CN1108379C - Method of extracting high-purity copper-zinc superoxide dismutase from animal's blood - Google Patents
Method of extracting high-purity copper-zinc superoxide dismutase from animal's blood Download PDFInfo
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- CN1108379C CN1108379C CN00117762A CN00117762A CN1108379C CN 1108379 C CN1108379 C CN 1108379C CN 00117762 A CN00117762 A CN 00117762A CN 00117762 A CN00117762 A CN 00117762A CN 1108379 C CN1108379 C CN 1108379C
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Abstract
The present invention relates to a method for extracting high-purity hepatocuprein from animal's blood, which comprises the following steps: an anticoagulant is added to animal's blood; precipitates are removed; temperature gradient treatment of 3 to 6 grades is applied to the supernatant liquid; the precipitates are removed again; salting out operation at two stages with different saturation is applied to the supernatant liquid by ammonium sulfate; the final precipitates are eluted by a buffer solution for removing the residual salting out liquid; finally, the SOD enzyme with high purity and specific activity is obtained. The present invention has the advantages of simple operation, low cost, high enzyme yield and high specific activity of enzyme.
Description
The present invention relates to a kind of extraction superoxide dismutase method of (Superoxide dismutase is called for short SOD), particularly relate to a kind of method of from animal blood, extracting the high-purity copper-zinc superoxide dismutase.
People such as McCord in 1969 find that a kind of pale blue in the ox blood red corpuscle contains cuproprotein and has enzymic activity, can catalysis superoxide anion generation disproportionation, called after superoxide dismutase thus.Till now, the researchist has carried out decades the research of SOD both at home and abroad, finds to have four quasi-SODs to exist, that is: Cu.Zn-SOD; Fe-SOD; Mn-SOD; The active factor of finding in mammiferous extracellular fluid of a kind of SOD of having is called for short Ec-SOD, i.e. the outer SOD of born of the same parents.
Superoxide dismutase is because its special effect, can single-minded removing superoxide anion, so all illness that too much causes owing to superoxide anion, available SOD prevents and treats.The existing one side favourable to object of superoxide anion also has disadvantageous one side.It can make virally inactivated, causes lipid peroxidation, destroys cytolemma, killer cell, and change the viscosity that lubricating fluid is located in joint etc.Superoxide anion and some disease are closely related, it can cause radiation disease and lupus erythematosus and other immunological diseases such as tissue is inflamed, reperfusion injury syndromes, senile cataract, radiocystitis.Evidence gives exogenous sod and can alleviate and delay these diseases, also can cure some diseases.When body aging, superoxide anion is to make cell suffer one of reason of frequent damage, so that SOD can be used for is anti-ageing.Lipofuscin (being age pigment) is the snperoxiaized product of tenuigenin unsaturated fatty acids, and SOD can be used as the additive of makeup, in order to preventing or to reduce the generation of senile plaque in skin, and has sunscreen effect and antiphlogistic effects simultaneously.SOD has a wide range of applications in industries such as medicine, food, makeup at present.
Now, people have extracted SOD from different sources, and the SOD of animal-origin, the SOD of plant origin and microbe-derived SOD are arranged.Have from the SOD of animal-origin: various blood SOD, internal organs SOD; Have from the SOD of plant origin: soybean SOD, corn SOD etc.; Have from microbe-derived SOD: yeast SOD etc.Though have the SOD of multiple class like this to occur, really can reach the considerably less of large-scale production.
At present, through years of researches, the method of extracting SOD both at home and abroad from the blood of animal is very many, the method that adopts often generally is with the centrifugal collection erythrocyte of fresh animal blood, vigorous stirring makes its haemolysis, adding ammonium sulfate behind the haemolysis saltouts, general all is the precipitation of collection 50% to 90%, utilize the most of foreign protein of the centrifugal removal of supercentrifuge, use trichloromethane again, ethanol, acetone and other organic solvent precipitates, and further removes foreigh protein removing, through desalination, remove residual ammonium sulfate, pass through column chromatography system (generally all through twice column chromatography) at last again, could separate obtaining the SOD alive than height ratio.This technology more complicated, because used expensive column chromatography system, column packing and supercentrifuge, and, through behind the column chromatography, the yield of product is reduced greatly, so just significantly improved the cost of product, the cycle of purifying technique is also very long.
Purpose of the present invention just provides a kind of production technique method of extracting the high-purity copper-zinc superoxide dismutase from animal blood simple, with short production cycle, with low cost.
