CN107365830A - A kind of oxidoreductase activity method of testing - Google Patents
A kind of oxidoreductase activity method of testing Download PDFInfo
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- CN107365830A CN107365830A CN201610312284.7A CN201610312284A CN107365830A CN 107365830 A CN107365830 A CN 107365830A CN 201610312284 A CN201610312284 A CN 201610312284A CN 107365830 A CN107365830 A CN 107365830A
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Abstract
The present invention relates to technical field of biomedical detection, monitors redox enzyme activity in real time using the color change of new single sulfonic acid tetrazolium detection reagent, there is provided a kind of detection method of dehydrogenase activity；From single sulfonic acid tetrazolium detection architecture, good linear dose-relationship is presented in dehydrogenase Enzyme activity assay；By reactant in 450nm absorbance, EC50 curves are obtained, carry out the Bush Vitality's of dehydrogenase.The detection method of the present invention is a kind of High sensitivity, the Dehydrogenase activity detection method of simple operation, it is applied widely, cover the dehydrogenase of various life entities, including for archeobacteria (Thermophilic Bacterium, Natrinema altunense sp), bacterium (lactic acid bacteria, nitrobacteria, Escherichia coli, Diplococcus pneumopniae etc.), various types of cells structure-biological actinomyces, blue-green algae (algae that quivers, basketball algae, nostoc etc.), most unicellular algas (chlamydomonas, green alga etc.), fungi (edible mushroom, saccharomycete, mould etc.), animals and plants.
Description
Technical field
The present invention relates to technical field of biomedical detection, more particularly to utilize single sulfonic acid tetrazolium detection reagent
Color change monitor in real time NAD (P) dependence redox enzyme activity, there is provided the Activity determination of this fermentoid
Method.
Background technology
Oxidoreducing enzyme is to have known one kind most in enzyme class, wherein oxidizing ferment (oxidase；oxydase)
The effect that energy catalytic specie is aoxidized by oxygen；Dehydrogenase (dehydrogenase) can be catalyzed from material molecule
The effect of hydrogen is sloughed, more to be catalyzed alcohol groups (- CHOH) in donor, aldehyde, ketone groups (- HCO or-RCO)
And alkyl is because of the redox of (- CH2-CH2-)., as class protein, it is some special to activate for it
Hydrogen atom, these hydrogen atoms are made to be shifted and aoxidized the starting substance by appropriate hydrogen acceptor.Natural receptor master
There is NADH (NAD+) and nicotinamide-adenine dinucleotide phosphate (NADP+) and
Cytochromes.
Oxidoreducing enzyme participates in a variety of vital movements, including sugar, fat, amino acid, nucleotide metabolism, ammonia
Base acid synthesis and degraded, the oxidation of pyruvic acid, photosynthesis, pentose phosphate pathway (HMP), oxidative phosphorylation,
Oxidation and synthesis of fat etc., and it is in energy transfer and material circulation and indispensable.Organism
Oxidoreductase activity largely reflects the activated state of organism, can directly represent biological cell
To the power of its matrix degrading capabilities.Therefore, oxidoreductase activity detection is widely used in biochemical sewage
Research and the application fields such as processing, total number of bacterial colonies inspection, water quality toxicity inspection, Soil Contamination Evaluation.
Detection for oxidoreductase activity can be by adding artificial hydrogen acceptor, including TTC (2,3,5- chlorinations three
Phenyl tetrazole), resazurin, methylene blue, INT (iodonitrotetrazolium purple), by the reduction of artificial hydrogen acceptor
Discoloration rate determines the intensity of certain embodiments, wherein, that be most widely used is TTC.TTC- Dehydrogenase activtities
Property detecting method operating process need to add organic reagent, and solution colour developing is shallow, easily fades, reaction time length.Accordingly,
It is this to study a kind of Determining Method of Dehydrogenase Activity easy to operate, fast and effective, safe, applied widely
Field urgent problem to be solved.
