CN1376718A - Process for separating and purifying haematoglobin by liquid-solid extracting system - Google Patents
Process for separating and purifying haematoglobin by liquid-solid extracting system Download PDFInfo
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- CN1376718A CN1376718A CN 02115705 CN02115705A CN1376718A CN 1376718 A CN1376718 A CN 1376718A CN 02115705 CN02115705 CN 02115705 CN 02115705 A CN02115705 A CN 02115705A CN 1376718 A CN1376718 A CN 1376718A
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Abstract
A process for separating haematoglobin from blood and purifying it by a liquid-solid extracting system features that the PEG modifier is mixed with the polymer-salt-water as liquid-solid extracting system and the mixture is used to extract haematoglobin from the blood of pig, ox, or man, and includes extracting, back extracting and dialysing. Its advantages are high purity (more than 99%), simple equipment, high speed, low cost, and high selectivity.
Description
Technical field
The present invention relates to a kind of method with liquid-solid extraction system separation and purification of hemoglobin from blood.More particularly, be from blood, to extract oxyphorase by polyoxyethylene glycol (PEG) modifier mixed polymer-salt-liquid-solid affinity extraction method of water.
Background technology
Extraction process, the precipitator method, chromatography, electrophoretic method, liquid phase chromatography etc. are generally adopted in proteinic separation and purification.The precipitator method are divided into organic solvent precipitation method and isoelectric point precipitation, the organic solvent precipitation method that Preparation and Properties of Serum and Plasma Proteins.IV.A System for the Separation into Fractions of the Protein andLipoprotein Components of Biological Tissues and Fluids. (J.Am.Chem.Soc.1964:459-475) introduces easily causes protein denaturation, Shen together, " biological chemistry " (second edition) first volume of Wang Jingyan chief editor, (Higher Education Publishing House, Beijing, 1994, p.210-222.) isoelectric point precipitation of Jie Shaoing can only be used to remove iso-electric point at a distance of bigger foreign protein, the electrophoretic method of submitting a written statement to a higher authority and introducing, though the liquid phase chromatography goods purity height that chromatography and Chinese patent ZL 94190708 provide, but treatment capacity is little, time-consuming, effort is mainly used in quantitative analysis of protein, detect and make with extra care.
Extract the oxyphorase extraction process, mainly contain aqueous two-phase extraction and reverse micelle extraction, wherein mix the two phase aqueous extraction system research that the back forms with two kinds of hydrophilic polymers or polymer salt, become the Appropriate technology of biomaterials such as laboratory or technical scale isolated protein, but Purification of Recombinant Protein A by Aqueous Two-phaseExtraction Intergrated with Affinity Precipitation.Biotechnol.Bioeng. (1992, the polymkeric substance that 40:1381-1387) provides/salt two phase aqueous extraction system, this system is separated to operate and will uses separating funnel, complex operation; And in the polymer/polymer two phase aqueous extraction system, because two phase densities are close, be that viscosity is very big mutually, cause the difficulty that is separated, and because the character of superpolymer, biomacromolecule exists stronger absorption and emulsifying effect at two-phase interface, causes proteinic two-phase to be detained." (the biotechnology journal ", 1997,13 (4): the reverse micelle extraction technology that 430-432. introduces is in " (bioseparation engineering " that Sun Yan in 1998 writes, the book that Beijing Chemical Industry Press publishes is spoken of tensio-active agent and the protein that this method must add and very easily form mixture under the electrostatic attraction effect, be deposited in phase interface, cause proteinic sex change.
Summary of the invention
The objective of the invention is to overcome be separated difficulty and reverse micelle technology of double water-phase technology extraction oxyphorase and extracted the defective that oxyphorase causes proteinic sex change; a kind of method of liquid-solid affinity extraction system separation and purification of hemoglobin is provided; required equipment is simple; the separation and purification cost is low; speed is fast; it is mutually easy to become; Cheng Xianghou directly inclines and liquid phase liquid-solid two-phase is separated; centrifugal in need not aqueous two-phase extraction waited operation, without organic solvent, and nontoxicity; become gathering compound and salt pair protein that stable and provide protection are arranged; the extracting and separating selectivity is good, and loading capacity is big, is to be easy to the new technology that scale is amplified separation and purification of hemoglobin.
