Preface
The Chinese name of SOD is superoxide dismutase, and its English name is Superoxide dimutase, molecular weight (bovine red blood cells) of about 32,000 is a widely existed in the organism of the metalloprotein alcohols, alias: orgotein, ormetein, ontosein; Cu.Zn-SOD; Palosein, etc., SOD is known as the biological body of the metalloprotein alcohols. Ormetein, ontosein; Cu.Zn-SOD; Palosein, etc. SOD is regarded as the most promising medicinal enzyme in the 21st century by scientists.
I. This process is a metalloenzyme isolated from erythrocytes, liver and other mammalian tissues; it contains two subunits, each containing one copper atom and one zinc atom, and SOD can catalyze the disproportionation of O2 to H2O and O2, and its properties not only depend on the enzyme protein, but also depend on metal ions in the active site. SOD and Fe-SOD, Mn-SOD. Zn-SOD, Fe-SOD and Mn-SOD. Under normal conditions, SOD is more stable. Different sources of Cu.Zn--SOD, its ultraviolet absorption spectrum is slightly different, such as bovine blood SOD in the 258nm at the maximum absorption, while the maximum absorption peak of pig blood SOD for 265nm, in specific conditions, the enzyme activity can be reduced, or even completely lost, such as cyanide can inhibit SOD activity, hydrogen peroxide to make the decline in the activity of SOD, guanidinium chloride can significantly inhibit the activity of SOD, and so on.
The use of SOD is mainly based on SOD is a superoxide anion scavenger, superoxide anion (O2 ~) is a toxic substance in the body, it can cause a variety of diseases, so it can remove the inflammatory process accompanied by the generation of superoxide ions, and show a strong anti-inflammatory effect.
In the pharmaceutical industry: SOD can treat a variety of diseases caused by superoxide ion, such as diabetes, cataracts, rheumatoid arthritis, etc., but also anti-inflammatory drugs, but also anti-aging, anti-tumor, anti-radiation, anti-autoimmune diseases, modern research shows that: SOD can also cure hypertension, coronary heart disease, cholecystitis, emphysema and other incurable diseases. Daily-use chemical industry: Because SOD has the effect of preventing cell aging and detoxification, therefore, in cosmetics and skin care agents and other industrial products, after adding SOD, it has the effect of sunscreen, blemish removal, and making the skin soft. It is an indispensable additive in the cosmetic industry, and a large number of products have appeared in the domestic market. Food industry: in the food additives plus SOD, after the enhancement of nutrition, lifting the toxicity, prolonging the life of cells in the body, the domestic has SOD nutrient solution products, and through the Shanghai scientific research unit of the relevant experts' appraisal.
Three, the development of SOD prospects: SOD in the country only a few scientific research units and biochemical pharmaceutical factories research and development of this product, but the use of SOD products manufacturers are increasing, far from meeting market demand . In order to encourage and vigorously develop SOD new products, the state has been included in the key scientific and technological development projects, and put a large number of funds, has been successful, began to transfer to the enterprise production, the market outlook is very broad.
Four, Note: (1) I warmly welcome friends from all walks of life on-site learning, by biochemical engineers to your lectures, through on-site operation, guidance, training, so that you master the technology as soon as possible. (2) The process, technical information, etc. without the consent of my company, shall not be transferred without authorization, reprinted, sold to any unit or individual, violators of the law.
New method of refining technology
I. Main equipment: (1) industrial centrifuge; (2) thermostatic water bath (10L - 50L) (can be made) 1; (3) freezer 1; (4) blender 1; (5) glassware: 1000 ml, 500 ml beaker each of the three, 50 ml beaker 2, 50 ml, 500 ml measuring cups each 1; (6) a number of plastic buckets.
Two, the main reagents: (industrial purity) sodium citrate, citric acid, glucose, ethanol 90%, chloroform, acetone, sodium chloride, disodium phosphate, sodium dihydrogen phosphate, deionized or distilled water, glucose gel SephadexG - 100 and DEAE - cellulose.
Three, the preparation of reagents:
1. Anticoagulant formula: (1) 30g of sodium citrate, citric acid 10g glucose 25g with water to 1 liter, generally such is every 7ml of blood, plus 1ml (anticoagulant). (2)40g/liter sodium citrate solution: add 0.9g of sodium chloride to 1 liter of water.
