CN101936930B - Method for determining superoxide anion radicals in tape grass leaves - Google Patents
Method for determining superoxide anion radicals in tape grass leaves Download PDFInfo
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Abstract
The invention discloses a method for measuring superoxide anion radicals in tape grass leaves, belonging to the measuring field of the measuring of superoxide anion radicals. The method comprises the following steps of: firstly, collecting and cleaning the tip parts of fully exposed small and tender tape grass leaves; carrying out Tiron captureing on the Tiron of radicals in a closed operation box in a closed nitrogen environment; and finally measuring by using an EPR (Electron Paramagnetic Resonance) technology. The invention has the advantages of simpler operation and easy grasping, and can measure the superoxide anion radicals of the tape grass leaves in a very short time;, find that and the measured radicals are found to be the superoxide anion radicals according to the superfine structure constant and, the spectrogram shape analysis of an EPR spectrogram and the combination of the remarkable decreases of the radical strength of the leaves pretreated by SOD (Super Oxide Dismutase), and therefore the invention is the most direct, accurate and effective method for measuring the superoxide anion radicals. Superoxide anions are sensitive to pollutants and have a favorable dosage effect relation. The invention is also suitable for other aquatic plant and terrestrial plant species and can be suitable for measuring the radicals of the plant root tissue.
Description
Technical field
The present invention relates to a kind of assay method of radical anion, a kind of method of measuring ultra-oxygen anion free radical in the eel grass blade of saying so more specifically.
Background technology
Active oxygen (ROS) is the very general name of the oxygen radical of high bioactivity that has that produces in the aerobic metabolism process in exogenous oxygenant or the cell, comprises superoxide anion (O
2 -), hydroxy radical (OH), hydroperoxy (HO
2 -), hydrogen peroxide (H
2O
2), singlet oxygen (
1O
2) etc.The photosynthesis of plant tissue, respiration, oxidative phosphorylation, fatty acid-beta oxidation and many oxidasic metabolic processes all can produce ROS.More and more many research confirms; ROS is the normal part of plant metabolism; Can be used as signaling molecule inducing plant body stress respond to environment-stress; And specified conditions (like coercing of pollutant) can cause the accumulation of ROS down, and then cause the oxidative modification or the degraded of membrane lipid peroxidating, protein molecular, and the oxidative damage of dna molecular etc.Therefore, the variation of the generation of research freedom base, accumulation and metabolism, can reflect the oxidative stress of plant under adverse environmental factor with stress level of response, the early diagnosis that can be environmental contaminants provides early warning signal.Therefore, generation and the accumulating level of accurately measuring plant tissue ROS have important theoretical meaning and application prospect.
O
2 -Be the free radical that produces among the various ROS the earliest, accept an electronics and form by oxygen molecule.Though O
2 -Oxidability in vivo and harmfulness are not as OH, but very important a kind of free radical.It can be converted into various free radicals as precursor, such as O
2 -Disproportionation reaction can take place generate H
2O
2And O
2 -And H
2O
2Can react through Fenton, processes such as Haber-Weiss reaction, Winterbourn reaction and photolysis convert OH to.Therefore to O in the plant tissue
2 -Detection by quantitative especially seem significant.But, directly measure very difficulty in plant because the half life period of free radical is very of short duration, and exists concentration extremely low.The researcher often adopts indirect method to remove to detect the O that produces in the plant at present
2 -Such as passing through O in spectrophotometry specific compound and the plant homogenate
2 -The intermediate product that azanol reaction produces takes place, and calculates O in the plant
2 -Generation speed, but must be noted that the disturbing factor that influences indirect method is a lot.In addition, the O of plant tissue
2 -Also can directly catch, such as realizing O through nitroblue tetrazolium (NBT) development process
2 -Direct colour developing on plant leaf blade, thus O obtained
2 -Distributed intelligence on leaf tissue, obviously not enough but this method exists on accurately quantitatively, and also the same with indirect method, the disturbing factor of this method is many, and applicable floristics is very restricted.