The present invention is achieved in that in color-comparison tube adding concentration is 0.5-3.0molL
-1Inorganic salt, the concentration of inorganic salt and the kind of polymkeric substance, concentration have dependency, and pH is controlled at 3.0～9.0, and adding concentration is that 3～20% one-tenth gathering compound solution (polymer concentration is different and variant according to kind, the concentration of salt) and concentration are 0.2～2.0gmL
-1Polyoxyethylene glycol (PEG) modifier solution, add blood sample then, with secondary water constant volume, place on the electronic concussion machine of Kang Shi horizontal vibration 2-10 minute, preferred 4-6 minute, take off and be inverted 10-20 minute, preferred 12-16 minute, system is divided into polymeric solid phase and salt solution liquid phase, and oxyphorase is extracted into solid phase;
After above-mentioned solid phase added the secondary water dissolution, adding concentration was 0.5 * 10
-3-2.0 * 10
-3MolL
-1Ethylenediamine tetraacetic acid (EDTA) and concentration are 0.5-3.0molL
-1Inorganic salt place on the electronic concussion machine of Kang Shi horizontal vibration 2-10 minute, and preferred 4-6 minute, take off and be inverted 10-20 minute, preferred 12-16 minute, system was divided into polymeric solid phase and salt solution liquid phase again, and oxyphorase is gone into liquid phase by back extraction;
Above-mentioned collection is gone into the oxyphorase of liquid phase and is dialysed in dialysis tubing, and micromolecular salt, small amount of polymer can be passed through dialysis tubing, and stay oxyphorase macromole in the dialysis tubing, and its purity can reach more than 99%.
Become the gathering compound to have: Tweens, polyethylene pyrrolinone, polyoxyethylene glycol, polyoxyethylene lauryl ether, triton x-100, gelatin etc., become the phase inorganic salt that ammonium sulfate, potassiumphosphate, Trisodium Citrate, sodium phosphate, salt of wormwood etc. are arranged, modifier has polyoxyethylene glycol 8000 modifiers, polyethylene glycol 6000 modifier.
The present invention extracts oxyphorase in pig blood, ox blood, the human blood with PEG modifier mixed polymer-salt-water liquid-solid extraction system, and through single extraction, a back extraction and dialysis can obtain the aqueous solution of purity greater than 99% oxyphorase.In this system, bovine hemoglobin, human hemoglobin, PINPROL mixed allocation COEFFICIENT K (hemoglobin concentration in the K=solid phase: hemoglobin concentration in the liquid phase) all reach 1000; Single extraction solid phase recovery rate R (amount of solid phase oxyphorase after the R=extraction phase-splitting: the total amount that adds the oxyphorase of system) greater than people such as 99%[Wuenschell G.E. at paper Aqueous Two-phase MetalAffinity Extraction of Heme Proteins. (Bioprocess Eng.1990; 5:199～202) the double-aqueous phase system extraction oxyphorase partition ratio in is 14; the PINPROL partition ratio is 6]; a stripping rate can reach 75%, and (people such as Liu Jinglin is at " SCI "; 1997; its the 8th stripping rate of reverse micelle system in 20:1122 one literary composition is 73.2%), through high performance liquid chromatography; the disc gel electrophoresis; light-intensity method checking purity is not less than U.S. sigma reagent that company purchases.
Embodiment
Embodiment 1: directly oxyphorase in the pig blood is carried out separation and purification with polyethylene glycol 6000 modifier mixing tween 80-ammonium sulfate liquid-solid extraction system.