2. Saline: (0.9%) Add 0.9 g of sodium chloride to 91 g of water.
3. Phosphate buffer solution preparation: PH7.6, 0.2M phosphate buffer solution, usually with Na2HPO4.H2O, 35.61 g / liter and NaH2PO4.2H2O 31.21 g / liter, the former add 87.0 ml, the latter add 13.0 ml after mixing that is PH = 7.6 buffer solution, if you use K2HPO4.3 H2O is 45.644g/liter, NaH2PO4.2H2O is 31.21g/liter, still 87.0ml of the former, the latter 13.0ml mixed that is.
4. coolant: in this process there is the use of -15 degrees low temperature, generally use the following formula: 33 grams of salt to add 100 grams of snow or crushed ice, this proportion of the mixture can reach -21.3 degrees.
Four, the process
(a) in addition to hemoglobin
1. blood anticoagulation treatment, in fresh bovine, equine and porcine blood quickly added to the ACD1 anticoagulant and slowly stirred, every 7 milliliters of blood added to the anticoagulant 1 milliliters or anticoagulant No. 2.
2. Put the anticoagulated blood into the centrifuge, centrifuge at 3000r/min for about 15--20 minutes, use the pipette to suck the supernatant (yellow serum) carefully and throw it off, and the lower red blood cells are washed with 2--3 times of saline for 2--3 times, and in each washing, centrifuge at 3000r/min for 7--8 minutes, and get the washing after repeated operation. After repeated operations, the washed hemoglobin cells can be preserved for half a year by freezing at -15 degrees Celsius without affecting the yield and specific activity, which is necessary when the blood volume is large and the processing is incomplete.
3. Add an equal volume of deionized water to the washed red blood cells and stir vigorously for 30 minutes to break down the cell walls of the red blood cells so as to leach out the SOD in them. It is best to put the lysed blood for organic extraction after standing overnight at 0--4 degrees.
4. Then slowly add 0.2 - 0.25 times the volume of pre-cooled (-15 degrees) 95% ethanol and 0.1 - 0.15 times the volume of pre-cooled chloroform to the lysed blood, stirring for 10-15 minutes, the original lysed blood was a slurry, static 1 - 2 hours, with a filter cloth to remove the coagulation of hemoglobin, and then will be 2500r/min centrifugation of the filtrate for about 1-5 minutes to leave supernatant. Throw away the precipitate, at this time if the supernatant is slightly yellow, can be filtered with fast filter paper, you can get clear and transparent crude enzyme solution with a slight blue color.
(B) preliminary purification of crude enzyme solution:
1. acetone alcohol: to the enzyme solution plus 0.8 times the amount of pre-cooled (-15 degrees) acetone, generating a large number of white precipitate, centrifuged at 3000r/min for 2 minutes to collect the precipitate, the supernatant can be absorbed without direct dumping.
2. Thermal denaturation: (in addition to heterogeneous proteins) to prepare a balanced temperature bath, in this process, 30 minutes in advance, filled with water to turn on the power supply, so that it is heated to 55 - 60 degrees in order to save time, the amount of precipitation about 50 times the amount of volume ratio to join the 0.2MPH = 7.6 sodium phosphate buffer solution, the water bath is heated to 60 degrees, 15 minutes after the rapid cooling to room temperature.3000r/min Centrifuge for 2 minutes to get the supernatant, in the supernatant, slowly add 0.6 times the volume of pre-cooled (-15 degrees Celsius) acetone dropwise, to produce a white precipitate.
3. Initial purity of SOD: add deionized water to the above precipitate, make 20--30% SOD solution, divided into sample bottles, put into the freeze dryer, after the start to 0.2--0.1Torr vacuum, about 1.5--2 hours, to get the make-up or food SOD, the specific activity of the product is greater than 3000u/mg, the appearance of the appearance of a slightly bluish white lyophilized product. The product has a specific activity of more than 3000u/mg and a white lyophilized product with a bluish appearance.