At present, searching is a kind of fast, accurate, efficient, directly the free radical detection method has become the key of research freedom base biological action.Electron paramagnetic resonance (EPR) combine the spin trapping technology be the current research free radical the most directly, effective method, be widely used in the research in fields such as animal toxicology, biomedicine recently.The ultimate principle of the method is to utilize trapping agent and active free radical reaction to form more stable free radical, i.e. spin adduct, and then detect with EPR.Spin agent for capturing relatively more commonly used at present has 5,5-dimethyl-1-pyrrolin-N-oxide (DMPO), the special butane (NtB) of nitroso-, α-phenyl-N-tert-butyl group azomethine-N-oxide (PBN) etc.Wherein because PBN is more stable relatively; The spin adduct life-span is long; In measuring animal body, use aspect the free-radical contents comparatively extensive, but this trapping agent and be not suitable for the particularly detection of free radical in the hydrophyte tissue of various plants, and can not stablize itself and O are provided
2 -The characteristic spectral line of spin adduct has limited its popularization.
In aquatic ecosystem, aquatic higher plant is to keep the stable crucial ecogroup of water body, and submerged plant the reaction that makes it coerce water pollution of aquatic characteristics is the most direct fully.Eel grass (Vallisnerianatans (Lour.) Hara) is heavy this plant of pasture and water of the perennial acaulescence of Hydrocharctaceae Vallisneria, is distributed widely in the waters such as river and shallow lake of China low altitude area.Become at present the hydrophyte species that often adopt in the toxicological experiment, the more all contaminations that concentrates on of correlative study produces the oxidative stress aspect to it, but the rarely seen report of positive evidence of pollutant stress-inducing free-radical generating.Yin Ying etc. (2007) once shredded the eel grass blade after the freeze drying; Directly detect the blade free-radical contents with EPR; The EPR spectrum peak that obtains is one unimodal, but this method fail the use characteristic trapping agent and directly catch the live body free radical, the free radical of mensuration is difficult to accurately quantitatively; The spectrum peak that obtains is lack of specific also, has influenced its popularization.Therefore, research and development a kind of efficient directly and the free radical capture technology of high specificity seem significant.
Summary of the invention
The technical matters that 1, will solve
The present invention mainly is the deficiency to existing free radical assay method; A kind of method of measuring ultra-oxygen anion free radical intensity in the common large-scale submerged plant eel grass blade is provided; Adopt 1,2-dihydroxy benzenes-3,5-sodium disulfonate (Tiron) is as the spin trapping agent; Use ultra-oxygen anion free radical in the EPR technical measurement eel grass blade; Can realize fast, accurately, directly carrying out free radical and detect, for pollutant stress-inducing plant leaf blade produces free radical positive evidence is provided, this method also can be applicable to O in laboratory inside and outside other aquatic and terrestrial plant monoid blade
2 -Quantitative examination.
2, technical scheme
3, a kind of method of measuring eel grass blade ultra-oxygen anion free radical comprises following several steps:
The first step; Gather the most advanced and sophisticated position of the eel grass young leaflet tablet that fully exposes, clean;
Second step: the Tiron of free radical catches;
The 3rd step: EPR measures.
First step wherein, the blade collection, clean: clip cleans through pure water through the tip portion of the eel grass young leaflet tablet of exposure rapidly, blots the blade surface residual moisture fast with filter paper.
Second step, the Tiron of free radical catches: take by weighing the plant leaf blade of 0.1～0.3g rapidly, place in the porcelain system mortar; Water liquid nitrogen and make its rapid cooling, break blade fast into pieces and be ground into powder with pestle, treat that remaining liquid nitrogen volatilization is clean after; Immediately in leaf weight g: trapping agent liquor capacity mL is that 1: 8～1: 10 ratio adding Tiron trapping agent solution (includes 10～20mM catechol-3,5-sodium disulfonate (Tiron, CAS 149-45-1; Sigma; The U.S.), 50mM 2-cyclohexylamino ethyl sulfonic acid (CHES) and 0.5% (v/v) Tween-20, pH 8.4～8.6) and fully homogenate.This homogenate is 10,000～12,000rpm, 4 ℃, centrifugal 15～20min.Get supernatant and inject the 3mm quartz ampoule, put into liquid nitrogen rapidly and keep, measure in order to EPR.Catching preceding half an hour, in the closed-loop operation case, importing nitrogen with thorough removal oxygen, to prevent that producing methoxyl disturbs the result.Above whole operation process is carried out in the closed-loop operation case of nitrogen environment.
