CN105866053A - Method for two-enzyme detection of content of gamma-aminobutyric acid - Google Patents
Method for two-enzyme detection of content of gamma-aminobutyric acid Download PDFInfo
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- CN105866053A CN105866053A CN201610221748.3A CN201610221748A CN105866053A CN 105866053 A CN105866053 A CN 105866053A CN 201610221748 A CN201610221748 A CN 201610221748A CN 105866053 A CN105866053 A CN 105866053A
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- aminobutyric acid
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- gamma aminobutyric
- lanthanum
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- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 title claims abstract description 130
- 229960003692 gamma aminobutyric acid Drugs 0.000 title claims abstract description 65
- OGNSCSPNOLGXSM-UHFFFAOYSA-N (+/-)-DABA Natural products NCCC(N)C(O)=O OGNSCSPNOLGXSM-UHFFFAOYSA-N 0.000 title claims abstract description 64
- 238000000034 method Methods 0.000 title claims abstract description 27
- 238000001514 detection method Methods 0.000 title claims abstract description 24
- 239000007788 liquid Substances 0.000 claims abstract description 30
- 102000004190 Enzymes Human genes 0.000 claims abstract description 28
- 108090000790 Enzymes Proteins 0.000 claims abstract description 28
- 239000006228 supernatant Substances 0.000 claims abstract description 15
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 13
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 12
- 101710088194 Dehydrogenase Proteins 0.000 claims abstract description 12
- 239000007983 Tris buffer Substances 0.000 claims abstract description 10
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims abstract description 10
- 229910052746 lanthanum Inorganic materials 0.000 claims abstract description 9
- 241001465754 Metazoa Species 0.000 claims abstract description 8
- 239000003513 alkali Substances 0.000 claims abstract description 8
- 238000002156 mixing Methods 0.000 claims abstract description 6
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 6
- XJLXINKUBYWONI-NNYOXOHSSA-O NADP(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-NNYOXOHSSA-O 0.000 claims abstract 3
- KPGXRSRHYNQIFN-UHFFFAOYSA-N 2-oxoglutaric acid Chemical compound OC(=O)CCC(=O)C(O)=O KPGXRSRHYNQIFN-UHFFFAOYSA-N 0.000 claims description 34
- 238000012360 testing method Methods 0.000 claims description 22
- HWXBTNAVRSUOJR-UHFFFAOYSA-N alpha-hydroxyglutaric acid Natural products OC(=O)C(O)CCC(O)=O HWXBTNAVRSUOJR-UHFFFAOYSA-N 0.000 claims description 17
- 229940009533 alpha-ketoglutaric acid Drugs 0.000 claims description 17
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 12
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 11
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 8
- FZLIPJUXYLNCLC-UHFFFAOYSA-N lanthanum atom Chemical compound [La] FZLIPJUXYLNCLC-UHFFFAOYSA-N 0.000 claims description 8
- ICAKDTKJOYSXGC-UHFFFAOYSA-K lanthanum(iii) chloride Chemical compound Cl[La](Cl)Cl ICAKDTKJOYSXGC-UHFFFAOYSA-K 0.000 claims description 8
- 102000008214 Glutamate decarboxylase Human genes 0.000 claims description 7
- 108091022930 Glutamate decarboxylase Proteins 0.000 claims description 7
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 6
- 230000000694 effects Effects 0.000 claims description 5
- XKUYOJZZLGFZTC-UHFFFAOYSA-K lanthanum(iii) bromide Chemical compound Br[La](Br)Br XKUYOJZZLGFZTC-UHFFFAOYSA-K 0.000 claims description 5
- 229910021529 ammonia Inorganic materials 0.000 claims description 4
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 claims description 3
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 3
- CTTDRZXJNHOVMU-UHFFFAOYSA-K I(=O)[O-].[La+3].I(=O)[O-].I(=O)[O-] Chemical compound I(=O)[O-].[La+3].I(=O)[O-].I(=O)[O-] CTTDRZXJNHOVMU-UHFFFAOYSA-K 0.000 claims description 2
- 238000002161 passivation Methods 0.000 claims description 2
- 229940044652 phenolsulfonate Drugs 0.000 claims description 2
- 239000000376 reactant Substances 0.000 claims description 2
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 2
- BYMUNNMMXKDFEZ-UHFFFAOYSA-K trifluorolanthanum Chemical compound F[La](F)F BYMUNNMMXKDFEZ-UHFFFAOYSA-K 0.000 claims description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 2
- 230000001934 delay Effects 0.000 claims 1
- 239000000463 material Substances 0.000 abstract description 3
- -1 lanthanum halide Chemical class 0.000 abstract description 2
- 238000001291 vacuum drying Methods 0.000 abstract description 2
- 239000000203 mixture Substances 0.000 abstract 2
- 230000001373 regressive effect Effects 0.000 abstract 2
- 239000002253 acid Substances 0.000 abstract 1
- 229940124277 aminobutyric acid Drugs 0.000 abstract 1
- 239000012295 chemical reaction liquid Substances 0.000 abstract 1
