CN1057607C - Double functional chromatographic material and preparing method and use - Google Patents
Double functional chromatographic material and preparing method and use Download PDFInfo
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- CN1057607C CN1057607C CN95114252A CN95114252A CN1057607C CN 1057607 C CN1057607 C CN 1057607C CN 95114252 A CN95114252 A CN 95114252A CN 95114252 A CN95114252 A CN 95114252A CN 1057607 C CN1057607 C CN 1057607C
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- chromatographic material
- carrier
- double functional
- kallikrein
- phenyl
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Abstract
The present invention relates to a double-function chromatography material which has a structure: X-OCH2CH(OH)CH2O-R-O-C(NH)NH***, X=C6H5-, Y=NHC6H5C(NH)NH2, R represents a substrate material which comprises various kinds of agarose. The chromatography material adopts a special preparation method, and phenol and benzene pyrimidine are crosslinked with the same carrier and simultaneously have affinity and hydrophobic effects. The special adsorption for large biological molecules is increased. The present invention also provides a method for removing human urinary kallikrein from urine trypsinase and purifying kallikrein by using the chromatography material.
Description
The present invention relates to biochemical field.
The affinity chromatography technology is a kind of technology of carrying out separation and purification according to the biological characteristics and the chemical constitution of biomolecule, be about to aglucon that a kind of and separated material has specific adsorption and combine with carrier by direct or indirect method and make affinity adsorbent, be used for certain enzyme of separation and purification or protein.The hydrophobic chromatography technology is the method for carrying out separation and purification according to biomolecule surface hydrophobicity difference.
Phenol and epichlorokydrin reaction product and agarose (as: Sepharose) under given conditions is crosslinked, can make a kind of chromatographic material (as: Phenyl Sepharose, Pharmacia company product) with hydrophobic effect.
It is crosslinked by amino n-caproic acid of 6-and agarose that benzene is pricked pyrimidine (Benzamidine), can make a kind of affinity chromatographic material (as: Benzamidine SepharoseCL, Pharmacia company product).
Above-mentioned two kinds of chromatographic materials are existing product, but all have limitation as a kind of chromatographic material its specificity and selectivity in to the separation and purification of many components biological substance respectively.
The objective of the invention is to propose a kind of double functional chromatographic material and preparation method, and adopt this chromatographic material to remove the method for people's UK and purifying kallikrein in urinary trypsin inhibitor at above-mentioned defective.
Double functional chromatographic material of the present invention, satisfy following structural requirement:
Here X=C
6H
5_
Y＝_NHC
6H
5C(NH)NH
2
R represents a kind of agarose.
Preparation method's step of double functional chromatographic material of the present invention is as follows:
1) epoxy compound is synthetic:
Get phenol and epichlorokydrin and reflux under alkali condition, it is fully reacted, remove water layer, decompression distillation obtains 1,2-epoxy-3-phenoxypropane;
2) preparation of phenyl-carrier cross-linked composite:
Any water-fast agarose is as carrier material, and with carrier and an amount of 1,2-epoxy-3-phenoxypropane is the direct crosslinked phenyl-carrier complexes that makes at ambient temperature;
3) phenyl-carrier-benzene is pricked the preparation of pyrimidine cross-linked composite:
Phenyl-carrier complexes with cyanogen bromide or the activation of other activators, is added amino n-caproic acid of 6-and p-aminophenyl respectively and pricks the pyrimidine hydrochloride, make benzene and prick the pyrimidine double functional chromatographic material.
Adopt double functional chromatographic material of the present invention, to the suction-operated of people's UK, can successfully remove people's UK in the urinary trypsin inhibitor according under given conditions; And under certain PH and ionic strength conditions, carry out wash-out, be purified into people's UK.
Embodiment I
1. epoxy compound is synthetic:
Get 376 gram phenol and 748 gram epichlorokydrin, put in the three neck round-bottomed flasks that 2000ml is equipped with reflux condensing tube, tap funnel, reflux also drips 20% sodium hydroxide solution, 600 grams, refluxes 2 hours, puts cold.Pour the separating funnel branch vibration layer into, organic layer divides three washings with 200ml distilled water (adding 20% sodium hydroxide solution 1.0ml), removes water layer, adds the jolting of 50ml ether again and quickens layering.Organic layer with 100ml distillation moisture secondary washing, is removed water layer again, and organic layer adds 150 gram anhydrous sodium sulfates again and absorbs remaining moisture content.Organic layer is poured in the vacuum distillation apparatus, collected the cut liquid of boiling point about 110 ℃ in decompression distillation under the 5mmHg condition.Make 1,2-epoxy-3 phenoxypropane.
Reaction equation:
2. the preparation of phenyl-carrier cross-linked composite:
Get the sepharose 4B (Spharose 4B, Pharmacia company product) of about 3000ml precipitation volume, use the sintered filter funnel suction filtration,, use a large amount of distilled water flushings again, remove wherein protective agent and antiseptic with the washing of 0.5mol/L sodium chloride solution.1 of the NaOH of the 1mol/L of adding 2000ml, 240ml, 2-epoxy-3-benzene oxygen phenylpropyl alcohol alkane and 3 gram tetrahydro boron sodiums stirred 12 hours down in room temperature (30 ℃) at least.Be heated to 50 ℃ again, the tetrahydro boron sodium that adds 3 grams stirred 5 hours.Suction filtration after reaction finishes, the adding distil water washing.Put in 95% ethanol and soaked 12 hours, filter, be kept in 95% ethanol.Make phenyl-sepharose 4B compound.