(C) SOD further purification:
SOD fine is generally SOD products through column chromatography, column chromatography of SOD without small molecules and other impurities, chromatography of SOD macromolecules with high activity. SOD fine price is higher, it is mainly used as a biochemical reagent, laboratory standard reagent trial. Currently there are two kinds of SOD column chromatography, the two can be used alone, can also be used in combination, generally DEAE - cellulose chromatography and SephadcxG - 100 dextran gel chromatography, will be (ii) 2, heat denaturation of the SOD acetone precipitate after the solution with PH7.6, 0.2MNaH2PO4-NaHPO4 buffer, there are insoluble heterogeneous proteins, when the precipitate is removed by centrifugation, clearing and removing the precipitate, and then the precipitate is removed from the column, and then the precipitate is removed from the column by centrifugation. Centrifugation to throw off the precipitate, the supernatant on the DEAE - cellulose or SephadsxG - 100 chromatography column (1 * 20cm or 2.5 * 30cm), gradient elution , eluent PH7.6, 0.2M, NaH2PO4 - NaHPO4 buffer, downstream speed control at 0.2 - 0.5ml/min, in the spectrophotometer with UV spectra 325nm in the spectrophotometer to measure the effluent, the merger of the vitality peaks, and then repeated dialysis with distilled water, dialysis thoroughly, put the sample dish in the freeze dryer after drying that is the SOD boutique.
V. Notes:
1. Master the appropriate temperature and time: Although SOD is more stable to heat, but in the process of separation and purification is best to use a lower temperature, generally 0 - 10 degrees is appropriate, and the time should not be longer than 3 days.
2. Pay attention to the extraction, purification methods: the use of organic solvents or heat can be effective in precipitating proteins, but the combination of appropriate solvents and heating can achieve the best results.
Six, SOD test method: activity test, a more convenient way is to use the Lianhuanxuanol autoxidation method, first measured Lianhuanxuanol autoxidation rate, and then add SOD and then measured after the addition of SOD oxidation rate, according to the definition of the enzyme activity unit, i.e., in the optimal conditions, the amount of the enzyme per minute inhibits Lianhuanxuanol autoxidative decomposition rate of up to 50% of the enzyme is defined as a unit of enzyme. The total value of the activity can be determined sequentially, and the purity can also be determined by polyacrylamide gel electrophoresis to see whether a band with a molecular weight of about 32,000 is consistent with the guidance of the relevant literature.
Superoxide dismutase (superoxide dimutase abbreviated as SOD) is an important scavenger of oxygen free radicals, as a medicinal enzyme, causing great concern in biochemistry and medicine at home and abroad.SOD can be used in the treatment of a variety of diseases, rheumatoid arthritis, myocardial infarction, etc., and has a positive effect on anti-radiation, anti-aging, anti-tumor, etc., and is widely used in cosmetics. It also has a positive effect on anti-radiation, anti-aging, anti-tumor, etc. And it is widely used in cosmetics and food additives.
Since 1969, Mccord and Fridovich found superoxide dismutualization for the first time in bovine blood cells, so far, dozens of scientific research units have made a lot of research and trial production of SOD, and some have been mass production. In April 1989 in Kaifeng, Henan Province, "the third pharmaceutical enzyme conference", SOD is listed as a key development project, 90 years in July in Dalian, "the second national SOD conference", SOD has been developed as a pharmaceutical enzyme faster, will become a promising biochemical products.
SOD is a kind of biological enzyme which widely exists in animals, plants and microorganisms, foreign medicinal SOD is extracted from blood, domestic SOD has developed more than 20 kinds of varieties, which proves that China's research and application of SOD has entered a new period.
China's SOD from cattle, horses, pigs, chickens, dogs and other animals in the blood extracted from a rich source of low cost, extraction and purification is more convenient, the drug cell membrane is easy to break, with conventional separation and purification method can be obtained with a high degree of purity of SOD.
This technology adopts thermal denaturation, ultrafiltration of the new technology, not only greatly simplifies the process, and the quality of the product and the yield than the old process has been a greater increase in the quality of the extracted SOD is CuFePO4, which is a new technology of ultrafiltration. The extracted SOD are CuZu-SOD.
