CN1865942A - Method for fast detecting and positioning plant superoxide dismutase - Google Patents
Method for fast detecting and positioning plant superoxide dismutase Download PDFInfo
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- CN1865942A CN1865942A CN 200610019167 CN200610019167A CN1865942A CN 1865942 A CN1865942 A CN 1865942A CN 200610019167 CN200610019167 CN 200610019167 CN 200610019167 A CN200610019167 A CN 200610019167A CN 1865942 A CN1865942 A CN 1865942A
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- superoxide dismutase
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- potassium permanganate
- ferric ion
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Abstract
The related method to screen, quantize and locate the SOD and qSOD comprises: with the strong reducibility, such fading KMnO4 solution and F33+ solution with speed direct proportion to the enzyme activity, dropping plant juice into KMnO4 and Fe3+ or other solution, and detecting the enzyme precisely; while sticking plant tissue on carrier contained KMnO4 ion fit to location.
Description
Technical field
The present invention relates to from plant identify active principle, quantitatively, the active method of position tissue, specifically be the screening of plant superoxide dismutase and class superoxide dismutase, quantitatively, tissue contains the method for enzyme spots localization.
Background technology
Ubiquity superoxide dismutase (SOD) and analog (title quasi-SOD) in the plant have very strong elimination free radical function, the strong murder by poisoning of elimination activity oxygen pair cell, to anti-ageing, anti-senile dementia is effective, can prophylaxis of tumours, anticancer; Yet measure the superoxide dismutase and class superoxide dismutase utmost point trouble, need expensive instrument and condition, many, the long flow path of whole determination step needs half a day at least.The assay method of some document record superoxide dismutases has:
1, the research of [autograph] mistletoe polyphenoils is measured [author] and is killed bright [1] Gong Zhunan [mechanism] [1] life science institutes of Nanjing Normal University such as [2], bio-science technology system of [2] Nanjing University, [periodical name] Chinese herbal medicine .2001,32 (12).1081-1083, article report SOD determination of activity adopts the NBT light of Stewert and Bewley to suppress method; The luis oxygen electrode method is adopted in hydrogen peroxidase (CAT) determination of activity, with the size that oxygen meter shows enzymatic activity of putting of per minute; The guaiacol development process is adopted in peroxidase (POD) determination of activity, represents with the variable quantity of unit interval internal optical density; Glutathione peroxidase (GSHPx) is represented enzymatic activity with reference to the dtnb assay of LeopoldFlohe etc. with per minute absorbance changing value.The mensuration of anti-oxidant non-enzyme material: the VC Determination on content, with reference to the method for Yi Xianfeng etc.The GSH Determination on content is with reference to the method for Ellman.With the DTNB colour developing, survey absorbance at the 422nm place, make typical curve with GSH and calculate GSH content.Determination of chlorogenic acid is pressed methods such as Hu Jingjiang, and the flavonoids assay is built methods such as the people with reference to punishment.
2, Chinese patent＜application number〉CN92107634.7＜title〉assay method＜application (patent right) people of activity of super-oxide dismutase〉No. 7 47 406 Room＜summaries of the new exemplary road in [address] Nanjing, Jiangsu Province〉the invention discloses a kind of method of measuring superoxide dismutase activity, this method genus nitrosomonas salt forming method utilizes XO to add the XOD reactive system and produces 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 aqueous slkali that azanol is dissolved in the weak acid, and adopt new damping fluid p H value and new developer proportioning.The present invention compared with prior art, the test fluid absorbance 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 examination is used.[principal claim] a kind of superoxide dismutase activity assay method, belong to the nitrite forming method, it utilizes xanthine (XO) to add xanthine oxidase (XOD) reaction system and produces ultra-oxygen anion free radical O, xanthine oxidase oxidation azanol forms nitrite, adding developer (oxygen base benzene sulfonic acid is added N-first naphthyl two amido ethene) again makes test fluid be aubergine, test process carries out in the phosphoric acid buffer liquid system, it is characterized in that: 1.1 phosphate buffer pH values are 8.0, and wherein EDTA concentration is 2 * 10
-5Ml/l; 1.2XO be dissolved in the alkaline solution, fully dissolving; 1.3 azanol is dissolved in the weak acid; Make developer 1.4 adopt 1.65mg/ml sulfanilic acid and 0.75mg/ml N-first naphthyl diamino-vinyl and 20% glacial acetic acid mixed liquor.