- 229940065734 gamma-aminobutyrate Drugs 0.000 abstract 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 abstract 1
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 15
- 239000000872 buffer Substances 0.000 description 12
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 description 9
- 241000196324 Embryophyta Species 0.000 description 8
- 239000007787 solid Substances 0.000 description 6
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 6
- 239000000049 pigment Substances 0.000 description 5
- 239000012086 standard solution Substances 0.000 description 5
- 235000001014 amino acid Nutrition 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 238000013459 approach Methods 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 235000013311 vegetables Nutrition 0.000 description 4
- 230000003287 optical effect Effects 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 206010002660 Anoxia Diseases 0.000 description 2
- 241000976983 Anoxia Species 0.000 description 2
- 206010021143 Hypoxia Diseases 0.000 description 2
- 241000227653 Lycopersicon Species 0.000 description 2
- 241000209094 Oryza Species 0.000 description 2
- 235000007164 Oryza sativa Nutrition 0.000 description 2
- 230000007953 anoxia Effects 0.000 description 2
- 238000004737 colorimetric analysis Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000004816 paper chromatography Methods 0.000 description 2
- 235000009566 rice Nutrition 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000004809 thin layer chromatography Methods 0.000 description 2
- 102000018832 Cytochromes Human genes 0.000 description 1
- 108010052832 Cytochromes Proteins 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 239000005708 Sodium hypochlorite Substances 0.000 description 1
- XSTXAVWGXDQKEL-UHFFFAOYSA-N Trichloroethylene Chemical compound ClC=C(Cl)Cl XSTXAVWGXDQKEL-UHFFFAOYSA-N 0.000 description 1
- 230000036579 abiotic stress Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 230000002490 cerebral effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 108010055793 gabase Proteins 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000010413 mother solution Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000011265 semifinished product Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- NESLWCLHZZISNB-UHFFFAOYSA-M sodium phenolate Chemical compound [Na+].[O-]C1=CC=CC=C1 NESLWCLHZZISNB-UHFFFAOYSA-M 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
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- Physics & Mathematics (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a method for two-enzyme detection of the content of gamma-aminobutyric acid. The method comprises the following steps: grinding a sample to be detected in liquid nitrogen, and adding alcohol; carrying out vacuum drying, adding lanthanum halide, uniformly mixing above materials, centrifuging the obtained mixture, and collecting obtained supernatant; adding an alkali liquid to the supernatant, uniformly mixing the alkali liquid with the supernatant, and centrifuging the obtained mixture to obtain a gamma-aminobutyric acid extract liquid; adding NADP<+>, a Tris buffer liquid and a gamma-aminobutyrate aminotransferase-succinic semialdehyde dehydrogenase two enzyme liquid to the obtained extract liquid in order to obtain a mixed liquid; determining the initial value of the mixed liquid at a wavelength of 340nm by using an ultraviolet-visible spectrophotometer; adding alpha-oxpentanedioic acid to the mixed liquid, reacting, determining the final value of the obtained reaction liquid at the wavelength of 340nm, drawing a standard curve, and acquiring a primary regressive curve; and calculating the content of gamma-aminobutyric acid according to the initial value, the final value and the primary regressive curve. The method effectively reduces interferences, and can simply, rapidly and accurately detect the content of gamm-aminobutyric acid in plant and animal materials.
Description
Technical field
The present invention relates to gamma aminobutyric acid detection technique field, particularly relate to a kind of double enzyme detection gamma aminobutyric acid
The method of content.
Background technology
Gamma aminobutyric acid (GABA) is a kind of non-protein amino acid being widely present, be animal and plant cells from
By the important component in amino acid pool.In plant tissue, the content of GABA is low, about 0.03～2.00 μm ol/gFW,
But at the different environmental stimuli of response, can be multiplied after mechanical damage, anoxia, Salt Strees Condition etc..Correctly
Measure animal vegetable tissue GABA content, be by treating correlative diseases, analyze the sound that plant stimulates to external world
Should be with an important indicator of the ability of adaptation.
