodiment 4 serine stretch protein attribute experiment
Hx-C freeze-dried powders isolated in embodiment 1 are dissolved with deionized water, and it is 1U/ to be diluted to enzymatic activity
ml。
With the BFG of normal saline 1%（Sigma companies）Solution.
Phenylmethylsulfonyl fluoride is dissolved with isopropanol（PMSF, Merck company）, solution concentration 4mg/ml.
Laboratory operating procedures are as follows：
（1）Take 1% BFG solution 2ml, constant temperature 5 minutes at 37 DEG C.
（2）Three small test tubes are taken, mark 1#, 2#, 3# respectively, often pipe adds 200 μ l blood clotting enzyme solutions.
（3）10 μ l distilled water are added to 1# test tubes respectively, 2# test tubes add 10 μ l isopropanols, and 3# test tubes add 10 μ l
PMSF, 5 minutes are incubated in 37 DEG C of water-baths.
（4）Agglutination test observation is individually carried out by test tube number order.1% N of good fibre of constant temperature is added into test tube
The μ l of fibrillarin original solution 200, timing immediately, while mixing is gently shaken, stood in 37 DEG C of water-baths, it is anti-to observe aggegation in test tube
Situation about answering.Timing is terminated with solution white floc sedimentation.
It the results are shown in Table 2
The influence of table 2, PMSF to the aggegation time
| Test tube number | 1% fibrinogen solution | Blood clotting enzyme solutions 1U/ml | Examine or check content | The aggegation time |
| 1# | 200μl | 200μl | 10 μ l distilled water | 56 seconds |
| 2# | 200μl | 200μl | 10 μ l isopropanols | 58 seconds |
| 3# | 200μl | 200μl | 10μl PMSF | >600 seconds |
According to the experimental result in table one, draw to draw a conclusion：1. the PMSF of 100ppm concentration completely inhibit the blood clotting
Enzymatic activity, it was demonstrated that the Agkistrodon acutus hemocoagulase atrox is serine protease.2. micro isopropanol is on this agglutinating reaction without influence.
Annex
Agkistrodon acutus hemocoagulase atrox unit definition and activity determination method
1. BFG determination methodTake 1.0% BFG of normal saline（Sigma companies）Solution
1ml is put in small test tube, and 37 ± 0.5 DEG C of water-baths are incubated 3 minutes, add the enzyme solutions 1ml to be measured of 37 ± 0.5 DEG C of preheatings, count immediately
When, fibrinogen solution shook in 120 ± 30 seconds there is white floc sedimentation, then the enzyme solutions are 1U/ml.
2. standard human plasma's determination methodTake standard human plasma 1ml to put in small test tube, put in 37 DEG C of ± 0.5 DEG C of water-baths and preheat 3
Minute, the enzyme solutions 1ml to be measured of 37 ± 0.5 DEG C of preheatings is added, timing immediately, it is white that human plasma shook appearance in 60 ± 20 seconds
Floc sedimentation, then the enzyme solutions are 1U/ml.
Note：It need to be diluted when determining unknown enzymatic activity high solution with deionized water, be used to determine until reaching 1U/ml；
Its extension rate is the enzyme-activity unit number in every milliliter of protoenzyme solution.
Sequence table
<110>Beijing Konruns Pharmaceutical Co., Ltd.