Third step, EPR measures: EPR measures the EMX 10/12 type electron paramagnetic resonance appearance that uses Bruker company.EPR operating parameter: probe temperature: room temperature; Central magnetic field: 3476G; Scan width: 50G; Modulating frequency: 100KHz; Microwave power: 20mW; Modulation amplitude: 1.0G; Receiver gain:2 * 10
4Scan number of times: 1 time.The signal intensity that obtains is used to characterize free-radical contents.
4, beneficial effect
The present invention adopts EPR to combine the spin trapping technology, discloses a kind of method of direct mensuration eel grass leaf tissue ultra-oxygen anion free radical, and with compared with techniques in the past, the present invention has the following advantages:
(1) fast, efficient.The present invention's operation is comparatively simple, easy master, and can record the ultra-oxygen anion free radical in the eel grass blade in a short period of time.
(2) directly, accurately.Can confirm that according to the hyperfine structure parameter of EPR wave spectrogram the free radical of being measured is a ultra-oxygen anion free radical, and not strong unlike chemical method and the such selectivity of chromatography.
(3) responsive and have a dose-effect relationship preferably.The multiple pollutant of result of study can significantly induce superoxide anion to produce under low concentration; Its intensity and exposure exist dose-effect relationship preferably in the finite concentration scope, show that free radical can be used as the biomarker that responsive indication pollutant is coerced.
(4) practicality is wider.The present invention is suitable for other hydrophyte and terrestrial plant kind too, and applicable to root tissue.Ultra-oxygen anion free radical can both be directly measured in pollutant exposure for different, can be applicable to open-air original position experiment simultaneously.
Description of drawings
Fig. 1 coerces down the EPR wave spectrum of eel grass blade ultra-oxygen anion free radical for the variable concentrations microcapsule phycotoxin MC-LR
Fig. 2 is the relation of MC-LR exposure concentrations and ultra-oxygen anion free radical intensity.Numeric representation is mean value ± standard error (n=3);
*With
*Represent that respectively processed group has conspicuousness (p＜0.05) and utmost point significant difference (p＜0.01) with respect to control group
Fig. 3 coerces down the EPR wave spectrum of eel grass blade ultra-oxygen anion free radical for the variable concentrations ammonium nitrogen
Fig. 4 is the relation of ammonium nitrogen exposure concentrations and ultra-oxygen anion free radical intensity.Numeric representation is mean value ± standard error (n=3);
*The expression processed group has significant difference (p＜0.05) with respect to control group
Fig. 5 is Cd in the bed mud
2+The relation of concentration and eel grass blade ultra-oxygen anion free radical intensity.Numeric representation is mean value ± standard error (n=3);
*The expression processed group has utmost point significant difference (p＜0.01) with respect to control group
Fig. 6 is the variation of ultra-oxygen anion free radical intensity behind SOD enzyme pre-service eel grass blade.Numeric representation is mean value ± standard error (n=3);
*The expression processed group has significant difference (p＜0.05) with respect to control group
Embodiment
Below further specify of the present invention practicing through instance
Selecting the common submerged plant eel grass of China's fresh water environment (Vallisneria natans (Lour.) Hara) is biological subject.Plant is obtained by commercial eel grass cultivating seeds, and nutrient solution adopts improved Hoagland nutrient solution (0.1 *), disposes with the tap water behind the aeration.Cultivation temperature is 25 ℃, and intensity of illumination is 4000lux, and the photoperiod is 12/12h.The plant seedling of choosing the about 10cm of height that grows fine moves into the glass jar contain 5cm silica sand, includes the Hoagland dilution, raise and train a couple of days after, add variable concentrations MC-LR, exposure concentrations is 0,0.1,0.5,1.0,5.0,10.0 μ g L
-1, 3 of each concentration group are parallel, expose 14d, change water every other day and add nutrient solution and MC-LR.Expose the tip portion of clip eel grass young leaflet tablet after accomplishing, clean, blot the blade surface residual moisture fast with filter paper through pure water.Take by weighing the plant leaf blade of about 0.2g rapidly; In the porcelain system of the placing mortar, water liquid nitrogen and make its rapid cooling, break blade fast into pieces and be ground into powder with pestle; Add 2mL trapping agent solution and (include 10mM Tiron (Sigma; The U.S.), 50mM 2-cyclohexylamino ethyl sulfonic acid (CHES) and 0.5% (v/v) Tween-20, pH 8.6) and fully homogenate.This homogenate is 10,500rpm, 4 ℃, centrifugal 20min.Supernatant is used for EPR and measures.Above whole operation process is carried out in the closed-loop operation case of nitrogen environment.