Present method step is as follows:
1., add the sodium citrate anticoagulant of 0.2-0.5% (weight ratio) in will fresh animal blood (pig, ox, sheep, chicken etc.), low temperature (4-8 ℃) was placed 30-100 minute, treated the solution layering, removed most of supernatant;
2., precipitation heated rapidly to 50 ℃ by room temperature, be incubated and reduces to room temperature rapidly after 15 minutes, removes precipitation after centrifugal 15 minutes for 4000 rev/mins, must supernatant liquor; Or precipitation is heated rapidly to 55 ℃ by room temperature, be incubated and reduces to room temperature rapidly after 10 minutes, removes precipitation after centrifugal 15 minutes for 4000 rev/mins, must supernatant liquor;
3., thermograde is handled: supernatant liquor is carried out 3-6 level thermograde handle, elementary temperature is 40-50 ℃, the level temperature is 70-80 ℃ eventually, per two inter-stage thermograde differences are 5-10 ℃, it all is to be warming up to certain grade of temperature rapidly, to be kept reducing to room temperature more rapidly after 10-30 minute by room temperature earlier that every grade of thermograde of supernatant liquor is handled, low-speed centrifugal (4000-6000 rev/min) 10-30 minute all after every grade of cooling is removed precipitation;
4., supernatant liquor carried out two-stage saltout: at first add ammonium sulfate, making the liquid saturation ratio of saltouing is 45-70%, abundant stirring and dissolving, low temperature (4-8 ℃) was placed after 12-36 hour, low speed (4000-6000 rev/min) centrifugal 10-30 minute is removed precipitation; Continuation adds ammonium sulfate in supernatant liquor to make the liquid saturation ratio of saltouing be 85-95%, abundant stirring and dissolving, and low temperature (4-8 ℃) was placed after 12-36 hour, low speed (4000-6000 rev/min) centrifugal 10-30 minute, collecting precipitation (being designated as precipitation 1); Supernatant liquor low temperature (4-8 ℃) was again placed 12-36 hour, carried out suction filtration with G4 frosted funnel, collecting precipitation (being designated as precipitation 2);
5., be respectively two group precipitations of dissolving step 4 of potassium phosphate buffer of 0.05-0.2M, pH=7.0 with concentration, ultrafiltration (10000M) is removed ammonium sulfate and is promptly got than the two group SOD enzymes slightly different with purity of living.
The raw material of following experimental data is an ox blood.Show that through experimental test the staging life of the high purity SOD enzyme freeze-drying prods that the present invention makes reached more than 3 years.
The small white mouse feeding study: 20 male and female half and half, body weight 18-22 gram is pressed 0.5ml/10 gram body weight, and 1 filling stomach was observed 7 days, and none death does not as a result see that spirit, fur etc. are unusual yet, proves that SOD nontoxicity or toxicity are extremely low.
The ultra-violet absorption spectrum of SOD:
Through the UV scanning of Shanghai analytical instrument factory 730 type spectrophotometers to SOD, the obtained the maximum absorption that records ox blood Cu.Zn-SOD is 261nm, A
261/ A
280=1.34.
SOD enzyme and relevant document (McCord, J.M. ﹠amp that above digital proof obtains with this kind method; Fridovich, I.Superoside dismutase, J.Biol.Chem, 1969,244,6049) the SOD enzyme of report is consistent.
The cataphoretic determination of SOD:
One, agarose plate gel electrophoresis result: two points appear in amino black 10B dyeing, and five bright spot territories appear in active coloring.Active coloring: the gel flat board behind the electrophoresis, in 1.225 * 10 of strict lucifuge
-3Ol/L tetrazole nitro indigo plant (NBT) solution (includes Riboflavin Tetrabutyrate .8 * 10
-3Mol/L) and 2.8 * 10
-3Mol/L Tetramethyl Ethylene Diamine solution (is used PH=7.8,3.6 * 10
-3Mol/L potassium phosphate salt damping fluid preparation) soaked 15-20 minute in, it is clear and legible to blue background and colourless bright spot territory that anti-then flat board is put in the fluorescent box illumination.
Two, acrylamide disk electrophoresis result: two bands appear in amino black 10B, and obvious spot territory appears in active coloring.
The results in electrophoresis shows that the SOD enzyme that the present invention obtains is that electrophoresis is pure, thereby has reached goal of the invention.
