CN1418952A - Process for preparing superoxide dismutase - Google Patents
Process for preparing superoxide dismutase Download PDFInfo
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- CN1418952A CN1418952A CN 02147888 CN02147888A CN1418952A CN 1418952 A CN1418952 A CN 1418952A CN 02147888 CN02147888 CN 02147888 CN 02147888 A CN02147888 A CN 02147888A CN 1418952 A CN1418952 A CN 1418952A
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- blood cell
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- dismutase
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Abstract
The preparation method of superoxide dismutase includes the following steps: 1). separating blood cell; 2). washing blood cell; 3) dissolving and breaking blood cell; and 4). removing hemoglobin to obtain crude enzyme liquor. It is characterized by that the crude enzyme liquor is heated at 60-65 deg.C for 20 min. to obtain high purity superoxide dismutase, and the crude enzyme liquor is heated at 60-65 deg.C for 20 min., the high purity superoxide dismutase with small heteroprotein content and large specific activity of enzyme can be obtained.
Description
Technical field
The present invention relates to a kind of method that superoxide-dismutase in the pig blood is extracted.
Background technology
In the prior art, the extraction of carrying out superoxide-dismutase in pig blood is adopted separation usually, extracted with ethanol and chloroform then, obtain crude enzyme liquid.Because pig blood poor heat resistance, thereby the crude enzyme liquid of obtaining usually contains more foreign protein.
Summary of the invention
At above-mentioned deficiency, the object of the present invention is to provide a kind of preparation method who in pig blood, extracts highly purified superoxide-dismutase.
To achieve these goals, technical scheme of the present invention is: the preparation method of superoxide-dismutase, carry out according to the following steps:
1), separate blood cell: get fresh pig blood 250ml, add 1g trisodium citrate (antithrombotics) immediately, stir, under 0-4 ℃ of condition, 3000 rev/mins centrifugal 15 minutes, blood cell; 2), washing blood cell: 0.9% physiological saline that adds with volume washs blood cell, and centrifugal with above-mentioned similarity condition, triplicate; 3), dissolve the blood cell that breaks: the blood cell after the washing is collected in the beaker, adds then and the distilled water of blood cell, in ice-water bath, stirred haemolysis 30 minutes, and in 0-4 ℃ refrigerator, place with volume; 4), dehemoglobinize: with adding 95% ethanol of precooling of 0.25 times of volume and the precooling chloroform of 0.15 times of volume in the haemolysis, stirred 15 minutes, left standstill 30 minutes, 5600 rev/mins centrifugal 10 minutes, get supernatant liquor, and recentrifuge, must crude enzyme liquid; It is characterized in that crude enzyme liquid was heated 20 minutes under 60-65 ℃ of condition, obtain highly purified superoxide-dismutase.
The present invention heats crude enzyme liquid 20 minutes under 60-65 ℃ of condition, can obtain the high purity superoxide-dismutase that heteroproteins content is few, the specific activity of enzyme is big.This method is simple, and cost is few, and the efficient height has greatly improved the purity of enzyme.
Description of drawings
Fig. 1 is that differing temps is handled back superoxide-dismutase specific activity change curve to slightly carrying enzyme liquid.