Specific implementation method: in color-comparison tube, adding 4.0mL concentration is 4.5molL
-1Ammonium sulfate is regulated pH to 6.5, and adding 2.0mL concentration is that 30% polymkeric substance tween 80 solution and 1.0mL concentration are 5.0gmL
-1Polyethylene glycol 6000 modifier solution adds 2mL pig blood sample then, is settled to 10.0mL with secondary water, place on the electronic concussion machine of Kang Shi horizontal vibration 5 minutes, take off and be inverted 15 minutes, system is divided into polymeric solid phase and salt solution liquid phase, and oxyphorase is extracted into solid phase;
After above-mentioned solid phase added the secondary water dissolution, adding 1.0mL concentration was 10.0mmolL
-1Ethylenediamine tetraacetic acid (EDTA) and 4.0mL concentration are 4.5molL
-1Ammonium sulfate places on the electronic concussion machine of Kang Shi horizontal vibration 4 minutes, takes off and is inverted 15 minutes, and system is divided into polymeric solid phase and salt solution liquid phase once more, and oxyphorase is gone into liquid phase by back extraction;
Above-mentioned collection is gone into the oxyphorase of liquid phase and is dialysed in dialysis tubing, and micromolecular salt, small amount of polymer can be passed through dialysis tubing, and stay oxyphorase macromole in the dialysis tubing, and its purity can reach more than 99%.
With above-mentioned gained PINPROL spectrphotometric method for measuring, its result is as shown in table 1.The yield PINPROL of once just coming together (PHb) reaches 99.93%, stripping rate PHb and reaches 69.99%.
Table 1: the result of polyethylene glycol 6000 modifier mixing tween 80-ammonium sulfate liquid-solid extraction system separation and purification of hemoglobin from the pig blood sample
| Test number (TN) | The total egg content of blood sample (mg) | Blood sample Hb content (mg) | The back solid phase Hb that just coming together measures (mg) | Salt water Tot Prot (mg) after the back extraction | Salt water Hb amount (mg) after the back extraction | The Hb recovery rate of just coming together (%) | Stripping rate (%) | Hb purity (%) after the back extraction | The purifying multiple |
| ????1 | ? 6.754 | ? 5.842 | ????5.837 | ????4.117 | ????4.086 | ||||
| ????2 | ????5.840 | ????4.122 | ????4.088 | ||||||
| ????3 | ????5.835 | ????4.119 | ????4.085 | ||||||
| ????4 | ????5.839 | ????4.119 | ????4.087 | ||||||
| ????5 | ????5.838 | ????4.120 | ????4.086 | ||||||
| Mean value | ????5.838 | ????4.120 | ????4.086 | ????99.93 | ????69.99 | ????99.17 | ????19.28 |
Embodiment 2: directly respectively oxyphorase in the bovine blood is carried out separation and purification with polyethylene glycol 6000 modifier mixing tween 80-ammonium sulfate liquid-solid extraction system.
Specific implementation method is identical with embodiment 1.With above-mentioned gained bovine hemoglobin spectrphotometric method for measuring, its result is as shown in table 2.The yield bovine hemoglobin of once just coming together (BHb) reaches 99.91%, stripping rate PHb and reaches 70.00%.
Table 2: the result of polyethylene glycol 6000 modifier mixing tween 80-ammonium sulfate liquid-solid extraction system separation and purification of hemoglobin from the bovine blood sample
| Test number (TN) | The total egg content of blood sample (mg) | Blood sample Hb content (mg) | The back solid phase Hb that just coming together measures (mg) | Salt water Tot Prot (mg) after the back extraction | Salt water Hb amount (mg) after the back extraction | The Hb recovery rate of just coming together (%) | Stripping rate (%) | Hb purity (%) after the back extraction | The purifying multiple |
| ??1 | ?? 6.734 | ?? 5.792 | ??5.784 | ??4.084 | ??4.049 | ||||
| ??2 | ??5.786 | ??4.085 | ??4.051 | ||||||
| ??3 | ??5.789 | ??4.088 | ??4.053 | ||||||
| ??4 | ??5.785 | ??4.083 | ??4.050 | ||||||
| ??5 | ??5.790 | ??4.086 | ??4.053 | ||||||
| Mean value | ??5.787 | ??4.085 | ??4.051 | ??99.91 | ??70.00 | ??99.17 | ??19.50 |
Embodiment 3: directly respectively oxyphorase in the human blood is carried out separation and purification with polyethylene glycol 6000 modifier mixing tween 80-ammonium sulfate liquid-solid extraction system.