Adopt the pyrogallol method of improvement to measure, concrete measuring method is seen document: biological chemistry biophysics progress, 1996,4,71-73.
1, temperature gradient method dehematize red eggs are white
| Condition | Enzyme concn U/ml | Protein concentration mg/ml | Total activity ten thousand U | Than U/ml alive | Cumulative volume ml |
| 50 ℃ of centrifugal back supernatant liquors | 6282.79 | 74.93 | 5843 | 83.85 | 9300 |
| 60 ℃ of centrifugal back supernatant liquors | 6677.01 | 31.40 | 5809 | 212.63 | 8700 |
| 70 ℃ of centrifugal back supernatant liquors | 6965.85 | 15.52 | 5712 | 448.72 | 8200 |
| 75 ℃ of centrifugal back supernatant liquors | 7244.03 | 9.21 | 5650 | 786.54 | 7800 |
As can be seen, in the whole thermograde processing process, the ratio work of enzyme has improved nearly 10 times from above table, and the yield of enzyme has reached 96.70% (5650/5843).
2, the two-stage ammonium sulfate precipitation is surveyed the supernatant liquor test result:
Group 1: the one-level 50% saturation ratio ammonium sulfate of saltouing, the secondary 90% saturation ratio ammonium sulfate of saltouing
| Enzyme concn U/ml | Protein concentration mg/ml | Total activity U | Than U/mg alive | Vigor reclaims % | The output mg of enzyme | |
| Thermally denature | 7244.03 | 9.21 | 7244030 | 786.54 | 100 | 9210 |
| Precipitation 1 | 12519.96 | 0.16 | 2687536 | 7452.36 | 37.1 | 34.3 |
| Precipitation 2 | 7322.05 | 0.07 | 1571954 | 9634.28 | 21.7 | 15.03 |
Group 2: the one-level 55% saturation ratio ammonium sulfate of saltouing, the secondary 90% saturation ratio ammonium sulfate of saltouing
| Enzyme concn U/ml | Protein concentration mg/ml | Total activity U | Than U/mg alive | Vigor reclaims % | The output mg of enzyme | |
| Thermally denature | 7244.03 | 9.21 | 7244030 | 786.54 | 100 | 9210 |
| Precipitation 1 | 16192.62 | 0.21 | 2644071 | 8261.54 | 36.5 | 34.29 |
| Precipitation 2 | 8720.37 | 0.103 | 1424176 | 10023.4 | 19.66 | 16.8 |
Group 3: the one-level 60% saturation ratio ammonium sulfate of saltouing, the secondary 95% saturation ratio ammonium sulfate of saltouing
| Enzyme concn U/ml | Protein concentration mg/ml | Total activity U | Than U/mg alive | Vigor reclaims % | The output mg of enzyme | |
| Thermally denature | 7244.03 | 9.21 | 7244030 | 786.54 | 100 | 9210 |
| Precipitation 1 | 21675.47 | 0.24 | 3122176 | 10321.6 | 43.1 | 34.57 |
| Precipitation 2 | 13317.61 | 0.114 | 1876203 | 12106.9 | 25.9 | 16.8 |
Group 4: the one-level 65% saturation ratio ammonium sulfate of saltouing, the secondary 90% saturation ratio ammonium sulfate of saltouing
| Enzyme concn U/ml | Protein concentration mg/ml | Total activity U | Than U/mg alive | Vigor reclaims % | The output mg of enzyme | |
| Thermally denature | 7244.03 | 9.21 | 7244030 | 786.54 | 100 | 9210 |
| Precipitation 1 | 15637.6 | 0.201 | 2843281 | 8687.56 | 39.25 | 36.54 |
| Precipitation 2 | 8096.29 | 0.085 | 1470538 | 10120.3 | 20.3 | 15.4 |
From four above tables as can be seen, the precipitation 1 of group 3 (60% saturation ratio ammonium sulfate to 95% saturation ratio ammonium sulfate) is lived with the ratio of precipitation 2 and the vigor recovery all is higher than other three groups, the foreign protein of explanation group 3 is removed thoroughly, and precipitate 2 ratio work apparently higher than precipitation 1, but precipitate 2 yield and do not precipitate 1 height, come to occur easily aborning contradiction outwardly, in fact, we can produce simultaneously for SOD product double-duty.SOD lives at application need high purity, the height ratio of pharmaceutical industries, and does not just need very height ratio SOD product alive at makeup and health care of food product industry, and our present Technology just in time can satisfy the needs of these two industries simultaneously.