A kind of compounds with single sulphenyl tetrazole structure of patent CN201510757078.2- and its application
The synthetic method of single sulfonic acid tetrazotized zole compound is provided, in short, by 4- nitrophenyl hydrazines at 65 DEG C
(0.958g) is dissolved in methanol (22ml), and 2- formylbenzenesulfonic acids sodium (1.188g) is used at 50 DEG C
Processing 2 hours.After processing, Orange red solid hydrazine, yield 87% are obtained.In second step, by gained
Hydrazine (0.614g) be dissolved in 20ml methanol, with freshly prepared 2- methoxyl groups -4- nitrobenzenediazoniums (0.36
G) reacted 2 hours at 0 DEG C with 1mol/L sodium hydroxides (0.9ml), then heat to 25 DEG C of reactions 2
hrs.After post processing, formazan, yield 85% are obtained.In the third step, at 0 DEG C Jiang formazan (0.522g)
Methanol (18ml) is dissolved in, adds 37% HCl (2g), with hydrogen peroxide (2.27ml, 20mM) oxygen
Change 2hrs, then in 25 DEG C of reaction overnights.Post processing and silicagel column after purification, have obtained tetrazolium list
Sulfonic acid detection reagent, yield 99%, purity 45%.
The content of the invention
Single sulfonic acid tetrazolium is as a kind of new single sulfonic acid tetrazotized zole compound, experimental principle and WST-8 classes
Seemingly：, can be by Intramitochondrial dehydrogenase in the presence of electronics coupled reagent 1-methoxy PMS
The orange-yellow formazan product (formazan) of reduction generation high water soluble, 450nm is determined by ELIASA
Wavelength OD values.Living cells quantity is more, then absorbance is higher.Single corresponding formazan product of sulfonic acid tetrazolium
It is highly water soluble, cytotoxicity is low, high sensitivity, to not producing false positive with inoxidizability drug screening,
Therefore in drug screening, cell proliferating determining, CTA, large intestine carcinoma and biotic factor
The application such as Activity determination above have broad prospects.
Oxidation redox enzyme activity is monitored in real time using the color change of new single sulfonic acid tetrazolium detection reagent
A kind of power, there is provided detection method of redox enzyme activity.Examined from single sulfonic acid tetrazolium detection reagent
Good linear dose-relationship is presented in survey system, oxidoreducing enzyme Enzyme activity assay；By reactant 450nm's
Absorbance, EC50 curves are obtained, carry out the Bush Vitality's of oxidoreducing enzyme.
The detection method of the present invention is a kind of High sensitivity, the oxidoreducing enzyme Enzyme activity assay method of simple operation,
It is applied widely, cover the oxidoreducing enzyme of various life entities, including for archeobacteria (Thermophilic Bacterium,
Natrinema altunense sp), bacterium (lactic acid bacteria, nitrobacteria, Escherichia coli, Diplococcus pneumopniae etc.), various types of cells
Structure-biological actinomyces, blue-green algae (algae that quivers, basketball algae, nostoc etc.), most unicellular alga (clothing
Algae, green alga etc.), fungi (edible mushroom, saccharomycete, mould etc.), animals and plants.
The present invention in view of the shortcomings of the prior art, such as cytotoxicity, anti-to the false positive with inoxidizability medicine
Should, there is provided a kind of oxidoreducing enzyme vigor testing methods；Including following content：
A kind of oxidoreductase activity method of testing, single sulfonic acid tetrazolium detection examination is used in the detection method
Agent；
Described single sulfonic acid tetrazolium detection reagent includes single sulfonic acid tetrazolium or single sulphur containing culture medium
Sour tetrazolium and 1-methoxy PMS；
The chemical formula of described single sulfonic acid tetrazolium is：
Preferably, it is single in single sulfonic acid tetrazolium detection reagent in described oxidoreductase activity method of testing
The concentration of sulfonic acid tetrazolium is 0.1~10mM, and 1-methoxy PMS concentration is 5~25 μM.
Preferably, described oxidoreductase activity method of testing comprises the following steps：
Step 1: appropriate oxidoreducing enzyme is corresponded into substrate, NAD (P)+, single sulfonic acid tetrazolium detection reagent mixes
In Tris-NaCl buffer solutions or phosphate buffer；
Step 2: by the solution to be measured containing oxidoreducing enzyme according to 1:It is molten that the volume ratio of (0.01~1) adds buffering
Liquid, it is well mixed, is placed under the enzyme optimal reactive temperature and reacts 0.5~4 hour, detect 450nm suction
Luminosity；
Preferably, the source of the oxidoreducing enzyme in described oxidoreductase activity method of testing include people source,
The bacterial origins such as the mammal sources such as mouse source, insect source, Escherichia coli, actinomyces, thermophilic salt are thermophilic
Hot archeobacteria source, originated from fungus.