I. Drugs and Instruments:
(1) Trisodium citrate: Na3C6H6O7 (2) Industrial acetone CaH6O
(3) o-benzyl benzoin (4) Acid phosphoric acid potassium K3Poe4
(5) Weakly alkaline anion-exchanger DEAE Sephadsxa-50 Appearance 40-120ubrd or DEAE-cell-ujose (DE-32) diethyl cellulose. (6) Freeze dryer (own set)
(7) Laminar folding column (7.5*30cm) (8) Centrifuge
The above reagents and other agents and instruments can be purchased from chemical reagent stores and medical equipment stores in various provinces and cities.
Two, Cu, Zu-SOD extraction and purification (cattle, horse blood as an example)
1, the industrial process:
Add anticoagulant Salt washing
Fresh bovine blood --------------------->red blood cells ------------------------>
<Centrifugation to remove yellow plasma Add NaCL
Thermal denaturation 1 Thermal denaturation 2
Deposition of erythrocytes -------------->yellowish mixture --------------------->
68 degrees for 30 mins 60-65 degrees for 20 mins
Add Acetone Add Acetone Chromatography <
Light blue clear solution ----------->flocculent precipitate -------------->precipitate ------------>
Freeze-drying
Desalting by dialysis
Elution ----------------->dialysate ---------& gt;SOD(lyophilized)
2, extraction method
(1) collection of erythrocytes: 100 liters of fresh bovine blood, add 3. 8% trisodium citrate anticoagulant mixing well, stand, so that erythrocytes naturally settle, remove most of the plasma on the upper layer, then centrifuged to remove the rest of the plasma, and then add three times the amount of 0.9% sodium chloride (refined table salt) wash twice, centrifugation can be The pressure-accumulated erythrocytes (40 liters) were collected by centrifugation.
(2) thermal denaturation 1: the collection of red blood cells and then add 4 kg of sodium chloride, 40 grams of copper chloride, 100 liters of distilled water stirred, the water bath heated to 68 degrees Celsius constant temperature for 30 minutes, quickly cooled to room temperature, filtration in addition to the precipitate, collect the filtrate.
(3) thermal denaturation 2: filtrate at 65 degrees under a constant temperature water bath, slowly into the filtrate 40 grams of copper chloride, until the filtrate clear to light blue until, hold 20 minutes, cooled quickly to room temperature, centrifugation to precipitate the supernatant.
(4)SOD crude: ultrafiltration of the supernatant add 0.75 times the volume of acetone precipitation, centrifugation to collect the precipitate in away from water, centrifugation to remove insoluble matter to get the clear liquid, the clear liquid add 0.75 times the volume of acetone precipitation, centrifugation to collect the precipitate, the precipitate by the anhydrous acetone washing and dehydration of the two times, vacuum drying of the light blue powdery SOD crude
(5)SOD crude purification: SOD crude dissolved in 2.5mmol / L, PH value of 7.6 potassium phosphate buffer, the mixture on the 7.5 * 30cm layer column, ion exchanger for the DEAE-SphadexA-50 (the column beforehand with the buffer equilibrium), to 100 ~ 200ml / hr on the speed of the sample, after the completion of the same buffer A280mm value, less than 0.02 and then 25 ~ 200mmol / L, PH value, less than 0.02 and then use the same buffer A280mm value, less than 0.02 and then use 25 ~ 200mmol / L, PH value, less than 0.02. 200 mmol/L, pH 7.6 potassium phosphate buffer for linear gradient segmental elution at a flow rate of 100 ml/hour. Collected with a partial collector, the eluate was desalted by dialysis to get the concentrated dialysate, freeze-dried to get the finished SOD product, (light green).
Three, SOD activity determination:
SOD determination of many methods, common chemical method, immunoassay, such as the electric spot focusing method, the principle of chemical method is mainly the use of some compounds, such as o-o-benzenetriol, in the process of oxidation of the colored intermediates will be produced in the descending superoxide free radicals (O2) so that the use of the SOD sub-example (O2). The enzyme activity is measured by preventing the accumulation of intermediates.
1, reagents and instruments
(1) catechol analytically pure, with 10mmol / LECL to 50mmol / L solution.
(2) UV-754 ultraviolet spectrophotometer.
2. Determination method:
(1) Determination of the autoxidation rate of o-phenyltriol.