For measuring the GABA content of animal vegetable tissue, people have been developed that multiple detection method.Common are
Thin layer chromatography, paper chromatography, paper electrophoresis, amino-acid analyzer method, high performance liquid chromatography etc..Its
In, GABA thin layer chromatography and paper chromatography detection method, with low cost, method is simple, easy to operate,
But both approaches exists error is big, repeatability is poor problem.Use amino-acid analyzer and high-efficient liquid
Chromatography detection GABA, the detection sensitivity of both approaches, stability and accuracy are the highest, but
Detecting instrument is expensive, objectively limits promoting the use of of both approaches.
Therefore, develop spectrophotometry GABA technology accurately and rapidly, become the mesh of people's research
Mark.Wherein, phenol-sodium hypochlorite colorimetric method and the double enzyme reagent kit colorimetry of Gabase are by compatriots institute
Know.Both approaches is simple to operate, with low cost, can directly measure the pure goods of GABA.Not enough
Being the GABA semifinished product for extracting from animal vegetable tissue, blood of human body, it measures easily by internal glutamic acid
Decarboxylase and the interference of cytochrome, cause detecting data higher.
Summary of the invention
It is an object of the invention to overcome above-mentioned the deficiencies in the prior art, it is provided that a kind of double enzyme detection γ
The method of Gamma-propalanine content, the method is simple and accurate, few dry by glutamate decarboxylase activity and pigment again
Disturb.
The present invention by the following technical solutions, the method for a kind of pair of enzyme detection gamma aminobutyric acid content, including with
Lower step:
(1) treat test sample at pulverized under liquid nitrogen, add alcohol, treat the activity of test sample Glutamic Acid decarboxylase with passivation；
(2), after being vacuum dried, add lanthanum, mixing, to precipitate water solublity phenol sulfonate, be centrifuged,
Collect supernatant；
(3) in the supernatant that step (2) obtains, alkali is added, mixing, centrifugal, collect gamma aminobutyric acid
Extracting solution；
(4) in step (3) gained extracting solution, NADP is added+, Tris buffer and gamma aminobutyric acid turn
The double enzyme liquid of ammonia enzyme-succinic semialdehyde dehydrogenase, obtains mixed liquor；
(5) use the mixed liquor that ultraviolet-uisible spectrophotometer determination step (4) obtains at 340nm wavelength
The initial value at place；
(6) the mixed liquor addition α-ketoglutaric acid obtained to step (4) is reacted, and measures reactant liquor and exists
End value at 340nm wavelength；
(7) draw standard curve, try to achieve a regression curve, according to initial value, end value and once return
Curve, calculates the content of gamma aminobutyric acid.
Further, in step (1), treat that test sample is plant tissue or animal tissue.
Further, in step (1), alcohol be the one in methanol, ethanol, normal propyl alcohol and n-butyl alcohol or
Several；Treat that test sample is 1:4.0～1:8.0 with the mass ratio of alcohol.
Further, in step (2), lanthanum is in lanthanum fluoride, lanthanum chloride, lanthanum bromide and lanthanum iodite
One or more；Treat that test sample is 1:0.2～1:2.0 with the mass ratio of lanthanum.
Further, in step (3), lanthanum is 1:1.5～1:3 with the mol ratio of alkali.
Further, in step (3), alkali is in sodium hydroxide, potassium hydroxide, sodium bicarbonate and ammonia
One or more.
Further, in step (4), test sample and NADP are treated+Mass ratio be 1:0.005～1:0.3.
Further, in step (4), the pH value of Tris buffer is 8.6, and Tris buffer concentration is
0.2M, treats that test sample is 1:0.05～1:2.82 with the mass ratio of Tris buffer.
Further, in step (4), the double enzyme liquid of GABA transaminase-succinic semialdehyde dehydrogenase
It is 1:1～1:10 with the mass ratio treating test sample.
Further, in step (5), treat that test sample is 1:0.0016～1:0.094 with the mass ratio of α-ketoglutaric acid.
Further, in step (5), after adding α-ketoglutaric acid, the response time is 45-80 minute.
Further, standard curve making method is as follows:
Prepare the GABA standard solution of a series of variable concentrations, utilize ultraviolet-uisible spectrophotometer to measure not
With the absorption at wavelength 340nm of the GABA standard solution of concentration, with optical density as vertical coordinate, concentration is
Abscissa Criterion curve, tries to achieve a regression curve.