3, Chinese patent＜application number〉200420015686＜denomination of invention〉photochemistry TRAP illumination meter＜applicant〉Shandong Province Biology Engineering Research Center Information, Yao Xiaoyu＜contact address〉No.12 Zhangzhou Road, Zhangdian District, Zibo City, Shandong Province Shandong Technology Univ＜claim〉a kind of photochemistry TRAP illumination meter, comprise casing and light source, it is characterized in that: cabinet wall periphery and bottom surface are provided with specular layer; At least two light sources are set up in parallel in box cavity; Sample stage is set between light source.＜summary〉a kind of photochemistry TRAP illumination meter, be applicable to that (Superoxide dismutase, SOD) photochemistry enlarges the instrument of measuring the spectrochemical analysis of using to superoxide dismutase.It comprises casing and light source, and feature is that cabinet wall periphery and bottom surface are provided with specular layer; At least two light sources are set up in parallel in box cavity; Sample stage is set between light source.External instrument has similar products, and not only high price, and structure is a light source beam, and detection limit is little, and one batch can only be detected three cuvettes, causes between group between sum of errors batch error big, poor reproducibility.The utility model has the advantages that: a large amount of cuvettes colorimetric under the same conditions in the same time, can the elimination group between error between sum of errors batch, favorable reproducibility, detection limit is big simultaneously.
4, Chinese patent＜application number〉200310109579＜denomination of invention〉a class tea tree superoxide dismutase specific expressino sequence label and a biochip＜applicant thereof〉Tea Inst., Chinese Academy of Agricultural Sciences＜contact address〉Hangzhou, Zhejiang province city Xihu District cloud No. 1, the road＜summary of dwelling〉the invention discloses a class tea tree superoxide dismutase specific expressino sequence label and a biochip thereof.Expressed sequence tag has the sequence shown in SEQ ID No.1～SEQ ID No.3; Shown in one or several combination in 3 sequences; The complementary series of every sequence or homologous sequence; 8～100 sequence or its complementary seriess that continuous nucleotide is a probe in every sequence.The utilization expressed sequence tagging method carries out DNA sequence to tea tree complementary DNA (cDNA) (c DNA) library that makes up and measures, the expressed sequence tag that obtains is by rejecting redundant sequence, internet database is retrieved, classification, rich long-pending nonredundancy sequence, obtained 3 tea tree superoxide dismutase expressed sequence tag, the present invention is applied to the characterization and evaluation of crop germplasm resource, the early prediction of crop heterosis, the generation of crop breeding and new varieties and evaluation, the research of stress resistance of plant, the calibrating of plant single nucleotide polymorphism (SNP), the security of transgene agricultural product detects, the screening of new herbicides and novel agrochemical etc.
5, Chinese patent＜application number〉87101427＜denomination of invention〉colour immunization test board for determinating Cu and Zn-SOD (super-oxide dismutase) in human blood＜applicant〉General Hospital of the PLA Navy＜contact address〉No. 6＜claim in Fucheng Road, Haidian District, Beijing City〉the determinating Cu and Zn-SOD (super-oxide dismutase) in human blood colour exempts from not have assay plate, be to utilize antigen-antibody reaction to measure the assay plate of people's blood superoxide dismutase total content, the human copper zinc superoxide dismutase that it is characterized in that using purifying mixes as tire antiserum and agar chaff of the height of antigen preparation, is layered on the plastic plate and makes.＜summary〉colour immunization test board for determinating Cu and Zn-SOD (super-oxide dismutase) in human blood is the special measuring plate that is used to measure human red cell Cu, Zn-SOD total content.This plate is recorded with the 1% ion agarose that contains protein dyestuff, indicator and neutral salt and high tire anti-people Cu, Zn-SOD serum and is formed.Eliminate the interference of haemoglobin to measuring in the blood sample, omitted the loaded down with trivial details step of conventional method.Blood sampling volume is few, and reaction result is clear, observes easily, and method is easy, is suitable for each medical treatment, R﹠D institution's use.