Specific implementation method is identical with embodiment 1.With above-mentioned income earner's oxyphorase spectrphotometric method for measuring, its result is as shown in table 3.The yield human hemoglobin (HHb) that once just coming together reaches 99.97%; One time stripping rate PHb reaches 75.00%.
Table 3: the result of polyethylene glycol 6000 modifier mixing tween 80-ammonium sulfate liquid-solid extraction system separation and purification of hemoglobin from the human blood sample
| Test number (TN) | The total egg content of blood sample (mg) | Blood sample Hb content (mg) | The back solid phase Hb that just coming together measures (mg) | Salt water Tot Prot (mg) after the back extraction | Salt water Hb amount (mg) after the back extraction | The Hb recovery rate of just coming together (%) | Stripping rate (%) | Hb purity (%) after the back extraction | The purifying multiple |
| ??1 | ?? 6.713 | ?? 5.742 | ??5.740 | ??4.342 | ??4.305 | ||||
| ??2 | ??5.740 | ??4.340 | ??4.304 | ||||||
| ??3 | ??5.741 | ??4.342 | ??4.306 | ||||||
| ??4 | ??5.739 | ??4.341 | ??4.305 | ||||||
| ??5 | ??5.739 | ??4.340 | ??4.304 | ||||||
| Mean value | ??5.740 | ??4.341 | ??4.305 | ??99.97 | ??75.00 | ??99.17 | ??20.00 |
Embodiment 4: directly oxyphorase in the pig blood is carried out separation and purification with polyoxyethylene glycol 8000 modifier mixing tween 80s-potassiumphosphate liquid-solid extraction system.
Specific implementation method: in color-comparison tube, adding 4.0mL concentration is 4.0molL
-1Potassium phosphate solution, acidity transfers to 7.2, and adding 2.0mL concentration is that 30% polymkeric substance tween 80 solution and 1.0mL concentration are 5.0gmL
-1PEG8000 modifier solution adds pig blood sample 2.0mL then, is settled to 10.0mL with secondary water, places on the electronic concussion machine of Kang Shi horizontal vibration 5 minutes, takes off and is inverted 15 minutes, and system is divided into polymeric solid phase and salt solution liquid phase, and oxyphorase is extracted into solid phase; After solid phase added the secondary water dissolution, adding 1.0mL concentration was 10.0mmolL
-1Ethylenediamine tetraacetic acid (EDTA) and 4.0mL concentration are 4.0molL
-1Potassiumphosphate places on the electronic concussion machine of Kang Shi horizontal vibration 4 minutes, takes off and is inverted 15 minutes, and system is divided into polymeric solid phase and salt solution liquid phase once more, and oxyphorase is gone into liquid phase by back extraction; Oxyphorase in the liquid phase is dialysed in dialysis tubing, and micromolecular salt, small amount of polymer can be passed through dialysis tubing, and stay oxyphorase macromole in the dialysis tubing, and its purity can reach more than 99%.
With above-mentioned gained PINPROL spectrphotometric method for measuring, its result is as shown in table 4.The yield PINPROL of once just coming together (PHb) reaches 99.90%, stripping rate PHb and reaches 74.98%.
Table 4: the result of polyoxyethylene glycol 8000 modifier mixing tween 80s-potassiumphosphate liquid-solid extraction system separation and purification of hemoglobin from the pig blood sample
| Test number (TN) | The total egg content of blood sample (mg) | Blood sample Hb content (mg) | The back solid phase Hb that just coming together measures (mg) | Salt water Tot Prot (mg) after the back extraction | Salt water Hb amount (mg) after the back extraction | The Hb recovery rate of just coming together (%) | Stripping rate (%) | Hb purity (%) after the back extraction | The purifying multiple |
| ??1 | ? 6.754 | ? 5.842 | ??5.839 | ??4.413 | ??4.379 | ||||
| ??2 | ??5.840 | ??4.412 | ??4.378 | ||||||
| ??3 | ??5.831 | ??4.407 | ??4.373 | ||||||
| ??4 | ??5.836 | ??4.411 | ??4.377 | ||||||
| ??5 | ??5.835 | ??4.409 | ??4.375 | ||||||
| Mean value | ??5.836 | ??4.410 | ??4.376 | ??99.90 | ??74.98 | ??99.23 | ??20.09 |
Embodiment 5: directly oxyphorase in the bovine blood is carried out separation and purification with polyoxyethylene glycol 8000 modifier mixing tween 80s-potassiumphosphate liquid-solid extraction system.