Compared with prior art, the beneficial effects of the present invention is: the invention provides a kind of double enzyme detection γ
The method of Gamma-propalanine content, it is provided that detection method quickly and easily and accurately, few by glutamate decarboxylase
Activity and pigment disturb, to the screening of natural GABA resource and utilization, human body GABA associated conditions
Mutual change between Accurate Diagnosis and treatment, understanding plant metabolism, especially plant is to multiple biology and non-life
The response that thing is coerced has great practical value and directive significance.
Detailed description of the invention
Below in conjunction with embodiment, technical solution of the present invention is further elaborated.
First making standard curve, the manufacture method of standard curve is as follows:
Weigh a certain amount of GABA (from SIGMA) and prepare 200nmol GABA standard solution.And
As mother solution, prepare 0,40,80,120,160,200nmol series GABA standard solution.Inhale
Take the GABA titer of 2.2ml, add the NADP of 4mM+The pH 8.6Tris of 0.6ml, 0.2M
Double enzyme liquid 0.2ml and 20mM of the GABA transaminase-succinic semialdehyde dehydrogenase of buffer 0.8ml, 2U/ml
α-ketoglutaric acid 0.2ml.Before and after adding α-ketoglutaric acid, utilize ultraviolet-uisible spectrophotometer, point
Ce Ding initial value at GABA standard solution 340nm wavelength and end value.And sit with optical density value for vertical
Mark, with concentration as abscissa, Criterion curve, then try to achieve a regression curve equation:
Y=0.9481X+0.004, R2=0.9956 (X represents concentration, and Y represents optical density, R2For once returning song
The determination coefficient of line).
According to the regression curve tried to achieve, and treat test sample initial value at 340nm wavelength and end value,
Can calculate and treat the content of gamma aminobutyric acid in test sample.
Embodiment 1
(1) instrument
High-speed refrigerated centrifuge；Vacuum desiccator；Ultraviolet-uisible spectrophotometer.
(2) reagent
PH 7.2 kaliumphosphate buffer (0.1M)；The Tris buffer (0.2M) of pH 8.6；NADP+(4
mM)；α-ketoglutaric acid (20mM)；Glycerol；Beta-mercaptoethanol；GABA transaminase-succinic acid semialdehyde
(2U/ml, dissolves the double enzyme liquid of dehydrogenase with pH 7.2 kaliumphosphate buffer of 0.1M, includes 12.5% the third
Triol and the beta-mercaptoethanol of 5mM)；Methanol；Lanthanum chloride (80mM)；KOH(1M).
(3) concrete steps
Normal tomato plants fresh leaf material is cleaned after blotting, and puts into rapidly in liquid nitrogen, grind powdery is solid
Body, takes 0.4g powdery solid and proceeds in Eppendorf pipe, adds 1.6ml methanol, de-to be passivated glutamic acid
Carboxylic acid；After 10 minutes, it is vacuum dried 2h.Add the lanthanum chloride that 4.0ml concentration is 80mM, to remove
Pigment is gone to disturb.After shaking up 15 minutes, 13000g is centrifuged 5 minutes, collects supernatant.Draw 3.2ml
Supernatant, joins in another Eppendorf pipe, adds the KOH that 0.64mL concentration is 1M,
With the lanthanum chloride of precipitation excess, after shaking up 5 minutes, 13000g is centrifuged 5 minutes, and supernatant is GABA
Extracting solution.Taking 5ml test tube, be separately added into the GABA extracting solution of 2.2ml, 0.6ml concentration is 4mM
NADP+, 0.8ml concentration is the pH 8.6Tris buffer of 0.2M, and 0.2ml concentration is 2U/ml's
The double enzyme liquid of GABA transaminase-succinic semialdehyde dehydrogenase and 0.2ml concentration are the α-ketoglutaric acid of 20mM.
Before adding α-ketoglutaric acid, measure the initial value at 340nm wavelength, after reacting 60 minutes, measure
End value at 340nm wavelength.According to initial value and the difference of end value, try to achieve the Y in a regression curve
Value, substitutes into regression curve (Y=0.9481X+0.004, a R2=0.9956), in, X value can be calculated,
The GABA content obtaining the fresh blade of normal tomato plants is 1.50 μm ol/gFW.
Embodiment 2
(1) instrument
High-speed refrigerated centrifuge；Vacuum desiccator；Ultraviolet-uisible spectrophotometer.