Preferably, the oxidoreducing enzyme in described oxidoreductase activity method of testing includes：Lactic dehydrogenase,
Grape -6- phosphate dehydrogenases, alcohol dehydrogenase, aldehyde ketone reductase, aldose reductase, carbohydrate dehydrogenase, carnitine
Dehydrogenase, glycerol-3-phosphate dehydrogenase, HMG-CoA reductase, isocitric dehydrogenase, hypoxanthine core
Thuja acid dehydrogenase, β -one acyl-ACP reductases, malic dehydrogenase, L-threonine dehydrogenase, sorbierite
Dehydrogenase, pyruvate dehydrogenase system, hydroxysteroid dehydrogenase, glutamte dehydrogenase.
Preferably, the different oxidoreducing enzyme in described oxidoreductase activity method of testing have different
Optimal reactive temperature, temperature range is at 25 DEG C~90 DEG C；Different oxidoreducing enzyme has different optimal pHs,
The scope of different oxidoreducing enzyme pH value is 4~11.
Preferably, surfactant is included in the cushioning liquid in described oxidoreductase activity method of testing
With defoamer；
Described surfactant include hexadecyltrimethylammonium chloride, stearic acid, neopelex,
It is quaternary ammonium compound, lecithin, amino acid pattern, betaine type, fatty glyceride, fatty acid sorbitan, poly-
Sorb ester；
Described defoamer includes silicone emulsion, the fatty acid ester compounded thing of higher alcohols, polyoxyethylene polyoxypropylene season
Penta 4 alcohol ethers, polyoxyethylene polyoxy propyl alcohol amidogen ether, polypropylene glycerol aether and polyoxyethylene polyoxypropylene glycerine
Ether, dimethyl silicone polymer.
Preferably, redox in solution to be measured in the step two in described oxidoreductase activity method of testing
Enzyme Enzyme activity assay scope is 0.25mU/L~1000U/L.
Preferably, described oxidoreductase activity method of testing includes glutamte dehydrogenase-urease coupling
Reaction determines the urea in serum and urine with leucine dehydrogenase-urease coupling reaction and with this method.
Preferably, it is even to include glutaminase-glutamte dehydrogenase for described oxidoreductase activity method of testing
Connection reaction.
Preferably, described oxidoreductase activity method of testing, for dehydrogenase in clinical iii vivo serum sample
Detection.
Specifically,
Appropriate oxidoreducing enzyme is corresponded into substrate, NAD (P)+, single sulfonic acid tetrazolium detection reagent is mixed in
Tris-NaCl buffer solutions or phosphate buffer；Solution to be measured containing oxidoreducing enzyme is according to 1:(0.01~1)
Volume ratio add cushioning liquid, be well mixed, be placed under oxidoreducing enzyme optimal reactive temperature reaction 0.5~
4 hours, 450nm absorbance is detected, obtains the enzyme activity in solution to be measured.
The method of above-mentioned detection oxidoreductase activity, it is corresponding reaction substrate, anti-according to the difference of enzyme class
Condition is answered also to have any different.
(1) lactic dehydrogenase：When Tris-NaCl buffer solutions or phosphate buffer pH7~8, reaction substrate is
1~50mM pyruvic acid, 25~38 DEG C of reaction temperature；Tris-NaCl buffer solutions or phosphate buffer pH8~10
When, reaction substrate is 1~50mM lactic acid, 25~38 DEG C of reaction temperature.
(2) grape -6- phosphate dehydrogenases (G6PD)：Tris-NaCl buffer solutions or phosphate buffer pH7~8,
Reaction substrate is 1~50mM G-6-Ps, 25~38 DEG C of reaction temperature.
(3) alcohol dehydrogenase：Alcohol dehydrogenase for coenzyme, is catalyzed enough with NADH (NAD)
Reversible reaction between primary alcohols and aldehydes：CH3CH2OH+NAD+→CH3CHO+NADH+H+, Substratspezifitaet is wider,
Also other alcohol are acted on；Optimun pH 7.0~10.0, temperature are 30~40 DEG C of
(4) aldehyde ketone reductase:Using 3- deoxyfructoses as substrate, coenzyme NADP 11 is consumed；Optimal pH 7.4~
7.9,25~38 DEG C of reaction temperature.
