CN1082612A - The measuring method of activity of super-oxide dismutase - Google Patents
The measuring method of activity of super-oxide dismutase Download PDFInfo
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- CN1082612A CN1082612A CN 92107634 CN92107634A CN1082612A CN 1082612 A CN1082612 A CN 1082612A CN 92107634 CN92107634 CN 92107634 CN 92107634 A CN92107634 A CN 92107634A CN 1082612 A CN1082612 A CN 1082612A
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- dissolved
- azanol
- xod
- measuring method
- test
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Abstract
The invention discloses a kind of method of measuring superoxide dismutase activity, this method genus nitrosomonas salt formation method is utilized XO to add the XOD reactive system and is produced ultra-oxygen anion free radical O
2, O
2The oxidation azanol forms nitrite, passes through chromogenic reagent.The invention is characterized in the test XO is dissolved in the alkaline solution that azanol is dissolved in the weak acid, and adopt new pH of buffer value and new developer proportioning.The present invention compared with prior art, the test fluid absorbancy is big and stable, test result is accurate, test speed is fast, and is highly sensitive, cost is low.The present invention can be widely used in the laboratory and Clinical Laboratory is used.
Description
The present invention relates to the measuring method of superoxide dismutase activity in the free radical Med Biol field.
Superoxide-dismutase (Superoxide dismutase. is called for short SOD) is a kind of important enzyme that body is removed ultra-oxygen anion free radical, and the oxidation and the anti-oxidant balance of body played crucial effects.This enzyme can be removed ultra-oxygen anion free radical (O
2), the protection cell is avoided damage.Thereby the height of SOD vigor has close getting in touch with aging, tumour, inflammation, autoimmune disease, blood, cardiovascular diseases, nephropathy etc., and is significant to nosetiology discussion, diagnosis, treatment and the observation etc. of disease.
At present, the method for testing the SOD vigor both at home and abroad is a lot, as electron paramagnetic spectral method and nuclear magnetic resonance method, since required plant and instrument costliness, complicated operation, and common laboratory and hospital are difficult to use, domestic general employing chemical colorimetry, but be subjected to extraneous factor to influence greatly the experiment effect instability; " Reevaluarion of Assay Methods and Establishment of Kit for Superoxide Dismutase Activity " literary composition of YOSHIHIKO OYANAGVL, publish in ANALYTICALBIOCHEMISTRY142,290-296(1984), introduced a kind of nitrite method of the SOD of mensuration vigor.This method is utilized xanthine (XO) to add XOD (XOD) reactive system and is produced ultra-oxygen anion free radical O
2, in this system, add electronics acceptor azanol, then O
2The oxidation azanol forms nitrite, adds developer (Sulphanilic Acid+N-first bitter edible plant base two amido ethene) again, and then test fluid is red-purple.Above-mentioned reaction is at KH
2PO
4With Na
2B
4O
7Carry out in the buffer solution system.The problem that aforesaid method exists is: 1, add 10 in the slow liquid of phosphoric acid
-4The EDTA of M/L, concentration is too high, can influence copper zinc-SOD vigor, makes test result inaccurate; 2, substrate xanthine XO and azanol are the KH that is dissolved in PH8.2
2PO
4And Na
2B
4O
7In, 1. in fact XO can only dissolve a part, and some precipitates, and makes the result very inaccurate; 2. and azanol very easily oxidation in this buffer system, the preservation period of reagent is shortened; 3. because the autoxidation of azanol makes reagent under the situation that does not add XOD, can produce nitrite in the reaction system, promptly approaching then this method mensuration of control tube and blank pipe colour generation is worth little; 3, the developer that adopts of this method is 300 μ g/ml Sulphanilic Acid and the Glacial acetic acid of the basic two amido ethene of the 5 μ g/mlN-first bitter edible plants and 16.7% mixes, it is extremely shallow that this mixes chromogenic reagent, the control tube absorbancy is between 0.080-0.090, and general instrument is difficult to differentiate.
The object of the present invention is to provide a kind of measuring method of superoxide dismutase activity,, and make testing method easy, quick, economical with the accuracy of raising mensuration.
The object of the present invention is achieved like this: superoxide dismutase activity measuring method genus nitrosomonas salt formation method of the present invention, it utilizes xanthine (XO) to add XOD (XOD) reaction system and produces ultra-oxygen anion free radical O
2XOD oxidation azanol forms nitrite, add developer (Sulphanilic Acid adds N-first bitter edible plant base two amido ethene) again, make test fluid be red-purple, test process carries out in the phosphoric acid buffer liquid system, it is characterized in that selecting pH value during mensuration for use is 8.0 phosphoric acid buffer, wherein EDTA concentration is 2 * 10
-5Mol/L; XO is dissolved in the basic solution, fully dissolving; Azanol is dissolved in the weak acid; Adopt the 1.65mg/ml Sulphanilic Acid and 0.75mg/mlN-first bitter edible plant base diamino-vinyl and 20% Glacial acetic acid mixed solution to make developer.