EPR measures the EMX 10/12 type electron paramagnetic resonance appearance that uses Bruker company.EPR operating parameter: probe temperature: room temperature; Central magnetic field: 3476G; Scan width: 50G; Modulating frequency: 100KHz; Microwave power: 20mW; Modulation amplitude: 1.0G; Receiver gain:2 * 10
4Scan number of times: 1 time.The signal intensity that obtains is used to characterize free-radical contents.
Fig. 1 is the signal collection of illustrative plates that the variable concentrations microcapsule phycotoxin MC-LR is coerced eel grass blade ultra-oxygen anion free radical behind the 14d.Explaining that eel grass is induced through MC-LR can produce ROS, is caught by Tiron and generates the Tiron adduct, and can be arrived by the EPR spectral detection.According to the Hyperfine Structure Constants of spectrogram and the shape analysis of spectrogram, confirm that the active oxygen that captures in this experiment is O
2 -
Can find out that from Fig. 2 MC-LR induces eel grass blade O
2 -Intensity increases and increases along with the concentration that exposes, but 10 μ g L
-1Signal intensity slightly descends under the exposure concentrations.When exposure concentrations is 1.0 μ g L
-1The time, O
2 -Signal intensity be 181% of control group, compare with control group and have utmost point significant difference (p＜0.01), when exposure concentrations is 5.0-10.0 μ g L
-1The time, O
2 -Signal intensity be the 182%-173% of control group, compare with control group and have significant difference (p＜0.05, p＜0.01).In conjunction with scanning and transmission electron microscope observing, higher concentration (5.0-10.0 μ g L
-1) MC-LR exposes eel grass mesophyll cell microstructure has been caused tangible destruction, therefore infers O
2 -Signal intensity does not obviously rise and destroys relevant with eucaryotic cell structure.Bright through linear regression analysis Table, do not causing under the MC-LR exposure concentrations of the remarkable pathology damage of blade, itself and induce between the free radical intensity of generation and be tangible linear dose-effect relationship: y=1.21
*10
4X+3.12
*10
4, R
2=0.944, y represents O
2 -The free radical signal intensity, x represents the MC-LR exposure concentrations.Result of study explanation MC-LR exposure can cause O
2 -Thereby the accumulation at the eel grass blade might cause the plant oxidative damage.
Embodiment 2 ammonium nitrogens are coerced down the mensuration of eel grass blade ultra-oxygen anion free radical
Selecting submerged plant eel grass (Vallisneria natans (Lour.) Hara) is biological subject.Germination of eel grass plant and cultivation are with embodiment 1, and the plant seedling of choosing the about 10cm of height that grows fine moves into the glass jar that contains 5cm silica sand, includes no nitrogen Hoagland dilution, adds variable concentrations ammonium chloride, makes NH
4 +-N exposure concentrations reaches 0,0.05,0.1,3.0mg L
-1, 3 of each concentration group are parallel, expose 14d, change water every day and add ammonium chloride mother liquor.Expose the tip portion of clip eel grass young leaflet tablet after accomplishing, clean, blot the blade surface residual moisture fast with filter paper through pure water.Take by weighing the plant leaf blade of about 0.3g rapidly; In the porcelain system of the placing mortar, water liquid nitrogen and make its rapid cooling, break blade fast into pieces and be ground into powder with pestle; Add 2.4mL Tiron trapping agent solution and (include 15mM Tiron (Sigma; The U.S.), 50mM 2-cyclohexylamino ethyl sulfonic acid (CHES) and 0.5% (v/v) Tween-20, pH 8.5) and fully homogenate.This homogenate is 12,000rpm, 4 ℃, centrifugal 15min.Supernatant is used for EPR and measures.Above whole operation process is carried out in the closed-loop operation case of nitrogen environment.EPR measures the EMX 10/12 type electron paramagnetic resonance appearance that uses Bruker company.The EPR operating parameter is with embodiment 1.