(5) aldose reductase:The a variety of aldehyde of enzymatic, letones are reduced to alcohol supplemented by NADPH, most suitable bottom
The polymer of thing 4- hydroxyls aldehyde C-9 (4-HNE) and its glutathione, Optimun pH 7.0~10.0, temperature
For 30~40 DEG C of
(6) carbohydrate dehydrogenase：D-Glucose+NAD (P) D- gluconic acids (lactone), coenzyme are NADP or NAD,
Optimal pH 7.0~9.0,30~50 DEG C of temperature
(7) carnitin dehydrogenases：Reaction substrate is VBT, and coenzyme is NADP or NAD, optimal pH 7.0~
11.0,40~60 DEG C of temperature.
(8) glycerol-3-phosphate dehydrogenase：Reaction substrate glyceraldehyde 3-phosphate, coenzyme NAD, optimal pH 7.0~
9.0,20~40 DEG C of temperature.
(9) HMG-CoA reductase：In plant, catalysis is dependent on NADPH from 3- hydroxy-3-methyls penta 2
Acyl coenzyme A is to mevalonic acid (MVA)；In animal, the rate-limiting enzyme during liver cell synthetic cholesterol, catalysis
Generate mevalonic acid.Optimal pH 4.0~11.0,20~50 DEG C of temperature.
(10) isocitric dehydrogenase：Substrate is isocitric acid, coenzyme NAD/NADP, optimal pH 7.0~
9.0,20~40 DEG C of temperature.
(11) carnine acidohydrogenase：Substrate is inosinic acid, coenzyme NAD, most suitable
PH 6.0~10.0,25~40 DEG C of temperature.
(12) β -one acyl-ACP reductases：Substrate is β-hydroxyl acyl CoA, coenzyme NAD, optimal pH
6.0~10.0,25~40 DEG C of temperature.
(13) malic dehydrogenase：Substrate is malic acid or oxaloacetic acid, coenzyme NAD, NADP, most suitable
PH 6.0~10.0,35~60 DEG C of temperature.
(14) L-threonine dehydrogenase：Substrate L-threonine, coenzyme NAD, optimal pH 8.0~11.0；It is most suitable
55~90 DEG C of temperature.
(15) sorbitol dehydrogenase：Substrate D-glucitol, L- Chinese mugwort Du Chun, D- xylitol, D- galactitols etc.,
Coenzyme NAD, optimal pH 4.0~8.0；20~40 DEG C of optimum temperature.
(16) pyruvate dehydrogenase system：Substrate pyruvate, including (pyruvic dehydrogenase, dihydro sulphur are pungent for three kinds of enzymes
Sour CAT, dihydrolipoic acid dehydrogenase) and six kinds of confactors (diphosphothiamine, lipoic acid,
FAD, NAD, CoA and Mg ion), coenzyme NAD or NADP, optimal pH 4.0~10.0；20~60 DEG C of optimum temperature.
(17) hydroxysteroid dehydrogenase：1 beta hydroxysteroid dehydrogenase substrate is cortisol or cortisone；3α-
Hydroxysteroid dehydrogenase substrate is a variety of steroids matrix (such as bile acid).Coenzyme NAD or NADP, it is most suitable
PH4.0~10.0；20~60 DEG C of optimum temperature.
(18) glutamte dehydrogenase：Substrate is glutamic acid, coenzyme NAD or NADP, optimal pH 7.0~9.0；
20~40 DEG C of optimum temperature.
In the presence of electronics coupled reagent 1-methoxy PMS, single sulfonic acid tetrazolium detection reagent can
To be reduced by oxidoreducing enzyme in NAD (P) H systems or cell mitochondrial, the orange-yellow of high water soluble is generated
Formazans product (formazan), there is UV absorption at 450nm.
The detection method of the present invention is a kind of super-sensitive detection method based on cell.Used in the inventive method
Single sulfonic acid tetrazolium detection reagent has been arrived, can suitable for NAD (P) H systems and the basic test of living cells
For detecting the activity of oxidoreducing enzyme in cell.