The ratio of enzyme is lived and improved 10 times nearly in the whole heat-processed, and the yield of enzyme reaches more than 96%, illustrates that the red proteic effect of this temperature gradient method dehematize is reasonable, and is more far better than the effect that merely heats under a certain temperature.In the whole process,, only use low speed centrifuge (4000-6000 rev/min) to reach and remove precipitation and the purposes such as yield height of enzyme owing to adopted thermograde facture and two-stage to saltout.
In sum, the preparation method who from animal blood, extracts the high-purity copper-zinc superoxide dismutase of the present invention have that technology is easy, the cycle is short, cost is low, the yield of enzyme and than advantages of higher alive.
Further set forth the preparation method who reaches of the present invention below in conjunction with specific embodiment, embodiment adopts the level Four thermograde to remove precipitation process:
1, will add 38g citric acid antithrombotics among the fresh ox blood 10000ml, low temperature was placed 50 minutes for 4 ℃, treated the solution layering, removed most of supernatant, got the 4500ml precipitation;
2, will precipitate by room temperature and heat rapidly, and be incubated and reduce to room temperature rapidly after 15 minutes, remove precipitation after centrifugal 15 minutes for 4000 rev/mins to 50 ℃, the 4400ml supernatant liquor;
3, supernatant liquor is heated rapidly to 60 ℃, is incubated and reduces to room temperature rapidly after 10 minutes, removes precipitation after centrifugal 15 minutes for 4000 rev/mins, the 4300ml supernatant liquor;
4, supernatant liquor is heated rapidly to 70 ℃, is incubated and reduces to room temperature rapidly after 10 minutes, removes precipitation after centrifugal 15 minutes for 4000 rev/mins, the 4100ml supernatant liquor;
5, supernatant liquor is heated rapidly to 75 ℃, is incubated and reduces to room temperature rapidly after 5 minutes, removes precipitation after centrifugal 20 minutes for 4500 rev/mins, the 4000ml supernatant liquor;
6, add ammonium sulfate in the supernatant liquor and make saturation ratio reach 60%, fully stirring is dissolved ammonium sulfate fully, and low temperature (4 ℃) was placed 24 hours, and centrifugal (4500 rev/mins) 20 minutes remove precipitation;
7, add ammonium sulfate in the supernatant liquor again and make saturation ratio reach 95%, fully stirring is dissolved ammonium sulfate fully, and low temperature (4 ℃) was placed 24 hours, centrifugal (5000 rev/mins) 20 minutes, collecting precipitation (being designated as precipitation 1); Supernatant liquor low temperature (4 ℃) placement again carried out suction filtration with G4 frosted funnel after 24 hours, collecting precipitation (being designated as precipitation 2);
8, with 0.05M dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution 50ml dissolution precipitation 1 and precipitate 2 respectively, dissolving back ultrafiltration (10 fully, 000M) remove residual ammonium sulfate, obtain 345.7mg and 168mg purity and ratio two groups of slightly different highly purified SOD alive respectively, experimental data is the same.
Embodiment 2:
1, add 32g citric acid antithrombotics among the fresh pig blood 10000ml, low temperature was placed 30 minutes for 6 ℃, treated the solution layering, removed most of supernatant, the 4700ml precipitation;
2, will precipitate by room temperature and heat rapidly, and be incubated and reduce to room temperature rapidly after 10 minutes, remove precipitation after centrifugal 15 minutes for 4000 rev/mins to 55 ℃, the 4500ml supernatant liquor;
3, supernatant liquor is heated rapidly to 65 ℃, is incubated and reduces to room temperature rapidly after 10 minutes, removes precipitation after centrifugal 15 minutes for 4000 rev/mins, the 4350ml supernatant liquor;
4, supernatant liquor is heated rapidly to 75 ℃, is incubated and reduces to room temperature rapidly after 8 minutes, removes precipitation after centrifugal 15 minutes for 4000 rev/mins, the 4000ml supernatant liquor;
5, add ammonium sulfate in the supernatant liquor and make saturation ratio reach 55%, fully stirring is dissolved ammonium sulfate fully, and low temperature (6 ℃) was placed 18 hours, and centrifugal (4500 rev/mins) 15 minutes remove precipitation;
6, add ammonium sulfate in the supernatant liquor again and make saturation ratio reach 90%, fully stirring is dissolved ammonium sulfate fully, and low temperature (6 ℃) was placed 18 hours, centrifugal (5000 rev/mins) 15 minutes, collecting precipitation (being designated as precipitation 1); Supernatant liquor low temperature (6 ℃) placement again carried out suction filtration with G4 frosted funnel after 18 hours, collecting precipitation (being designated as precipitation 2);
7, with dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution 30ml of 0.1M dissolution precipitation 1 and precipitate 2 respectively, residual ammonium sulfate is removed in dissolving back ultrafiltration (10000M) fully, obtains 361.4mg and 156.5mg purity and ratio two groups of slightly different highly purified SOD alive respectively.