Reaction equation:
Wherein: HO-R-OH represents the carrier sepharose 4B
3. phenyl-carrier-benzene is pricked the preparation of pyrimidine cross-linked composite:
Get the phenyl-sepharose 4B cross-linked composite of 1000ml precipitation volume.Suction filtration, wash with water three times, add 600ml water and 100 gram cyanogen bromides and put the middle dropping of cryosel bath (10 ℃) 2mol/L sodium hydroxide solution, making the pH value move to the room temperature rotation between remaining on 11.0～11.5 after 10 minutes poured in the sintered filter funnel after 10 minutes, using cold 0.1mol/L sodium bicarbonate (pH9.5) buffer solution to take out immediately washes, in 2～3 minutes, wash the buffer solution of 5000～6000ml volume, stop activation.
Add 500ml distilled water and the amino n-caproic acid of 100 gram 6-, regulate pH value to 9.5 and stir after 24 hours, suction filtration adds water washing.Add 500ml distilled water and 4 gram p-aminophenyls bundle pyrimidine hydrochlorides adjusting pH to 4.5 again, add 50 gram EDC mixings, put under the room temperature and stir more than 12 hours.Suction filtration adds water washing, put in 95% ethanol preserve standby.Make phenyl-sepharose 4B-benzene and prick the pyrimidine compound.
Reaction equation
(2)
EDC
(3)M-O-R-OC(NH)NH(CH
2)
5COOH+H
2N-C
6H
5-C(NH)NH
2*HCL→
Wherein: HO-R-OH represents the carrier sepharose 4B
M represents C
6H
5-OCH
2CH (OH) CH
2-
Embodiment II
Repeat embodiment I, replace sepharose 4B to operate equally, make phenyl-agarose 2B-benzene and prick the pyrimidine compound with agarose 2B (Sepharose 2B, Pharmacia company product).
Embodiment III
Repeat embodiment I, replace sepharose 4B to operate equally, make phenyl-agarose 6B-benzene and prick the pyrimidine compound with agarose 6B (Sepharose 6B, Pharmacia company product).
Embodiment IV
1. column chromatography:
Get each 1000ml of double functional chromatographic material that adopts above-mentioned embodiment preparation respectively.Use phosphate buffer solution (pH8.0) the balance columns bed of 0.1mol/L in advance.Other gets urinary trypsin inhibitor, and (than vigor: 2500U/mg) make the solution that concentration is 100000U/ml (pH8.0) flushing post bed, kallikrein is adsorbed urinary trypsin inhibitor and then flows out with solution, collects effluent.
2. kallikrein assay:
(1) assay method of kallikrein: adopt chromophoric substrate S-2266 determination method [going into Jiang Zhangzi etc., clinical pharmacology (Japan) 29,419, (1981)].
Get 0.2mol/L trishydroxymethylaminomethane buffer solution (pH8.0) 500ul, 37 ℃ are incubated 5～10 minutes, other gets inspection product solution (sample solution is regulated pH to 8.0 and is diluted to 1 times) 400ul, mix 37 ℃ of insulations and add substrate solution [25mgS-2266 (the Japanese first chemical pharmacy strain formula can shut out product) adds the dissolved in distilled water of 28.8ml] after 2～5 minutes and mix 37 ℃ of insulations 30 minutes, add 50% acetic acid cessation reaction again.The trishydroxymethylaminomethane buffer solution (pH8.0) that other gets the 0.2mol/L that contains Aprotinin 10000～50000IU/L replaces the trishydroxymethylaminomethane buffer solution (pH8.0) of 0.2mol/L to operate equally, measure absorbance log (A) at wavelength 405nm place as blank, be calculated as follows out the content of kallikrein:
The content of kallikrein (mU/ml)=A * 9.55
(2) measure in the urinary trypsin inhibitor content of kallikreins (Kalikrein) before and after column chromatography as stated above, measurement result sees Table one:
Table one
| Lot number | Kallikrein content | Residual rate (%) | |
| Before the purifying (mU/ml) | Behind the purifying (mU/ml) | ||
| 950901 940902 | 0.8611 0.7273 | 0.0021 0.0045 | 0.24％ 0.62％ |
Embodiment V
1. the purifying of kallikrein:
Meet embodiment IV, will be adsorbed with sodium carbonate buffer solution (sodium chloride that contains 0.5mol/L) the pH10.0 wash-out of the chromatographic column of kallikrein, collect eluting peak with 0.1mol/L.Use efficient liquid phase chromatographic analysis, analyze the purity of kallikrein.
