CN104297185A - Method for detecting activity of catalase - Google Patents
Method for detecting activity of catalase Download PDFInfo
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- CN104297185A CN104297185A CN201410577230.4A CN201410577230A CN104297185A CN 104297185 A CN104297185 A CN 104297185A CN 201410577230 A CN201410577230 A CN 201410577230A CN 104297185 A CN104297185 A CN 104297185A
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Abstract
The invention relates to a method for detecting activity of catalase, belonging to the field of enzymatic activity detection methods. The method for detecting activity of catalase comprises the following steps: diluting a catalase standard substance into a standard solution with a series of catalase activity concentrations, adding 10mL of chlorine peroxide phosphate buffer solution in a 100mL iodine measuring flask, putting the iodine measuring flask in water bath at 25 DEG C and carrying out heat preservation, then adding 2mL of a standard enzyme solution, accurately carrying out heat preservation for 3 minutes, instantly adding 2mL of 0.5mol/L sulfuric acid to end reaction, adding 2mL of 0.5mol/L sulfuric acid into a contrast tube of a contrast reaction solution to end reaction, and then adding the enzyme solution; then extracting 0.1mL of the reaction solution, adding 2.9mL of a peroxidase-dianisidine solution in a test tube and shaking to be uniform, firstly carrying out zero adjustment by a blank test tube in 436microns by virtue of a spectrophotometer at 25 DEG C, then detecting absorbancy of each reaction solution test tube to obtain a curve about the relation between the standard enzymatic activity and the absorbancy. The test enzyme is processed by the same method, so that the enzymatic activity of the test enzyme can be obtained according to the absorbancy and the standard curve. The detection method has the characteristics of being accurate, high in practicability, simple in instrument requirement and suitable for operation in the conventional condition; the detection method belongs to one reliable method of color rendering methods for detecting the activity of the catalase.
Description
Technical field
The present invention relates to a kind of detection method of catalase activity, belong to method for detecting enzymatic activity field.
Background technology
Along with the reach of science and social progress, the purposes of hydrogen peroxidase (ECl.11.1.6) is more and more extensive.It is more is distributed in animal and plant cells, and aerobe metabolic process also has a large amount of hydrogen peroxidases and produces, and it produces and existence is a dynamic process.The hydrogen peroxide that animal's liver cellular metabolism produces is many, and this enzyme also has a large amount of savings, and extracting from animal's liver is the catalatic important channel of preparation; Bacterium and mould also have a large amount of hydrogen peroxidases during the fermentation and are released to outside born of the same parents, put aside in fermentation liquor, obtain the higher hydrogen peroxidase that can be applicable to industry and medical circle of purity by biochemical preparation method.
Hydrogen peroxide is a kind of strong oxidizer, and at present at textile industry more application bleaching, and unnecessary hydrogen peroxide can remove with hydrogen peroxidase, does the requirement according with very much environmental protection like this.Medically also can remove slough with low concentration hydrogen peroxide, and then with the unnecessary hydrogen peroxide of catalase reaction.Glucose oxidase and hydrogen peroxidase compound formulation also can be used for control animal intestinal, stomach by bacterial some diseases.Therefore catalatic enzyme activity determination seems very important for the field of this enzyme of investigation and application.
The more assay method of current application is titrimetry, Source of UV Rate Method etc., and this several method all can be subject to the restriction of certain condition in general operation application.Therefore study that a kind of accuracy is high, repeatability is high, easy and simple to handle, the detection method of the catalase activity of the operation be applicable under normal condition is necessary.
Summary of the invention
The present invention aims to provide that a kind of accuracy is high, repeatability is high, easy and simple to handle, the detection method of the catalase activity of the operation be applicable under normal condition.