Specific implementation method is identical with embodiment 4.With above-mentioned gained bovine hemoglobin spectrphotometric method for measuring, its result is as shown in table 5.The yield bovine hemoglobin of once just coming together (HHb) reaches 99.96%; One time stripping rate PHb reaches 75.01%.
Table 5: the result of polyoxyethylene glycol 8000 modifier mixing tween 80s-potassiumphosphate liquid-solid extraction system separation and purification of hemoglobin from the bovine blood sample
| Test number (TN) | The total egg content of blood sample (mg) | Blood sample Hb content (mg) | The back solid phase Hb that just coming together measures (mg) | Salt water Tot Prot (mg) after the back extraction | Salt water Hb amount (mg) after the back extraction | The Hb recovery rate of just coming together (%) | Stripping rate (%) | Hb purity (%) after the back extraction | The purifying multiple |
| ?1 | 6.734 | 5.792 | 5.784 | ?4.377 | ?4.342 | ||||
| ?2 | 5.791 | ?4.377 | ?4.343 | ||||||
| ?3 | 5.791 | ?4.379 | ?4.344 | ||||||
| ?4 | 5.790 | ?4.378 | ?4.343 | ||||||
| ?5 | 5.787 | ?4.375 | ?4.340 | ||||||
| Mean value | 5.790 | ?4.377 | ?4.342 | ?99.96 | ?75.01 | ?99.20 | ?20.18 |
Embodiment 6: directly oxyphorase in the human blood is carried out separation and purification with polyoxyethylene glycol 8000 modifier mixing tween 80s-potassiumphosphate liquid-solid extraction system.
Specific implementation method is identical with embodiment 4.With above-mentioned income earner's oxyphorase spectrphotometric method for measuring, its result is as shown in table 6.The yield bovine hemoglobin of once just coming together (HHb) reaches 99.97%; One time stripping rate PHb reaches 75.01%.
Table 6: the result of polyoxyethylene glycol 8000 modifier mixing tween 80s-potassiumphosphate liquid-solid extraction system separation and purification of hemoglobin from the human blood sample
| Test number (TN) | The total egg content of blood sample (mg) | Blood sample Hb content (mg) | The back solid phase Hb that just coming together measures (mg) | Salt water Tot Prot (mg) after the back extraction | Salt water Hb amount (mg) after the back extraction | The Hb recovery rate of just coming together (%) | Stripping rate (%) | Hb purity (%) after the back extraction | The purifying multiple |
| ????1 | ?? 6.713 | ?? 5.742 | ??5.740 | ??4.342 | ??4.305 | ||||
| ????2 | ??5.740 | ??4.340 | ??4.304 | ||||||
| ????3 | ??5.741 | ??4.342 | ??4.306 | ||||||
| ????4 | ??5.739 | ??4.341 | ??4.305 | ||||||
| ????5 | ??5.739 | ??4.340 | ??4.304 | ||||||
| Mean value | ??5.740 | ??4.341 | ??4.305 | ??99.97 | ??75.00 | ??99.17 | ??20.39 |
Claims (8)
1, a kind of method of separating and purifying haematoglobin by liquid-solid extracting system comprises the steps:
(1), in color-comparison tube, add inorganic salt, pH is controlled at 3.0～9.0, adds into gathering compound solution and polyoxyethylene glycol (PEG) 8000 or polyoxyethylene glycol (PEG) 6000 modifier solution, adds blood sample then, with secondary water constant volume, place on the electronic concussion machine of Kang Shi, horizontal vibration 2-10 minute is taken off and was inverted 10-20 minute, system is divided into polymeric solid phase and salt solution liquid phase, and oxyphorase is extracted into solid phase;
(2), after above-mentioned solid phase adds the secondary water dissolution, add ethylenediamine tetraacetic acid (EDTA) and inorganic salt, place on the electronic concussion machine of Kang Shi, horizontal vibration 2-10 minute, take off and be inverted 10-20 minute, system is divided into polymeric solid phase and salt solution liquid phase, and oxyphorase is gone into liquid phase by back extraction;
(3), oxyphorase that above-mentioned collection is gone into liquid phase places dialysis tubing to dialyse, micromolecular salt, small amount of polymer can be passed through dialysis tubing, and stay oxyphorase macromole in the dialysis tubing, its purity can reach more than 99%.