Embodiment
The preparation method of superoxide-dismutase, 1), separate blood cell carry out according to the following steps:: get fresh pig blood 250ml, add 1g trisodium citrate (antithrombotics) immediately, stir, under 0-4 ℃ of condition, 3000 rev/mins centrifugal 15 minutes, blood cell; 2), washing blood cell: 0.9% physiological saline that adds with volume washs blood cell, and centrifugal with above-mentioned similarity condition, triplicate; 3), dissolve the blood cell that breaks: the blood cell after the washing is collected in the beaker, adds then and the distilled water of blood cell, in ice-water bath, stirred haemolysis 30 minutes, and in 0-4 ℃ refrigerator, place with volume; 4), dehemoglobinize: with adding 95% ethanol of precooling of 0.25 times of volume and the precooling chloroform of 0.15 times of volume in the haemolysis, stirred 15 minutes, left standstill 30 minutes, 5600 rev/mins centrifugal 10 minutes, get supernatant liquor, and recentrifuge, must crude enzyme liquid; 5), crude enzyme liquid was heated 20 minutes, obtain highly purified superoxide-dismutase under 60-65 ℃ of condition.Example:
Get 8 and be numbered 1
40 ℃, 2
45 ℃, 3
50 ℃, 4
55 ℃, 5
60 ℃, 6
65 ℃, 7
70 ℃, 8
75 ℃Fill the crude enzyme liquid test tube, place differing temps respectively, be incubated 20 minutes, experimental result is as follows:
| Test tube number | Former crude enzyme liquid | ?1 40℃ | ?2 45℃ | ?3 50℃ | ?4 55℃ | ?5 60℃ | ?6 65℃ | ?7 70℃ | ?8 75℃ |
| SOD vigor u/ml | ?799.5 | ?733.5 | ?723 | ?538.5 | ?532.5 | ?517.5 | ?465 | ?216 | ?90 |
| Protein content mg/ml | ?2.95 | ?2.825 | ?2.45 | ?1.733 | ?1.365 | ?1.239 | ?1.147 | ?1.08 | ?0.815 |
| Specific activity u/mg | ?271 | ?259.5 | ?295 | ?310.7 | ?390 | ?417.6 | ?405.3 | ?200 | ?110.4 |
From as can be known above, between 40C-75 ℃ the main degree of any temperature handle 20 minutes all influential to enzymic activity, along with temperature rising gradually, enzymic activity reduces.From specific activity, maximum value appears between 60-65 ℃, as Fig. 1, illustrate that enzymic activity decline herein is less relatively, and the foreign protein removal is more, thereby extracts enzyme purity maximum at this section.
Claims (1)
1, the preparation method of superoxide-dismutase, 1), separate blood cell carry out according to the following steps:: get fresh pig blood 250ml, add the 1g trisodium citrate immediately, stir, under 0-4 ℃ of condition, 3000 rev/mins centrifugal 15 minutes, blood cell; 2), washing blood cell: 0.9% physiological saline that adds with volume washs blood cell, and centrifugal with above-mentioned similarity condition, triplicate; 3), dissolve the blood cell that breaks: the blood cell after the washing is collected in the beaker, adds then and the distilled water of blood cell, in ice-water bath, stirred haemolysis 30 minutes, and in 0-4 ℃ refrigerator, place with volume; 4), dehemoglobinize: with 95% ethanol of the precooling of 0.25 times of volume of adding in the haemolysis and the precooling chloroform of 0.15 times of volume, stirred 15 minutes, left standstill 30 minutes, 5600 rev/mins centrifugal 10 minutes, get supernatant liquor, and recentrifuge, get crude enzyme liquid: it is characterized in that crude enzyme liquid was heated 20 minutes, obtain highly purified superoxide-dismutase under 60-65 ℃ of condition.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02147888 CN1418952A (en) | 2002-12-19 | 2002-12-19 | Process for preparing superoxide dismutase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02147888 CN1418952A (en) | 2002-12-19 | 2002-12-19 | Process for preparing superoxide dismutase |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1418952A true CN1418952A (en) | 2003-05-21 |
Family
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 02147888 Pending CN1418952A (en) | 2002-12-19 | 2002-12-19 | Process for preparing superoxide dismutase |
Country Status (1)
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| CN (1) | CN1418952A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1300308C (en) * | 2004-07-29 | 2007-02-14 | 王建兴 | New method for picking-up superoxide dismutase from animal blood |
| CN100392076C (en) * | 2005-07-27 | 2008-06-04 | 周兴和 | Extraction and modification method for superoxide dismutase |
| CN102925416A (en) * | 2012-11-19 | 2013-02-13 | 景志刚 | Method for extracting superoxide dismutase from blood of mammal |
| CN103397003A (en) * | 2013-07-22 | 2013-11-20 | 南京宝迪农业科技有限公司 | New process of technology for extracting superoxide dismutase from animal blood |
-
2002
- 2002-12-19 CN CN 02147888 patent/CN1418952A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1300308C (en) * | 2004-07-29 | 2007-02-14 | 王建兴 | New method for picking-up superoxide dismutase from animal blood |
| CN100392076C (en) * | 2005-07-27 | 2008-06-04 | 周兴和 | Extraction and modification method for superoxide dismutase |
| CN102925416A (en) * | 2012-11-19 | 2013-02-13 | 景志刚 | Method for extracting superoxide dismutase from blood of mammal |
| CN103397003A (en) * | 2013-07-22 | 2013-11-20 | 南京宝迪农业科技有限公司 | New process of technology for extracting superoxide dismutase from animal blood |
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| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
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