The detection method of described catalase activity, comprises the following steps:
Hydrogen peroxidase standard items are diluted to the titer of a series of concentration of enzymatic activity, get 10mL chlorine peroxide phosphate buffer in 100mL iodine flask, put in 25 DEG C of water-baths and be incubated, then 2mL enzyme titer is added, accurate insulation 3min, add 2mL0.5mol/ sulfuric acid cessation reaction immediately, control reaction liquid control tube first adds enzyme-added liquid after 2mL0.5mol/L sulfuric acid cessation reaction.Then get above-mentioned reactant liquor 0.1mL to add 2.9mL peroxidase-dianisidine solution and shake up in testing tube, at 25 DEG C, after 436 μm of places are first with the zeroing of blank examination test tube, measure the absorbance of each reactant liquor testing tube with spectrophotometer again, obtain standard enzyme activity and absorbance relation curve.By above-mentioned identical method process tested enzyme, the enzymatic activity of tested enzyme can be obtained according to its absorbance and typical curve.
Preferably, hydrogen peroxide phosphoric acid buffer liquid making method of the present invention, for getting 1mL30% hydrogen peroxide, is diluted to 1000mL with 0.01mol/L, pH6.8 phosphate buffer.
Preferred, peroxidase of the present invention-dianisidine solution is 0.03% peroxidase and 1% dianisidine methanol solution.
Detection method of the present invention, its principle is that Catalases catalyze peroxide decomposition generates oxygen and water.Remaining hydrogen peroxide is with Catalyzed Synthesis By Peroxidase reaction under having oxygen donor to exist, and oxygen donor and o-(two) anisidine are oxidized to brown product, measures absorptance, and extrapolate enzymatic activity in 436 μm of places.
Detection method of the present invention, after measured, shows from the curve of standard back substrate concentration OD value, and enzymatic activity is within the scope of 23 ~ 69u/mL, and along with enzyme activity increases and the decline of OD value, in good linear relationship, curved line relation resolution is good.Enzyme activity declines very fast in more than 70u/mL, OD value, curved line relation resolution declines; Enzyme activity declined slow in below 22u/mL, OD value, and curved line relation resolution is very low.For this reason, detection method of the present invention is applicable to the Enzyme assay that enzymatic activity is the enzyme liquid between 23 ~ 69u/mL.It is 99.37% that its standard items measure the recovery.
That detection method of the present invention has is accurate, practical, instrument requirements simple, is applicable to the feature of the operation under normal condition, is to measure a kind of reliable method in activity of catalase development process.
Embodiment
Embodiment one:
Standard enzyme liquid is prepared: titer hydrogen peroxidase standard items being diluted to a series of concentration of enzymatic activity, for subsequent use.
Hydrogen peroxide phosphate buffer is prepared: get 1mL30% hydrogen peroxide, be diluted to 1000mL with 0.01mol/L, pH6.8 phosphate buffer, for subsequent use.
Peroxidase-dianisidine solution preparation: get 0.3g peroxidase and 10g dianisidine is dissolved in the methanol solution of 1000mL, for subsequent use.
Get 10mL chlorine peroxide phosphate buffer in 100mL iodine flask, put in 25 DEG C of water-baths and be incubated, then add 2mL enzyme titer, be accurately incubated 3min, add 2mL0.5mol/ sulfuric acid cessation reaction immediately, control reaction liquid control tube first adds enzyme-added liquid after 2mL0.5mol/L sulfuric acid cessation reaction.Then get above-mentioned reactant liquor 0.1mL to add 2.9mL peroxidase-dianisidine solution and shake up in testing tube, at 25 DEG C, after 436 μm of places are first with the zeroing of blank examination test tube, measure the absorbance of each reactant liquor testing tube with spectrophotometer again, obtain standard enzyme activity and absorbance relation curve.By above-mentioned identical method process tested enzyme, the enzymatic activity of tested enzyme can be obtained according to its absorbance and typical curve.