Add 10mmol/L of o-triol in 25 degrees, 45ml`50mmol/L, pH 8.3, K2HPO4-KH2PO4 buffer, shake quickly, pour into the colorimetric cup of 1cm optical diameter, measure the A-value every 30 seconds at the wavelength of 325mm, and ask the autoxidation rate to be controlled at 0.0700d/mm.
(2)Determination of SOD or crude enzyme extract activity:
The same method was used to measure the autoxidation rate of o-triol, and the SOD sample was added before adding o-triol, and the measured data were used to calculate the enzyme activity according to the following formula.
0.0700-A325mm/min
----------------------------------------＊100% degree
0.70
Sample dilution times Sample dilution times
Enzyme activity ( u/m1) = ------------ * total volume of reaction solution * ----------------
50% volume of sample solution
3, protein concentration (mg/ml) and protein calculation:
SOD or crude enzyme extract, after 280mm ultraviolet colorimetric measurement, check the standard curve of the bovine coupe white egg, which is calculated as follows The amount of ug protein per ml.
Protein concentration = ug protein/ml * sample dilution times * 10 minus 3 times = mg protein/ml total protein = mg protein/ml * total volume of the original liquid = mg protein.
4, the calculation of specific activity (u/mg protein):
Unit activity (u/ml) total activity
Specific activity = ----------------------- = ----------------- = u/mg protein
Concentration of protein (mg protein/ml) total protein
< p>Four, Cu, Zn-SOD properties:1, Cu, Z n-SOD is blue-green, molecular weight of 3200 or so, by two in a Cu and a Zn.
2, heat stable, natural bovine blood SOD, 75 degrees Celsius heated for a few minutes and its loss of enzyme activity is very small.
3, PH value on the impact of SOD, SOD in the PH5.3 ~ 10.5 range, its catalytic rate is not affected, PH3.6 when SODZn shed 95%, PH12SOD conformation of the non-deliverable conformation, resulting in the loss of enzyme activity.
4, SOD's ultraviolet absorption characteristics: SOD at 280mm no absorption peak.
5, metal coenzyme and enzyme activity. SOD is a metalloenzyme Zn is only related to the molecular structure, while Cu is related to the catalytic activity.
6, SOD is its has also and inactivation effect.
V. Drugs and instruments:
Trisodium citrate, analytically pure: Jiangsu Huaqiao Beigong four plants;
Industrial acetone: Shanghai Solvent Factory;
O-phenyltriol, analytically pure: Zunyi Chemical Factory, Guizhou;
Sixth, SOD's underwriting unit:
(1) Zhengzhou, Henan, the State-run St. Science and Technology Research Institute, Zhengzhou, 14 Nanyang Road, 450053, Henan, China. Zip code: 450053 "Henan Science and Technology Newspaper" October 3, 91
(2) Jintang County, Sichuan Province, craft products Technical School of Biochemistry Section Reception Room: Jintang quasi-execution of the courtyard, the Government Office Building, 1st Floor Zip code: 610404 Contact: Deng Yong Peng Li "Sichuan Rural Newspaper" March 1, 91
(3) Shenzhen City, Guangdong Province, on the step of the district Shixia East Ercang 14th Science and Technology Institute Zip Code: 518031 Contact: Huang Qiulin "Science and Technology News" July 10, 92
Description: 1, the information attached to the product sales address information are extracted from the last two years from the newspapers and magazines of the units advertisements, and indicate that there is a source.
2, you first read the information, line according to their own actual situation, the first small amount of test, and contact the product acquisition unit in advance, to obtain experience before mass production.
3, readers in contact with the acquisition of units, please be sure to contact in advance, with postage, to get a clear answer and then according to the situation to go to the people for, do not take the risk of going, so as not to cause unnecessary economic burden.
4, raw materials and instruments around the operation of the raw materials store supply, Guangzhou City, People's South Road, Shangjiu Road and other places are supplied.