6, Chinese patent＜application number〉92107634＜denomination of invention〉assay method＜applicant of superoxide dismutase activity〉Ji Yuejun, Yue Chengbin＜contact address〉No. 7 47 406 Room＜summaries of the new exemplary road in Nanjing, Jiangsu Province〉the invention discloses a kind of method of measuring superoxide dismutase activity, this method genus nitrosomonas salt forming method utilizes XO to add the XOD reactive system and produces 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 aqueous slkali 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 absorbance is big and stable, test result is accurate, test speed is fast, and is highly sensitive, cost is low.
7, Chinese patent＜application number〉200410050222＜denomination of invention〉utilize the method＜applicant of cereal kernel large-scale production superoxide dismutase complex enzyme〉Chen Xin＜contact address〉ShenYang, Liaoning Province city Shenhe District is auspicious in 60-1 6-1-1 chamber, gold Jue Si street, sub-district＜summary〉a kind of method of utilizing cereal kernel large-scale production superoxide dismutase complex enzyme, it is characterized in that: cereal kernel is screened, sterilizes, cleans; Seed soaked in warm water make its imbibition, insulation is germinateed in the germination groove; Germinated ceral is ground to form thick slurry, add damping fluid; To slightly starch and add cellulase and break into screened stock, adding lime chloride; After the solid-liquid layering, the filtrate press filtration, filtrate is centrifugal by hydro-extractor, collects centrifugate; Centrifugate is crossed ultra filtration membrane, adds carbohydrase, concentrates by neutral ultra filtration membrane again, makes it reach needed SOD concentration; Advantage of the present invention is a superoxide dismutase, and peroxidase and hydrogen peroxidase can both extract, and the product foreign protein is few, the vigor height of enzyme, and enzyme activity is controlled effective, long shelf-life; Article one, the basic technology route can produce series of products, and technology is simple, the automaticity height, and safety non-pollution, power consumption is few, and cost is low, high efficiency; Five kinds of all available these explained hereafter of cereal seed.
Above-mentioned document provides the assay method of some superoxide dismutases, the part but above-mentioned method also comes with some shortcomings, as reactions steps many as:＜number of patent application〉CN92107634.7 utilizes XO to add the XOD reactive system to produce ultra-oxygen anion free radical O
2, O
2The oxidation azanol forms nitrite, and by chromogenic reagent, the NBT light inhibition method that Stewert and Bewley are adopted in the SOD determination of activity is measured in the research of mistletoe polyphenoils for another example; The luis oxygen electrode method is adopted in hydrogen peroxidase (CAT) determination of activity, with the size that oxygen meter shows enzymatic activity of putting of per minute; The guaiacol development process is adopted in peroxidase (POD) determination of activity, represents with the variable quantity of unit interval internal optical density; Glutathione peroxidase (GSHPx) is represented enzymatic activity with reference to the dtnb assay of Leopold Flohe etc. with per minute absorbance changing value.The mensuration of anti-oxidant non-enzyme material: the VC Determination on content, with reference to the method for Yi Xianfeng etc.The GSH Determination on content is with reference to the method for Ellman.With the DTNB colour developing, survey absorbance at the 422nm place, make typical curve juice with GSH and calculate GSH content.Determination of chlorogenic acid is pressed method flavonoids assays such as Hu Jingjiang.Build method program times wherein such as the people with reference to punishment long, or make complicated as:＜application number〉87101427 need people's copper-zinc superoxide dismutase is made to extract purifying, the equipment multiple instruments expensive as:＜application number〉instrument of 200420015686 spectrochemical analysiss of using.It comprises casing and light source, and cabinet wall periphery and bottom surface are provided with specular layer; At least two light sources are set up in parallel in box cavity, and the method that in the past proposed does not all relate to the location in addition.
Summary of the invention
The purpose of this invention is to provide a kind of simple, sensitivity is high, being beneficial to the assay method of the superoxide dismutase class of field operation especially, is that plant superoxide dismutase and class superoxide dismutase are made Screening and Identification, quantitatively, tissue is contained the fast method that the enzyme position is intuitively determined.