Fig. 3 is the signal collection of illustrative plates that variable concentrations ammonium nitrogen MC-LR coerces eel grass blade ultra-oxygen anion free radical behind the 14d.Explaining that eel grass is induced through ammonium nitrogen can produce ROS, is caught by Tiron and generates the Tiron adduct, and can be arrived by the EPR spectral detection.According to the Hyperfine Structure Constants of spectrogram and the shape analysis of spectrogram, confirm that the active oxygen that captures in this experiment is O
2 -
From Fig. 4, can find out 0.05mg L
-1Ammonium nitrogen exposes O down
2 -Intensity decreases, and this is that low concentration N can keep plant and grow normally because N is the necessary nutrient of plant.Eel grass blade O afterwards
2 -Intensity is along with exposure concentrations begins to increase, and it is 3.0mg L that ammonium nitrogen is worked as exposure concentrations
-1The time, O
2 -Signal intensity be 152% of control group, compare with control group and have significant difference (p＜0.05).Result of study explanation ammonium nitrogen is in case above the needed concentration of the normal growth of plant, can cause O
2 -Thereby the accumulation at the eel grass blade might cause the plant oxidative damage.
Selecting submerged plant eel grass (Vallisneria natans (Lour.) Hara) is biological subject.Germination of eel grass plant and cultivation are with embodiment 1, and nutrient solution adopts improved Hoagland nutrient solution (0.1 *), disposes with the tap water behind the aeration.Cultivation temperature is 25 ℃, and intensity of illumination is 4000lux, and the photoperiod is 12/12h.The plant seedling of choosing the about 10cm of height that grows fine moves into adds different Cd
2+The glass jar of concentration bed mud, overlying water are the aeration tap water (test is picked up from the lake region, Taihu Lake with bed mud, artificially adds the variable concentrations chlorination at a distance from also aging 60d) of balance 10d; 3 of each concentration group are parallel, expose 14d.Expose the tip portion of clip eel grass young leaflet tablet after accomplishing, clean, blot the blade surface residual moisture fast with filter paper through pure water.Take by weighing the plant leaf blade of about 0.1g rapidly; In the porcelain system of the placing mortar, water liquid nitrogen and make its rapid cooling, break blade fast into pieces and be ground into powder with pestle; Add 1mL Tiron trapping agent solution and (include 20mM Tiron (Sigma; The U.S.), 50mM 2-cyclohexylamino ethyl sulfonic acid (CHES) and 0.5% (v/v) Tween-20, pH 8.4) and fully homogenate.This homogenate is 10,000rpm, 4 ℃, centrifugal 20min.Supernatant is used for EPR and measures.Above whole operation process is carried out in the closed-loop operation case of nitrogen environment.EPR measures the EMX 10/12 type electron paramagnetic resonance appearance that uses Bruker company.The EPR operating parameter is with embodiment 1.
Fig. 5 is Cd in the bed mud
2+The relation of concentration and eel grass blade ultra-oxygen anion free radical intensity.Can know by figure, add 1mg kg in the bed mud
-1Heavy metal Cd coerce and can extremely significantly induce O in the eel grass blade
2 -Generation, and coerce concentration and induce between the free radical intensity of generation and be tangible linear dose-effect relationship: y=2.87
*10
4X+4.58
*10
4, R
2=0.970.O
2 -Accumulation at the eel grass blade might cause lipid peroxidation, protein oxidation and dna damage etc., thereby plant is brought injury.