2,, it is characterized in that described one-tenth gathering compound is Tweens, polyethylene pyrrolinone, polyoxyethylene glycol, polyoxyethylene lauryl ether, triton x-100 or gelatin according to the method for the described separation and purification of hemoglobin of claim 1.
3,, it is characterized in that described inorganic salt are ammonium sulfate, potassiumphosphate, Trisodium Citrate, sodium phosphate or salt of wormwood according to the method for the described separation and purification of hemoglobin of claim 1.
4, according to the method for the described separation and purification of hemoglobin of claim 1, the concentration that it is characterized in that inorganic salt in the described liquid-solid extraction system is 0.5-3.0molL
-1
5,, it is characterized in that becoming the concentration of gathering compound in the described liquid-solid extraction system is 3～20% according to the method for the described separation and purification of hemoglobin of claim 1.
6, according to the method for the described separation and purification of hemoglobin of claim 1, the concentration that it is characterized in that modifier solution in the described liquid-solid extraction system is 0.2～2.0 gmL
-1
7, according to the method for the described separation and purification of hemoglobin of claim 1, the concentration that it is characterized in that ethylenediamine tetraacetic acid (EDTA) in the described liquid-solid extraction system is 0.5 * 10
-3-2.0 * 10
-3MolL
-1
8, according to the method for the described separation and purification of hemoglobin of claim 1, it is characterized in that color-comparison tube is placed on the electronic concussion machine of Kang Shi, horizontal vibration 4-6 minute is taken off and was inverted 12-16 minute.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1303413C (en) * | 2003-06-17 | 2007-03-07 | 余伟明 | Protein and virus quick-speed concentration method |
| CN101830979A (en) * | 2010-04-16 | 2010-09-15 | 中南民族大学 | Method for separating and purifying serum albumin by using liquid-solid extraction system |
| CN101294175B (en) * | 2008-06-20 | 2011-08-31 | 南京林业大学 | Method for producing low polyxylose with dual-aqueous phase hydrolyzation system |
| CN101575373B (en) * | 2009-06-12 | 2013-03-20 | 中国人民解放军第三军医大学野战外科研究所 | Preparation method of hemoglobin extract |
| CN113024664A (en) * | 2021-03-22 | 2021-06-25 | 北京航空航天大学 | Method for extracting earthworm hemoglobin |
| CN113087787A (en) * | 2021-05-19 | 2021-07-09 | 广西医科大学第二附属医院(广西医科大学第二临床医学院) | Earthworm hemoglobin separation and purification method |
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2002
- 2002-04-11 CN CNB021157057A patent/CN1169836C/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1303413C (en) * | 2003-06-17 | 2007-03-07 | 余伟明 | Protein and virus quick-speed concentration method |
| CN101294175B (en) * | 2008-06-20 | 2011-08-31 | 南京林业大学 | Method for producing low polyxylose with dual-aqueous phase hydrolyzation system |
| CN101575373B (en) * | 2009-06-12 | 2013-03-20 | 中国人民解放军第三军医大学野战外科研究所 | Preparation method of hemoglobin extract |
| CN101830979A (en) * | 2010-04-16 | 2010-09-15 | 中南民族大学 | Method for separating and purifying serum albumin by using liquid-solid extraction system |
| CN101830979B (en) * | 2010-04-16 | 2012-06-13 | 中南民族大学 | Method for separating and purifying serum albumin by using liquid-solid extraction system |
| CN113024664A (en) * | 2021-03-22 | 2021-06-25 | 北京航空航天大学 | Method for extracting earthworm hemoglobin |
| CN113087787A (en) * | 2021-05-19 | 2021-07-09 | 广西医科大学第二附属医院(广西医科大学第二临床医学院) | Earthworm hemoglobin separation and purification method |
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