The present invention is achieved in that
Potassium permanganate or ferric ion aqueous solution are pressed content 100,000/to millesimal concentration by the gradient preparation, move respectively in several test tubes, according to quantity water is splashed into respectively then, regularly, or accurately timing, the aubergine of observation potassium permanganate liquid or ferric ion and other reducible material color and lusters that fades take off.Or with potassium permanganate or ferric ion by content 100,000/place pipe to millesimal a certain concentration, according to quantity water is splashed into respectively, the optical density value difference and the Time Calculation of fading before and after accurately timing maybe will be reacted, and just compare and accurately to measure this enzyme active quantities, again plant tissue is affixed on and contains on potassium permanganate liquid or the ferric ion carrier, as seen on coloured carrier board, contain enzyme position color and luster accordingly with plant tissue and decorporate, just can orient the position of organizing superoxide dismutase and fermentoid.
Concrete grammar:
Potassium permanganate is mixed with 100,000/to five millesimal aqueous solution, ferric ion be mixed with ten thousand/to five per mille aqueous solution, split in the test tube by gradient, the juice that the plant that will screen is extruded splashes into, if superoxide dismutase and analog are arranged, the aubergine of potassium permanganate solution or ferric ion and other reducible material color and lusters that fades take off, its speed of fading increases with the activity of enzyme and accelerates, observe each pipe in the identical time, but also visual inspection goes out active height.The time of will fading accurately counts down, and the optical density value difference is done calculating with fade time and blank and all can be measured the enzymatic activity value before and after maybe will reacting.Plant tissue slice is affixed on the moistening carrier that contains potassium permanganate etc., can on the carrier board of colour generation, observes with the corresponding position of plant tissue color and luster and decorporate, and reach the accurate position of position tissue superoxide dismutase and fermentoid.
The carrier of described carrying potassium permanganate or ferric ion aqueous solution can be the tygon powder, the carboxymethyl cellulose powder, and any inert powder make, or make band look carboxymethyl cellulose gum plate, band color lake arogel plate, band look gelatin plate etc. and make this enzyme orientation tool with their glue, this instrument can be made into kit equally.
Principle of the present invention is: plant contains superoxide dismutase and class superoxide dismutase, they show extremely strong reductibility, the present invention utilizes superoxide dismutase and class superoxide dismutase to make the aubergine potassium permanganate solution, or ferric ion solution and other reducible material colour killings of fading, and differentiate, quantitatively locate this contained fermentoid of plant.The invention reside in, overcome in the past the inconvenience of taking a lot of work, taking reagent, needing expensive instrument consuming time that superoxide dismutase and superoxide dismutase analog are measured, adopt the low concentration potassium permanganate solution, or the cheap reagent of ferric ion aqueous solution and so on, just can be intuitively qualitative, quantitative to superoxide dismutase and tissue contain the enzyme position and do definite.
Compared with prior art, outstanding substantive distinguishing features of the present invention and obvious improvement are:
1, simple, reagent dosage is few, cheap, directly perceived, accurate, sensitive, be beneficial to field operation especially, can be quantitative to the superoxide dismutase class, tissue is contained the enzyme position to be done definite, it is high-speed that the implementation of the method allows this enzyme screening reach, and making a sample only needs just can finish several seconds, and the advantage of the standard of getting twice the result with half the effort is arranged.
2, utilize superoxide dismutase (SOD) and class superoxide dismutase (quasi-SOD) strong reducing property, it can make a large amount of reducible materials that fade fade, as make the potassium permanganate solution reducing decoloration, ferric ion aqueous solution reducing decoloration and other reducible materials that fades are faded, and speed increases proportional example with the activity of enzyme and it fades.Water is added drop-wise in potassium permanganate, ferric ion and other the reducible materials that fades, and plant superoxide dismutase and analog are determined, quantitatively, and plant tissue slice is affixed on the moistening carrier that contains potassium permanganate etc., can locate them.
3, different with art methods, this method step is few, and the time spent is short, need not expensive instrument, and can locate, and can be widely used in laboratory and clinical examination and use.
Embodiment
Example 1:
The potassium permanganate solution of 5ml ten thousand/(mass content) is put in the test tube, with portable squeezer fresh leaf of Herba Apii graveolentis is squeezed out juice, drip number and splash into this test tube, potassium permanganate promptly faded in about 30 seconds, leaf of Herba Apii graveolentis contains the superoxide dismutase class as can be known, this method is done contrast with nitroblue azoles (NBT) method, is equivalent to 600 (NBT) unit approximately.