At present, CCK-8 (or WST-8), MTT, XTT or MTS measure kit are had been developed that applied to cell
The measure of propagation and cytotoxicity.Wherein, CCK-8 (or WST-8) is most widely used, then single nitrogen of sulfonic acid four
Azoles salt detection reagent is compared with WST-8.
Experimental principle
Oxidoreducing enzyme catalyzing organic oxidative dehydrogenation and passes it to final hydrogen acceptor in biological cell.It
Hydrogen can be catalyzed to be transferred to from oxidized object on another object, i.e. the work of enzymatic organic substance dehydrogenation
With.Therefore, oxidoreductase activity can be detected by adding the method for artificial hydrogen acceptor.Detection examination
Single sulfonic acid tetrazolium detection reagent system is selected in agent, and produced under acceptor NAD (P)+effect has at 450nm
Absorption value material.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment
Or the required accompanying drawing used is briefly described in description of the prior art, it should be apparent that, in describing below
Accompanying drawing be only some embodiments of the present invention, for those of ordinary skill in the art, do not paying
On the premise of creative labor, other accompanying drawings can also be obtained according to these accompanying drawings.
Fig. 1 is the single sulfonic acid tetrazolium detection reagent of embodiment 1 to Escherichia coli glutamate dehydrogenase enzymatic activity
Linear dose response schematic diagram.
Fig. 2 is the glutamte dehydrogenase vigour of the separate sources of embodiment 1.With Escherichia coli dehydrogenase
Vigor is 100%.
Fig. 3 is inhibition assay result of the inhibitor compound of embodiment 1 to Escherichia coli glutamte dehydrogenase
Schematic diagram.
Fig. 4 is that the single sulfonic acid tetrazolium detection reagent of embodiment 2 is imitated to the linear dose response of lactic acid dehydrogenase activity
Fruit schematic diagram；Initial rate：Initial velocity(Vi).
Fig. 5 is the single sulfonic acid tetrazolium detection reagent of embodiment 2 to lactic acid dehydrogenase activity in different serum samples
Reaction effect schematic diagram
Fig. 6 is the single sulfonic acid tetrazolium detection reagent of embodiment 3 to the linear agent of grape -6- phosphate dehydrogenase activities
Quantitative response effect diagram.
Fig. 7 is the single sulfonic acid tetrazolium detection reagent of embodiment 4 to carnitin dehydrogenases active linear dose response
Effect diagram.
Fig. 8 is the linear dosage test effect diagram of the glutaminase active of embodiment 5.
Fig. 9 is the linear dosage test effect diagram of the aldose reductase activity of embodiment 6.
Embodiment
With reference to embodiment, the present invention is described in further detail, and following examples are the solutions to the present invention
Release and the invention is not limited in following examples.
Embodiment 1
As shown in Figure 1 to Figure 3, coding schedule reaches the cDNA genetic fragments source of glutamte dehydrogenase (GDH) albumen
Extensively, it is present in various life entities.
0~8nM of final concentration GDH, appropriate NADP+ are added in 100ul reaction Tris buffer solutions (pH7~9),
5~30mM glutamic acid, at room temperature 10~60min of concussion reaction.Then, single sulfonic acid tetrazolium inspection is added
Test agent, after mixing, 450nm absorbances (reference wavelength 620nm) are determined, obtain Escherichia coli
Source glutamte dehydrogenase linear equation is y=0.21x+0.075 (R2=0.999), x is glutamate dehydrogenase
Enzyme enzyme activity (nM).
In the experiment of glutamic acid inhibitor sifting, positive control selection HJ7 and HJ8.Negative control HJ1.
Concrete operations：0~30 μM of inhibitor of 50ul and 4nM GDH Tris-NaCl are added in 96 orifice plates
Buffer solution (pH7~9), concussion reaction 1~5 hour under room temperature environment.Add the appropriate NADP of 50ul+、
The cocktail buffer of glutamic acid and single sulfonic acid tetrazolium detection reagent, after mixing, determine 450nm absorbances
It is worth (reference wavelength 620nm).