<120>Agkistrodon acutus hemocoagulase atrox-C
<130>
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 129
<212> PRT
<213> Agkistrodon acutus
<400> 1
Asp Cys Ser Ser Gly Trp Ser Ser Tyr Glu Gly His Cys Tyr Lys
1 5 10 15
Val Phe Lys Gln Ser Lys Thr Trp Ala Asp Ala Glu Ser Phe Cys
20 25 30
Thr Lys Gln Val Asn Gly Gly His Leu Val Ser Ile Glu Ser Ser
35 40 45
Gly Glu Ala Asp Phe Val Gly Gln Leu Ile Ala Gln Lys Ile Lys
50 55 60
Ser Ala Lys Ile His Val Trp Ile Gly Leu Arg Ala Gln Asn Lys
65 70 75
Glu Lys Gln Cys Ser Ile Glu Trp Ser Asp Gly Ser Ser Ile Ser
80 85 90
Tyr Glu Asn Trp Ile Glu Glu Glu Ser Lys Lys Cys Leu Gly Val
95 100 105
His Ile Glu Thr Gly Phe His Lys Trp Glu Asn Phe Tyr Cys Glu
110 115 120
Gln Gln Asp Pro Phe Val Cys Glu Ala
125
<210> 2
<211> 123
<212> PRT
<213> Agkistrodon acutus
<400> 2
Asp Cys Pro Ser Asp Trp Ser Ser Tyr Glu Gly His Cys Tyr Lys
1 5 10 15
Pro Phe Asn Glu Pro Lys Asn Trp Ala Asp Ala Glu Asn Phe Cys
20 25 30
Thr Gln Gln His Thr Gly Ser His Leu Val Ser Phe Gln Ser Thr
35 40 45
Glu Glu Ala Asp Phe Val Val Lys Leu Ala Phe Gln Thr Phe Asp
50 55 60
Tyr Gly Ile Phe Trp Met Gly Leu Ser Asn Ile Trp Asn Gln Cys
65 70 75
Asn Trp Gln Trp Ser Asn Ala Ala Met Leu Lys Tyr Thr Asp Trp
80 85 90
Ala Glu Glu Ser Tyr Cys Val Tyr Phe Lys Ser Thr Asn Asn Lys
95 100 105
Trp Arg Ser Ile Thr Cys Arg Met Ile Ala Asn Phe Val Cys Glu
110 115 120
Phe Gln Ala
Claims (3)
1. Agkistrodon acutus hemocoagulase atrox-C, the enzyme is made up of two subunits of α, β, and wherein α subunits have the ammonia shown in SEQ ID No.1
Base acid sequence；β subunits have the amino acid sequence shown in SEQ ID No.2, are isolated from Chinese agkistrodon acutus (Agkistrodon
Acutus) its molecular weight is 29213.4D, and isoelectric point pI is 5.7, and it is obtained by following purification process：
1), Chinese agkistrodon acutus (Agkistrodon acutus) snake venom is pre-processed through ammonium sulfate precipitation；
2), by the DEAE-Sepharose Fast Flow anion exchange layers through pre-equilibration on pretreated snake venom solution
Post is analysed, post is washed with the PBS of 0.01M pH7.0~7.5, then with 0.01M pH7.0~7.5 of the NaCl containing 0.02M and 0.06M
PBS stepwise elutions, collect first eluting peak of 0.06M NaCl solutions；
3), NaCl is removed by dialysis after above-mentioned eluent suitably concentration or through diluting to be concentrated by ultrafiltration repeatedly；
4), by the solution after dialysis again on the DEAE-Sepharose Fast Flow chromatographic columns through pre-equilibration, use 0.01M
The PBS of pH7.0~7.5 washes post, then is eluted with the PBS of 0.01M pH7.0~7.5 of the NaCl containing 0.05M, collects 0.05M
Second eluting peak of NaCl solution；
5), it will be dialysed after above-mentioned collection eluent suitably concentration with deionized water or remove NaCl through Sephdex-G25 posts.
2. the medicine containing Agkistrodon acutus hemocoagulase atrox described in claim 1.
3. medicine as claimed in claim 2, it is freeze-dried powder, hemostatic plaster or liquid spray.
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| CN101560510A (en) * | 2008-09-27 | 2009-10-21 | 北京方策方程医药科技有限公司 | Agkistrodon acutus hemocoagulase atrox |
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| Title |
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| 尖吻蝮蛇类凝血酶的研究现状;欧光武 等;《中国医药导报》;20100430;第7卷(第12期);9-11 * |
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Address after: 101500 Miyun Economic Development Zone, Beijing, No. 11 South Road Applicant after: BEIJING KONRUNS PHARMACEUTICAL Co.,Ltd. Address before: 100085, A, building 7, building 9, Wah Wah building, 707 3rd Street, Beijing, Haidian District Applicant before: BEIJING KONRUNS PHARMACEUTICAL Co.,Ltd. |
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