2. efficient liquid phase chromatographic analysis:
Chromatographic condition: Beckman high performance liquid chromatograph
Chromatographic column: Tskgel 3000SW, 7.5*600mm (Beckman company product)
Item flows: the 0.1mol/L sodium dihydrogen phosphate
Detect wavelength: 280mm
Flow velocity: 0.6ml/min
Sample size: 10ul
This method is an exclusion chromatography, is that the size of molecular weight is per sample separated with its space conformation.The composition of different molecular weight separates successively in the sample under the effect of the item that flows, and what molecular weight was big flows out earlier.Can detect the composition and the purity of sample by detecting device.
Adopt the kallikrein of this double functional chromatographic purifying after testing, efficient liquid phase chromatographic analysis gel chromatography purity illustrates the effect that adopts this post layer material can reach the separation and purification kallikrein all above 80%.
Claims (4)
1, a kind of double functional chromatographic material is characterized in that satisfying following structural requirement:
Here X=C
6H
5_
Y＝_NHC
6H
5C(NH)NH
2
R represents a kind of agarose.
2, a kind of preparation method of double functional chromatographic material is characterized in that may further comprise the steps:
1) epoxy compound is synthetic, gets phenol and epichlorokydrin and refluxes under alkali condition, and it is fully reacted, and removes water layer, and decompression distillation obtains 1,2-epoxy-3-phenoxypropane;
2) preparation of phenyl-carrier cross-linked composite is got any water-fast agarose as carrier material, and with carrier and an amount of 1,2-epoxy-3-phenoxypropane is the direct crosslinked phenyl-carrier complexes that makes at ambient temperature;
3) phenyl-carrier-benzene is pricked the preparation of pyrimidine cross-linked composite, and phenyl-carrier complexes with cyanogen bromide or the activation of other activators, is added amino n-caproic acid of 6-and p-aminophenyl respectively and pricks the pyrimidine hydrochloride, makes benzene and pricks the pyrimidine double functional chromatographic material.
3, a kind of application of double functional chromatographic material is characterized in that adding urinary trypsin inhibitor with the described double functional chromatographic material dress of claim 1 post, regulate PH to 8.0, with PH is 8.0 phosphate buffer flushing post bed, and kallikrein is adsorbed, and the acid of urine tryptose is then flowed out with liquid.
4, the application of double functional chromatographic material according to claim 3, the layer post that it is characterized in that being adsorbed with kallikrein analysed the sodium phosphate buffer PH10.0 wash-out with the sodium chloride that contains 0.5mol/L of 0.1mol/L, collect eluting peak, promptly get the kallikrein of purifying.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN95114252A CN1057607C (en) | 1995-12-04 | 1995-12-04 | Double functional chromatographic material and preparing method and use |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN95114252A CN1057607C (en) | 1995-12-04 | 1995-12-04 | Double functional chromatographic material and preparing method and use |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1142984A CN1142984A (en) | 1997-02-19 |
| CN1057607C true CN1057607C (en) | 2000-10-18 |
Family
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN95114252A Expired - Lifetime CN1057607C (en) | 1995-12-04 | 1995-12-04 | Double functional chromatographic material and preparing method and use |
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| Country | Link |
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1100568C (en) * | 1999-04-26 | 2003-02-05 | 中国科学院大连化学物理研究所 | Process for synthesizing protein immunoadsorbent medium for removing pathogenic antibody and its compounds from plasm |
| CN101134953B (en) * | 2007-07-02 | 2011-02-09 | 广东天普生化医药股份有限公司 | Recombinant human pancreas kininogenase |
| CN101134952B (en) * | 2007-07-02 | 2011-02-09 | 广东天普生化医药股份有限公司 | Human urine kininogenase and method for making same |
| AU2014207409B2 (en) | 2013-01-20 | 2019-12-19 | Takeda Pharmaceutical Company Limited | Evaluation, assays and treatment of pKal-mediated disorders |
| CN104645949B (en) * | 2015-02-04 | 2017-01-25 | 浙江大学 | A kind of affinity chromatography medium with tetrapeptide as functional ligand and preparation method thereof |
| TW202104900A (en) | 2019-04-16 | 2021-02-01 | 美商戴氏公司 | Methods for quantitation of functional c1 esterase inhibitor (fc1-inh) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3886043A (en) * | 1972-08-24 | 1975-05-27 | Choay Sa | Process for preparing pure porcine beta-trypsin |
| EP0203049A1 (en) * | 1985-05-23 | 1986-11-26 | Pharmacia Ab | Method of cross-linking a porous polysaccharide gel |
| EP0295073A2 (en) * | 1987-06-08 | 1988-12-14 | Chromatochem, Inc. | Chromatographic material |
-
1995
- 1995-12-04 CN CN95114252A patent/CN1057607C/en not_active Expired - Lifetime
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3886043A (en) * | 1972-08-24 | 1975-05-27 | Choay Sa | Process for preparing pure porcine beta-trypsin |
| EP0203049A1 (en) * | 1985-05-23 | 1986-11-26 | Pharmacia Ab | Method of cross-linking a porous polysaccharide gel |
| EP0295073A2 (en) * | 1987-06-08 | 1988-12-14 | Chromatochem, Inc. | Chromatographic material |
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| Publication number | Publication date |
|---|---|
| CN1142984A (en) | 1997-02-19 |
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