Claims (3)
1. the detection method of catalase activity, comprises the following steps:
Hydrogen peroxidase standard items are diluted to the titer of a series of concentration of enzymatic activity, get 10mL chlorine peroxide phosphate buffer in 100mL iodine flask, put in 25 DEG C of water-baths and be incubated, then 2mL enzyme titer is added, accurate insulation 3min, add 2mL0.5mol/ sulfuric acid cessation reaction immediately, control reaction liquid control tube first adds enzyme-added liquid after 2mL0.5mol/L sulfuric acid cessation reaction;
Then get above-mentioned reactant liquor 0.1mL to add 2.9mL peroxidase-dianisidine solution and shake up in testing tube, at 25 DEG C, after 436 μm of places are first with the zeroing of blank examination test tube, measure the absorbance of each reactant liquor testing tube with spectrophotometer again, obtain standard enzyme activity and absorbance relation curve;
By above-mentioned identical method process tested enzyme, the enzymatic activity of tested enzyme can be obtained according to its absorbance and typical curve.
2. the detection method of catalase activity as claimed in claim 1, is characterized in that described hydrogen peroxide phosphoric acid buffer liquid making method is for getting 1mL30% hydrogen peroxide, is diluted to 1000mL with 0.01mol/L, pH6.8 phosphate buffer.
3. the detection method of catalase activity as claimed in claim 1, is characterized in that described peroxidase-dianisidine solution is 0.03% peroxidase and 1% dianisidine methanol solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201410577230.4A CN104297185A (en) | 2014-10-24 | 2014-10-24 | Method for detecting activity of catalase |
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| CN201410577230.4A CN104297185A (en) | 2014-10-24 | 2014-10-24 | Method for detecting activity of catalase |
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| CN104297185A true CN104297185A (en) | 2015-01-21 |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105498874A (en) * | 2016-01-29 | 2016-04-20 | 中国农业大学 | Chip, system and method for detecting peroxidase concentration, as well as chip production method |
| CN106706539A (en) * | 2016-12-01 | 2017-05-24 | 湖北大学 | Method of measuring activity of plant catalase |
| CN107255695A (en) * | 2017-07-14 | 2017-10-17 | 南通市产品质量监督检验所 | A kind of food additives catalase enzyme activity evaluation method |
| CN107449744A (en) * | 2017-07-14 | 2017-12-08 | 纤化（上海）生物化工股份有限公司 | The analysis method of activity of catalase in a kind of detection textile printing and dyeing industry |
| CN109975282A (en) * | 2019-04-04 | 2019-07-05 | 华侨大学 | A kind of spectrophotometry based on enzymatic potassium iodide oxidative color-developing detection content of hydrogen peroxide |
| CN113447607A (en) * | 2020-03-24 | 2021-09-28 | 电子科技大学中山学院 | Method for identifying piglet liver and adult pig liver by using catalase activity |
| CN113720961A (en) * | 2021-08-17 | 2021-11-30 | 武汉新华扬生物股份有限公司 | Method for detecting activity of glucose oxidase |
-
2014
- 2014-10-24 CN CN201410577230.4A patent/CN104297185A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105498874A (en) * | 2016-01-29 | 2016-04-20 | 中国农业大学 | Chip, system and method for detecting peroxidase concentration, as well as chip production method |
| CN106706539A (en) * | 2016-12-01 | 2017-05-24 | 湖北大学 | Method of measuring activity of plant catalase |
| CN107255695A (en) * | 2017-07-14 | 2017-10-17 | 南通市产品质量监督检验所 | A kind of food additives catalase enzyme activity evaluation method |
| CN107449744A (en) * | 2017-07-14 | 2017-12-08 | 纤化（上海）生物化工股份有限公司 | The analysis method of activity of catalase in a kind of detection textile printing and dyeing industry |
| CN109975282A (en) * | 2019-04-04 | 2019-07-05 | 华侨大学 | A kind of spectrophotometry based on enzymatic potassium iodide oxidative color-developing detection content of hydrogen peroxide |
| CN113447607A (en) * | 2020-03-24 | 2021-09-28 | 电子科技大学中山学院 | Method for identifying piglet liver and adult pig liver by using catalase activity |
| CN113720961A (en) * | 2021-08-17 | 2021-11-30 | 武汉新华扬生物股份有限公司 | Method for detecting activity of glucose oxidase |
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Application publication date: 20150121 |