Embodiment 4 superoxide dismutase SOD are to the influence of eel grass blade ultra-oxygen anion free radical content
Selecting submerged plant eel grass (Vallisneria natans (Lour.) Hara) is biological subject.Germination of eel grass plant and cultivation are chosen the plant seedling of the about 10cm of height that grows fine and are studied with embodiment 1.The fresh blade of clip eel grass immerses 0,10 immediately, 1000U mL
-1The SOD enzyme solutions (contain 10mMMnCl
2), solution places in the glass container of self-control vacuum-pumping.Vacuumize (5 minutes * 3 times), impel solution to penetrate into the leaf tissue cell, vacuum tightness obvious micro-bubble occurs with blade surface and is advisable.Behind the dark static 15min down, get the point of blade part, clean, blot the blade surface residual moisture fast with filter paper through pure water.Take by weighing the plant leaf blade of about 0.2g rapidly; In the porcelain system of the placing mortar, water liquid nitrogen and make its rapid cooling, break blade fast into pieces and be ground into powder with pestle; Add 2mL Tiron trapping agent solution and (include 12mMTiron (Sigma; The U.S.), 50mM 2-cyclohexylamino ethyl sulfonic acid (CHES) and 0.5% (v/v) Tween-20, pH 8.6) and fully homogenate.This homogenate is 10,500rpm, 4 ℃, centrifugal 20min.Supernatant is used for EPR and measures.Above whole operation process is carried out in the closed-loop operation case of nitrogen environment.EPR measures the EMX 10/12 type electron paramagnetic resonance appearance that uses Bruker company.The EPR operating parameter is with embodiment 1.
Fig. 6 shows the mL through 1000U
-1After the SOD enzyme solutions was handled, the blade free-radical contents significantly descended, and has confirmed that from another angle the free radical signal that the Tiron trapping agent is caught derives from superoxide anion really.Because it is limited that the efficient that makes SOD get into mesophyll cell is handled and vacuumized to the short time, so still can capture a certain amount of ultra-oxygen anion free radical signal after handling.
Claims (1)
1. method of measuring eel grass blade ultra-oxygen anion free radical comprises following several steps:
The first step; The most advanced and sophisticated position of the eel grass young leaflet tablet that collection fully exposes, clean: clip cleans through pure water through the tip portion of the eel grass young leaflet tablet of exposure rapidly, blots the blade surface residual moisture fast with filter paper;
Second step: the Tiron of free radical catches: take by weighing the plant leaf blade of 0.1 ~ 0.3 g rapidly, place in the porcelain system mortar, water liquid nitrogen and make its rapid cooling, break blade fast into pieces and be ground into powder with pestle; After treating that remaining liquid nitrogen volatilization is clean, immediately in leaf weight g: trapping agent liquor capacity mL is that the ratio of 1:8 ~ 1:10 adds Tiron trapping agent solution, wherein includes 10 ~ 20 mM catechols-3, the 5-sodium disulfonate; 50 mM 2-cyclohexylamino ethyl sulfonic acids and volume percent content are 0.5% Tween-20, and pH 8.4 ~ 8.6, and fully homogenate; This homogenate is 10,000 ~ 12,000 rpm; 4 ℃, centrifugal 15 ~ 20 min get supernatant and inject 3 mm quartz ampoules; Put into liquid nitrogen rapidly and keep, measure, catching preceding half an hour in order to EPR; In the closed-loop operation case, import nitrogen with thorough removal oxygen, to prevent that producing methoxyl disturbs the result, above whole operation process is carried out in the closed-loop operation case of nitrogen environment;
The 3rd step: EPR measures: EPR measures the EMX 10/12 type electron paramagnetic resonance appearance that uses Bruker company, EPR operating parameter: probe temperature: room temperature; Central magnetic field: 3476 G; Scan width: 50 G; Modulating frequency: 100 KHz; Microwave power: 20 mW; Modulation amplitude: 1.0 G; Receiving gain: 2 * 10
4Scan number of times: 1 time, the signal intensity of acquisition is used to characterize free-radical contents.
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