Example 2
With the tender bar crosscut of Morinda, then the cross section is affixed on the 100 order tygon powder plates of one of ten thousand (mass content) potassium permanganate solutions, after 2 minutes visible plate and the corresponding position of the tender bar of Morinda is from the center to the limit on aubergine from deeply to shallow to not having, visible superoxide dismutase mainly is contained in the tender bar green of Morinda marginal portion.The tender stem apex of Morinda is pressed juice, and the potassium permanganate solution that front and back is dripped juice is compared the activity that just can calculate it with known standard and is equivalent to 980 (NBT) unit.
Example 3
The tender bar crosscut of fresh dragon fruit that will be thicker, then the cross section is affixed on the 80 order methylol fiberboards of eight per milles (mass content) potassium permanganate solution, after 3 minutes visible plate face and the corresponding position of the tender bar of dragon fruit is from the center to the limit on aubergine from deeply to shallow to not having, visible superoxide dismutase mainly is contained in the tender bar green of dragon fruit marginal portion.The potassium permanganate solution that front and back is dripped juice just can be calculated its activity with the spectrophotometer colorimetric and with the standard specimen contrast.
Example 4
With the tender bar crosscut of new fresh asparagus, the cross section is affixed on immediately on the 0.2mm amylan wet plate that contains five per mille iron sulfate then, about 2 minutes visible plate faces and the corresponding position of tender bar on from the center to the limit yellow from deeply to shallow to not having.
Claims (3)
1, a kind of mensuration of plant superoxide dismutase, localization method, be quantitative to plant superoxide dismutase screening and superoxide dismutase analog, tissue contains the method for active site location, it is characterized in that: potassium permanganate or ferric ion are made into mass content 100,000/to millesimal aqueous solution, put in test tube or the container, the juice that the plant that will screen is extruded splashes into, the aubergine of observation potassium permanganate solution or ferric ion and other reducible material color and lusters that fades take off, the time of will fading accurately maybe will be reacted front and back optical density value difference and fade Time Calculation under the meter, compare, calculate content; Perhaps plant tissue is affixed on and contains on the isoionic inert carrier of potassium permanganate, can decorporate with the corresponding position of plant tissue color and luster, intuitively give expression to the accurate position of organizing superoxide dismutase and fermentoid in being on the mauve carrier board.
2, the mensuration of plant superoxide dismutase according to claim 1, localization method, it is characterized in that: the carrier of carrying potassium permanganate, ferric ion can be the tygon powder, the thin plate that methylol fiber or any inert powder or their colloid are made.
3, the mensuration of plant superoxide dismutase according to claim 1, localization method is characterized in that: the carrier sense plate of carrying potassium permanganate or ferric ion can be made into kit.
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| Application Number | Priority Date | Filing Date | Title |
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| CN200610019167A CN1865942B (en) | 2006-05-25 | 2006-05-25 | Method for fast detecting and positioning plant superoxide dismutase |
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| CN200610019167A CN1865942B (en) | 2006-05-25 | 2006-05-25 | Method for fast detecting and positioning plant superoxide dismutase |
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| CN1865942A true CN1865942A (en) | 2006-11-22 |
| CN1865942B CN1865942B (en) | 2010-05-12 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102426169A (en) * | 2011-11-09 | 2012-04-25 | 天津市天骄辐射固化材料有限公司 | Method for detecting content of peroxide in polyether polyol |
| CN109212015A (en) * | 2018-09-18 | 2019-01-15 | 泰山医学院 | A kind of material surface oxidation-reduction quality detection method |
-
2006
- 2006-05-25 CN CN200610019167A patent/CN1865942B/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102426169A (en) * | 2011-11-09 | 2012-04-25 | 天津市天骄辐射固化材料有限公司 | Method for detecting content of peroxide in polyether polyol |
| CN109212015A (en) * | 2018-09-18 | 2019-01-15 | 泰山医学院 | A kind of material surface oxidation-reduction quality detection method |
| CN109212015B (en) * | 2018-09-18 | 2021-02-23 | 山东第一医科大学(山东省医学科学院) | Method for detecting surface oxidation-reduction property of substance |
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| Publication number | Publication date |
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| CN1865942B (en) | 2010-05-12 |
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