(2) reagent
PH 7.2 kaliumphosphate buffer (0.1M)；PH 8.6Tris buffer (0.2M)；NADP+(6mM)；
α-ketoglutaric acid (40mM)；Glycerol；Beta-mercaptoethanol；GABA transaminase-succinic semialdehyde dehydrogenase
(4U/ml dissolves with 0.1M pH7.2 kaliumphosphate buffer, includes 12.5% glycerol and 5mM double enzyme liquid
Beta-mercaptoethanol)；N-butyl alcohol；Lanthanum bromide (0.1M)；NaOH(1M).
(3) concrete steps
Cleaned by the Rice Seedling Leaves of Anoxia stress after blotting, put into rapidly in liquid nitrogen, grind powdery is solid
Body, takes 0.4g powdery solid and proceeds in Eppendorf pipe, adds 2.4ml n-butyl alcohol, to be passivated glutamic acid
Decarboxylase；After 10 minutes, it is vacuum dried.Add the lanthanum bromide that 4.0ml concentration is 0.1M, remove
Pigment disturbs.After shaking up 15 minutes, 13000g is centrifuged 5 minutes, collects supernatant.Draw on 3.2ml
Clear liquid, joins in another Eppendorf pipe, adds the NaOH 0.76mL that concentration is 1M, with
The lanthanum bromide of precipitation excess, after shaking up 5 minutes, 13000g is centrifuged 5 minutes, and supernatant is GABA and carries
Take liquid.Taking 10ml test tube, be separately added into the GABA extracting solution of 2.2ml, 0.6ml concentration is 6mM
NADP+, 0.8ml concentration is the pH8.6Tris buffer of 0.2M, and 0.2ml concentration is 4U/ml's
The double enzyme liquid of GABA transaminase-succinic semialdehyde dehydrogenase and 0.2ml concentration are the α-ketoglutaric acid of 40mM.
Before adding α-ketoglutaric acid, measure the initial value at 340nm, after reacting 45 minutes, measure 340nm
The end value at place.According to initial value and the difference of end value, try to achieve the Y value in a regression curve, substitute into one
Secondary regression curve (Y=0.9481X+0.004, R2=0.9956), in, X value can be calculated, obtain by anaerobism
In the rice seedling leaf coerced, GABA content is 8.00 μm ol/gFW.
Embodiment 3
(1) instrument
High-speed refrigerated centrifuge；Vacuum desiccator；Ultraviolet-uisible spectrophotometer.
(2) reagent
PH 7.2 kaliumphosphate buffer (0.1M)；PH 8.6Tris buffer (0.2M)；NADP+(8mM)；
α-ketoglutaric acid (60mM)；Glycerol；Beta-mercaptoethanol；GABA transaminase-succinic semialdehyde dehydrogenase
(1U/ml dissolves with 0.1M pH7.2 kaliumphosphate buffer, includes 12.5% glycerol and 5mM double enzyme liquid
Beta-mercaptoethanol)；Ethanol；Lanthanum chloride (0.1M)；NaHCO3(5M)。
(3) concrete steps
White mouse brain is cleaned after blotting, and puts into rapidly in liquid nitrogen, grinds and obtains powdery solid.Take powdery solid
0.4g proceeds to Eppendorf pipe, adds 3.2ml normal propyl alcohol.After 15 minutes, vacuum drying；Add
4.0ml concentration is the lanthanum chloride of 0.1M.After shaking up 20 minutes, 13000g is centrifuged 5 minutes, collects supernatant
Liquid.In another Eppendorf pipe, add 3.2ml supernatant and 1mL concentration is the NaHCO of 5M3,
The lanthanum chloride of precipitation excess, after shaking up 10 minutes, 13000g is centrifuged 5 minutes, and supernatant is GABA
Extracting solution.Taking 15ml test tube, be separately added into the GABA extracting solution of 2.2ml, the concentration of 0.6ml is 8mM
NADP+, 0.6ml concentration is the pH8.6Tris buffer of 0.2M, and 0.4ml concentration is 1U/ml's
The double enzyme liquid of GABA transaminase-succinic semialdehyde dehydrogenase and 0.2ml concentration are the α-ketoglutaric acid of 60mM.
Before adding α-ketoglutaric acid, measure the initial value at 340nm, after reacting 80 minutes, measure 340nm
The end value at place.According to initial value and the difference of end value, try to achieve the Y value in a regression curve, substitute into one
Secondary regression curve (Y=0.9481X+0.004, R2=0.9956), in, X value can be calculated, obtain GABA
Content.White mouse cerebral tissue GABA content is 0.45mg/gFW.