Claims (2)
1, a kind of method of from animal blood, extracting the high-purity copper-zinc superoxide dismutase, step is as follows:
1. be the sodium citrate anticoagulant of 0.2-0.5% with adding weight ratio in the fresh animal blood, placed 30-100 minute for low temperature 4-8 ℃, treat the solution layering, remove most of supernatant;
2. precipitate by room temperature and heat rapidly, be incubated and reduce to room temperature rapidly after 15 minutes, remove precipitation after centrifugal 15 minutes for 4000 rev/mins to 50 ℃, supernatant liquor;
3. thermograde is handled: supernatant liquor is carried out 3-6 level thermograde handle, elementary temperature is 40-50 ℃, the level temperature is 70-80 ℃ eventually, per two inter-stage thermograde differences are 5-10 ℃, it all is to be warming up to certain grade of temperature rapidly, to be kept reducing to room temperature more rapidly after 10-30 minute by room temperature earlier that every grade of thermograde of supernatant liquor is handled, after every grade of cooling all low speed 4000-6000 rev/min centrifugal 10-30 minute, remove precipitation;
4. supernatant liquor being carried out two-stage saltouts: at first add ammonium sulfate, making the liquid saturation ratio of saltouing is 45-70%, and fully stirring and dissolving is placed after 12-36 hour for low temperature 4-8 ℃, low speed 4000-6000 rev/min centrifugal 10-30 minute, remove precipitation; Continuation adds ammonium sulfate in supernatant liquor to make the liquid saturation ratio of saltouing be 85-95%, and fully stirring and dissolving is placed after 12-36 hour for low temperature 4-8 ℃, low speed 4000-6000 rev/min centrifugal 10-30 minute, collecting precipitation is designated as precipitation 1; Supernatant liquor again low temperature 4-8 ℃ placed 12-36 hour, carry out suction filtration with G4 frosted funnel, collecting precipitation is designated as and precipitates 2;
5. be potassium phosphate buffer two group precipitations of dissolving step 4 respectively of 0.05-0.2M, pH=7.0 with concentration, the 10000M ultrafiltration is removed ammonium sulfate and is promptly got than two groups of SOD enzymes slightly different with purity of living.
2, a kind of method of extracting the high-purity copper-zinc superoxide dismutase from animal blood as claimed in claim 1, it is characterized in that: described animal blood can be from poultry and livestocks such as pig, ox, sheep, chickens.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101590223B (en) * | 2008-05-26 | 2012-01-18 | 刘安荣 | SOD complex capsule and preparation method thereof |
| CN101717756B (en) * | 2008-10-09 | 2012-05-09 | 哈尔滨仁皇药业股份有限公司 | Method for preparing superoxide dismutase |
| CN102181411A (en) * | 2011-04-01 | 2011-09-14 | 黑龙江宝迪肉类食品有限公司 | Method for extracting superoxide dismutase (SOD) from porcine blood red cells |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1034369A (en) * | 1987-01-08 | 1989-08-02 | 苏州医学院 | The method of purification of superoxide-dismutase |
| CN1038104A (en) * | 1989-02-02 | 1989-12-20 | 海军工程学院门诊部 | Extract the method for superoxide-dismutase |
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2000
- 2000-06-03 CN CN00117762A patent/CN1108379C/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1034369A (en) * | 1987-01-08 | 1989-08-02 | 苏州医学院 | The method of purification of superoxide-dismutase |
| CN1038104A (en) * | 1989-02-02 | 1989-12-20 | 海军工程学院门诊部 | Extract the method for superoxide-dismutase |
Non-Patent Citations (1)
| Title |
|---|
| 郑州轻工业学院学报，第10卷第3期 1995-09-01 王章存等，兔血红细胞超氧化物歧化酶提取方法研究 * |
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