Embodiment 2
1~50mM lactic dehydrogenases correspond to bottom lactic acid, NAD+, single sulfonic acid tetrazolium detection reagent is mixed in
Tr is-NaC l buffer solutions or phosphate buffer (pH8~10)；Solution to be measured containing lactic dehydrogenase according to
1:The volume ratio of (0.01~1) adds cushioning liquid, is well mixed, react 0.5 at a temperature of being placed in 25~38 DEG C~
4 hours, 450nm absorbance is detected, the enzyme activity of dehydrogenase in solution to be measured is obtained, is specifically shown in Fig. 4.
Dehydrogenase Content can be used as some indicators of disease detection in serum, by different serum sample serum-dilutions
Suitable multiple, then add 1~50mM lactic acid, NAD+, single sulfonic acid tetrazolium detection reagent be mixed in Tris-NaCl
Buffer solution or phosphate buffer (pH8~10)；Solution to be measured containing lactic dehydrogenase is according to 1:(0.01~1)
Volume ratio add cushioning liquid, be well mixed, react 0.5~4 hour at a temperature of being placed in 25~38 DEG C, inspection
450nm absorbance is surveyed, the enzyme activity of dehydrogenase in solution to be measured is obtained, is specifically shown in Fig. 5.
(lactic dehydrogenase normal value in serum：Male 80-280U/L, female 100-230U/L)
Embodiment 3
1~50mM G-6-Ps, NADP+, single sulfonic acid tetrazolium detection reagent be mixed in Tris-NaCl
Buffer solution or phosphate buffer (pH7~8)；The solution to be measured of the phosphate dehydrogenases of -6- containing grape according to
1:The volume ratio of (0.01~1) adds cushioning liquid, is well mixed, react 0.5 at a temperature of being placed in 25~38 DEG C~
4 hours, 450nm absorbance is detected, the enzyme activity of dehydrogenase in solution to be measured is obtained, is specifically shown in Fig. 5.
Embodiment 4
1~50mML- carnitines, NAD (P)+, single sulfonic acid tetrazolium detection reagent be mixed in Tris-NaCl buffer solutions
Or phosphate buffer (pH7~11.0)；Solution to be measured containing carnitin dehydrogenases is according to 1:The body of (0.01~1)
Product is well mixed, reacted 0.5~4 hour at a temperature of being placed in 40~60 DEG C, detected than adding cushioning liquid
450nm absorbance, the enzyme activity of dehydrogenase in solution to be measured is obtained, is specifically shown in Fig. 6.
Embodiment 5
Appropriate glutamine, glutaminase are mixed in phosphate buffer, reacted 0.5~3 hour, is produced
Glutamic acid.Glutaminase enzyme activity can be as obtained by the quantitative analysis of glutamic acid.Add appropriate paddy ammonia
Acidohydrogenase (4nM after optimization), NADP+, glutamic acid, 10~60min of concussion reaction at room temperature.Then,
Single sulfonic acid tetrazolium detection reagent is added, after mixing, determines 450nm absorbances (reference wavelength 620nm)
It is specifically shown in Fig. 7.
Embodiment 6
1~50mM aldose reductases correspond to substrate 4- hydroxyls aldehyde C-9 (4-HNE) and its glutathione polymer,
NAD+, single sulfonic acid tetrazolium detection reagent be mixed in Tris-NaCl buffer solutions or phosphate buffer (pH7~
10)；Solution to be measured containing aldose reductase is according to 1:The volume ratio of (0.01~1) adds cushioning liquid, mixing
Uniformly, reacted 0.5~4 hour at a temperature of being placed in 30~40 DEG C, detect 450nm absorbance, treated
Enzyme activity in solution is surveyed, is specifically shown in Fig. 8.
Furthermore, it is necessary to illustrate, the specific embodiment described in this specification, its compound named title
Etc. can be different, the equivalent or simple change that all principles according to described in inventional idea of the present invention are done, include
In in the protection domain of patent of the present invention.Those skilled in the art can be to described
Specific embodiment is made various modifications or supplement or substituted using similar mode, without departing from this hair
Bright structure surmounts scope defined in the claims, all should belong to protection scope of the present invention.