Attachment: detailed SOD acquisition information
1, Nanning City, Guangxi Biochemical Pharmaceutical Factory
Address: No. 1, Luban Road
2, Shanghai Biochemical Pharmaceutical Factory
Address: No. 513, Haihou Road
3, Xuzhou City, Jiangsu Province, Biochemical Pharmaceutical Factory
Address: No. 86, Hai Pei Road
4, Yanzhou City, Shandong Province, the raw materials and instruments are supplied by the raw material stores operating in various places, such as People's South Road, Shangjiu Road, Guangzhou City. p>4, Shandong Yanzhou City Biochemical Pharmaceutical Factory
Address: Meat factory
5, Hunan Changde Biochemical Pharmaceutical Factory
Address: Meat factory
6, Foshan City Biochemical Pharmaceutical Factory
Address: Foshan City
7, Guangzhou City, Mingxing Pharmaceutical Factory
Address: No. 48, North Industrial Avenue, Henan Rd, Guangzhou City
7, Guangzhou City, Mingxing Pharmaceutical Factory
Address: No. 48, North Industrial Avenue, Henan Rd. Industrial Avenue North No. 48
8, Guangzhou City, Baiyun Pharmaceutical Plant
address: from Guangzhou Yuexiu Park, take the 21 bus to the Sanyuanli North Exhibition get off along the side of the Mineral Springs Villa into the 200 meters
9, Harbin Biochemical Pharmaceutical Plant Harbin Meat Factory
address: Harbin, Daowai, Nanyi Street, No. 12 yard; Tel: 88423 to the pharmaceutical plant
10, Shenyang City, Shenyang Biochemical Pharmaceutical Plant
address: Harbin Huanggu District, Minglian Road No. 2;
11, Nanjing, Jiangsu Province, Biopharmaceutical Plant
address: Nanjing, Jiangsu Province, the lower part of the district near the Baota Bridge;
12, Jinhua, Zhejiang Province, Biochemical Pharmaceutical Plant
address: No. 51, Hepanqiao Road, Jinhua, Zhejiang Province
< p>13、Guangdong Biological Pharmaceutical FactoryAddress: Sanyuanli, Guangzhou City
14、Guangdong Shantou Biochemical Pharmaceutical Factory
Address: No.2, No.3, West Lane, Hushan Road; Tel: 666124
11、Nanjing Biological Pharmaceutical Factory, Jiangsu Province
Address: Near Baota Bridge, Xiaguan District, Nanjing City, Jiangsu Province
12. Jinhua Biochemical Pharmaceutical Factory, Zhejiang Province
Address: No.51, Hepanqiao Road, Jinhua City, Zhejiang Province
13. Guangdong Biopharmaceutical Factory
Address: Sanyuanli, Guangzhou City
14. Shantou Biochemical Pharmaceutical Factory, Shantou, Guangdong Province
Address: No.3, West Lane, Hushan Road
15. Shanghai Xiafei Daily-use Chemical Factory
16.Beijing Liyuan Daily-use Chemical Factory
17.Nanjing Cosmetic Factory
18.Shanghai No.2 Daily-use Chemical Factory
19Guangzhou Beauty and Cosmetic Factory
20Nanjing No.2 Pharmaceutical and Chemical Pharmaceutical Factory
Shanghai Yongfang Daily-use Chemical Factory
Guangzhou Cosmetic Factory
Hangzhou Fenghi Cosmetic Factory
Shanghai Daily-use Chemicals No.4 Factory
Tianjin Jinle Pharmaceutical Factory
Hangzhou No.1 Biochemical Pharmaceutical Factory
Shanghai Institute of Pharmaceutical Research Experimental Pharmaceutical Factory
Tianjin Nankai District Beauty and Cosmetics Factory
Suzhou City, Haoxian Biochemistry Factory
Shanghai Biochemical Pharmaceutical Factory
Tianjin Biomedical Technology Development Center
Shanghai Xianghai Cosmetic Factory
Beijing Daily-use Chemicals Factory
Wuhan Xin'andu Pharmaceutical Factory
Hubei Shashi Biochemical Pharmaceutical Factory
Beijing Sanlu Factory
Note: All the major pharmaceutical and cosmetic factories and beauty parlors are all the target of sales
Tel:010-68150495,6821495 68150495, 68212139
Fax: 010-68212015
Address: 3rd Floor, Huadian Group Building, Wujiacun, Shijingshan District, Beijing
Correspondence: P.O. Box 40-021, Shijingshan District, Beijing
Postal Code: 100040