As seen from the above embodiment, the method for a kind of pair of enzyme detection gamma aminobutyric acid content of the present invention, method
Simply, quickly；Avoid activity and the pigment interference of animal vegetable tissue Glutamic Acid decarboxylase；And light splitting light
Degree method instrument cost is cheap, easy to operate so that the method has the biggest application prospect.This detection method is to sky
The screening of right GABA resource and utilization, the Accurate Diagnosis of human body GABA associated conditions and treatment, understanding
Mutual change between plant metabolism, the response of multiple biology and abiotic stress is had great by especially plant
Practical value and directive significance.
The above is only the preferred embodiment of the present invention, is not limited to the present invention, it is noted that
For those skilled in the art, on the premise of without departing from the technology of the present invention principle, also
Can make some improvement and modification, these improve and modification also should be regarded as protection scope of the present invention.
Claims (9)
1. the method for double enzyme detection gamma aminobutyric acid content, it is characterised in that comprise the following steps:
(1) treat test sample at pulverized under liquid nitrogen, add alcohol, described in passivation, treat test sample Glutamic Acid decarboxylase
Activity；
(2), after being vacuum dried, add lanthanum, mixing, to precipitate water solublity phenol sulfonate, be centrifuged,
Collect supernatant；
(3) in the supernatant that step (2) obtains, alkali is added, mixing, centrifugal, collect gamma aminobutyric acid
Extracting solution；
(4) in step (3) gained extracting solution, NADP is added+, Tris buffer and gamma aminobutyric acid turn
The double enzyme liquid of ammonia enzyme-succinic semialdehyde dehydrogenase, obtains mixed liquor；
(5) use the mixed liquor that ultraviolet-uisible spectrophotometer determination step (4) obtains at 340nm wavelength
The initial value at place；
(6) the mixed liquor addition α-ketoglutaric acid obtained to step (4) is reacted, and measures reactant liquor and exists
End value at described 340nm wavelength；
(7) draw standard curve, try to achieve a regression curve, according to described initial value, end value and once
Regression curve, calculates the content of gamma aminobutyric acid.
The method of the most according to claim 1 pair of enzyme detection gamma aminobutyric acid content, it is characterised in that:
In step (1), described in treat that test sample is plant tissue or animal tissue.
The method of the most according to claim 1 pair of enzyme detection gamma aminobutyric acid content, it is characterised in that:
In step (1), described alcohol is one or more in methanol, ethanol, normal propyl alcohol and n-butyl alcohol；To be measured
Sample is 1:4.0～1:8.0 with the mass ratio of alcohol.
The method of the most according to claim 1 pair of enzyme detection gamma aminobutyric acid content, it is characterised in that:
In step (2), described lanthanum is the one in lanthanum fluoride, lanthanum chloride, lanthanum bromide and lanthanum iodite or several
Kind；The described mass ratio treating test sample and lanthanum is 1:0.2～1:2.0.
The method of the most according to claim 1 pair of enzyme detection gamma aminobutyric acid content, it is characterised in that:
In step (3), described lanthanum is 1:1.5～1:3 with the mol ratio of alkali.
The method of the most according to claim 1 pair of enzyme detection gamma aminobutyric acid content, it is characterised in that:
In step (3), described alkali is the one in sodium hydroxide, potassium hydroxide, sodium bicarbonate and ammonia or several
Kind.
The method of the most according to claim 1 pair of enzyme detection gamma aminobutyric acid content, it is characterised in that:
In step (4), described in treat test sample and NADP+Mass ratio be 1:0.005～1:0.3；Described Tris delays
The pH value rushing liquid is 8.6, and Tris buffer concentration is 0.2M, treats that test sample with the mass ratio of Tris buffer is
1:0.05～1:2.82；The double enzyme liquid of described GABA transaminase-succinic semialdehyde dehydrogenase and the matter treating test sample
Amount ratio is 1:1～1:10.
The method of the most according to claim 1 pair of enzyme detection gamma aminobutyric acid content, it is characterised in that:
In step (5), described in treat that the mass ratio of test sample and α-ketoglutaric acid is 1:0.0016～1:0.094.
The method of the most according to claim 1 pair of enzyme detection gamma aminobutyric acid content, it is characterised in that:
In step (5), after adding α-ketoglutaric acid, the response time is 45-80 minute.
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