Principle of the present invention: 1, xanthine XO dissolves in the alkaline solution, and dissolving fully; Be that reactant solution is even, then test result is satisfied; 2, azanol is dissolved in the weak acid, makes test fluid stable, and the shelf time is long; 3, add 2 * 10 in the phosphoric acid buffer
-5The EDTA concentration of mol/l is moderate can not to influence the SOD vigor simultaneously again by complexation of metal ions; Make measurement result accurately stable; 4, developer proportioning of the present invention can make color stability, and too high or too low meeting causes absorbancy too high or too low.
The present invention compared with prior art, its remarkable advantage is: 1, the test fluid absorbancy improves greatly, can reach 0.45, general 721 or 722 spectrophotometers can be tested; 2, test result is accurate, stable, and variation coefficient CV can reach 1.7%, and rate of recovery χ ± SD can reach 103% ± 2.63%(n=5); 3, measurement sensitivity height, ID
50Can reach 0.05 μ g/ml; 4, test speed is fast, per hour can survey 100 routine samples.The present invention can be widely used in laboratory and Clinical Laboratory.
Below in conjunction with embodiment the present invention is further described.
SOD of the present invention measures and adopts the XO+XOD reaction system to produce O
2The principle that adds azanol and developer again is determined in control tube and two test tubes of mensuration pipe and carries out, and measures in the pipe to add tested sample, do not add sample in the control tube, and according to following Step By Condition reagent preparation, taking a morsel adds respectively in the test tube:
1, Na
2HPO
320.5 gram+NaH
2PO
3752mg+EDTA38.7mg be dissolved in 37 ℃, in the 1000ml water;
2, the 70mg azanol is added in the 0.03N/L HCl solution of 100ml;
3,98mgXO is added to the Na of 0.1N/L
2HPO
4In the solution;
4, the XOD of 5 units is dissolved in the phosphoric acid buffer of 182ml;
5, the preparation of developer;
1. 0.66 gram Sulphanilic Acid is dissolved in 50 ℃, in the water of 150ml;
2. 0.3 gram N-first bitter edible plant base, two amido ethene are dissolved in 50 ℃, in the water of 150ml;
3. Glacial acetic acid 100ml;
Will be 1. 2. 3. three kinds of test solutions before test by 3: 3: 2 mixed, every test tube is got the 2ml colour developing.
Claims (3)
1, a kind of superoxide dismutase activity measuring method belongs to the nitrite forming method, and it utilizes xanthine (XO) to add XOD (XOD) reaction system and produces ultra-oxygen anion free radical O
2, XOD oxidation azanol forms nitrite, adds developer (oxygen base Phenylsulfonic acid is added N-first naphthyl two amido ethene) again and makes test fluid be red-purple, and test process carries out in the phosphoric acid buffer liquid system, it is characterized in that:
1.1 the phosphoric acid buffer pH value is 8.0, wherein EDTA concentration is 2 * 10
-5Ml/l;
1.2XO be dissolved in the basic solution, fully dissolving;
1.3 azanol is dissolved in the weak acid;
Make developer 1.4 adopt 1.65mg/ml Sulphanilic Acid and 0.75mg/mlN-first naphthyl diamino-vinyl and 20% Glacial acetic acid mixed solution.
2, superoxide dismutase activity measuring method according to claim 1 is characterized in that XO is dissolved in 0.1N/L Na
2HPO
4In.
3, superoxide dismutase activity measuring method according to claim 1 and 2 is characterized in that azanol is dissolved in the 0.03N/L HCl solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 92107634 CN1082612A (en) | 1992-07-09 | 1992-07-09 | The measuring method of activity of super-oxide dismutase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 92107634 CN1082612A (en) | 1992-07-09 | 1992-07-09 | The measuring method of activity of super-oxide dismutase |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1082612A true CN1082612A (en) | 1994-02-23 |
Family
ID=4942959
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 92107634 Pending CN1082612A (en) | 1992-07-09 | 1992-07-09 | The measuring method of activity of super-oxide dismutase |
Country Status (1)
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| CN (1) | CN1082612A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1111740C (en) * | 1998-09-10 | 2003-06-18 | 吉林省卫生防疫站 | Solid developer and manufacture method thereof and purposes |
| CN100494979C (en) * | 2006-06-02 | 2009-06-03 | 中国科学院沈阳应用生态研究所 | Analytical method for detecting hydroxyl amine reducing enzyme activity in soil |
| CN107884349A (en) * | 2017-10-13 | 2018-04-06 | 昆明理工大学 | The assay method of ultra-oxygen anion free radical in a kind of microbial body |
-
1992
- 1992-07-09 CN CN 92107634 patent/CN1082612A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1111740C (en) * | 1998-09-10 | 2003-06-18 | 吉林省卫生防疫站 | Solid developer and manufacture method thereof and purposes |
| CN100494979C (en) * | 2006-06-02 | 2009-06-03 | 中国科学院沈阳应用生态研究所 | Analytical method for detecting hydroxyl amine reducing enzyme activity in soil |
| CN107884349A (en) * | 2017-10-13 | 2018-04-06 | 昆明理工大学 | The assay method of ultra-oxygen anion free radical in a kind of microbial body |
| CN107884349B (en) * | 2017-10-13 | 2020-12-15 | 昆明理工大学 | Determination method of superoxide anion free radicals in microorganisms |
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| C01 | Deemed withdrawal of patent application (patent law 1993) | ||
| WD01 | Invention patent application deemed withdrawn after publication |