Claims (10)
- A kind of 1. oxidoreductase activity method of testing, it is characterised in that：Single sulfonic acid tetrazolium detection reagent is used in the detection method；Described single sulfonic acid tetrazolium detection reagent includes single sulfonic acid tetrazolium or single sulfonic acid tetrazolium and 1-methoxy PMS containing culture medium；The chemical formula of described single sulfonic acid tetrazolium is：
- 2. oxidoreductase activity method of testing according to claim 1, it is characterised in that：The concentration of single sulfonic acid tetrazolium is 0.1~10mM in described single sulfonic acid tetrazolium detection reagent, and 1-methoxy PMS concentration is 5~25 μM.
- 3. oxidoreductase activity method of testing according to claim 1, it is characterised in that：Comprise the following steps：Step 1: appropriate oxidoreducing enzyme is corresponded into substrate, NAD (P)+, single sulfonic acid tetrazolium detection reagent be mixed in Tris-HEPES buffer solutions, Tris-NaCl buffer solutions or phosphate buffer；Step 2: by the solution to be measured containing oxidoreducing enzyme according to 1:The volume ratio of (0.01~1) adds cushioning liquid, is well mixed, is placed under oxidoreducing enzyme optimal reactive temperature and reacts 0.5~4 hour, detect 450nm absorbance.
- 4. oxidoreductase activity method of testing according to claim 3, it is characterised in that：The source of described oxidoreducing enzyme includes the bacterial origins such as people source, mouse source mammal source, insect source, Escherichia coli, actinomyces, thermophilic salt Pyrococcus furiosus source, originated from fungus.
- 5. oxidoreductase activity method of testing according to claim 3, it is characterised in that：Described oxidoreducing enzyme includes：Lactic dehydrogenase, grape -6- phosphate dehydrogenases, alcohol dehydrogenase, aldehyde ketone reductase, aldose reductase, carbohydrate dehydrogenase, carnitin dehydrogenases, glycerol-3-phosphate dehydrogenase, HMG-CoA reductase, isocitric dehydrogenase, carnine acidohydrogenase, β -one acyl-ACP reductases, malic dehydrogenase, L-threonine dehydrogenase, sorbitol dehydrogenase, pyruvate dehydrogenase system, hydroxysteroid dehydrogenase, glutamte dehydrogenase.
- 6. oxidoreductase activity method of testing according to claim 3, it is characterised in that：Described different oxidoreducing enzyme have different optimal reactive temperatures, and temperature range is at 25 DEG C~90 DEG C；Different oxidoreducing enzyme has different optimal pHs, and the scope of different oxidoreducing enzyme pH value is 4~11.
- 7. oxidoreductase activity method of testing according to claim 3, it is characterised in that：Include surfactant and defoamer in described cushioning liquid；Described surfactant includes hexadecyltrimethylammonium chloride, stearic acid, neopelex, quaternary ammonium compound, lecithin, amino acid pattern, betaine type, fatty glyceride, fatty acid sorbitan, polysorbate；Described defoamer includes silicone emulsion, the fatty acid ester compounded thing of higher alcohols, polyoxyethylene polyoxypropylene pentaerythrite ether, polyoxyethylene polyoxy propyl alcohol amidogen ether, polypropylene glycerol aether and polyoxyethylene polyoxypropylene glycerin ether, dimethyl silicone polymer.
- 8. oxidoreductase activity method of testing according to claim 3, it is characterised in that：The Enzyme activity assay scope of oxidoreducing enzyme is 0.25mU/L~10U/L in solution to be measured in described step two.
- 9. according to the purposes of the oxidoreductase activity method of testing described in claim 3, it is characterised in that：It can be used for the viability examination of other non-redox enzymes, the product that the non-redox enzyme can be catalyzed generation is the substrate of oxidoreducing enzyme, and signal is produced by the further effect of oxidoreducing enzyme；Glutamte dehydrogenase-urease coupling reaction determines the urea in serum and urine with leucine dehydrogenase-urease coupling reaction and with this method；Glutamine either in glutaminase and glutamte dehydrogenase coupling reaction measure serum and urine, or the alanine in ALT and lactic dehydrogenase coupling reaction measure serum and urine.
- 10. the purposes of oxidoreductase activity method of testing according to claim 3, it is characterised in that：This method can be used for the content detection of dehydrogenase in clinical iii vivo serum sample.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110887933A (en) * | 2019-11-28 | 2020-03-17 | 中国科学院天津工业生物技术研究所 | Enzyme activity determination method based on real-time absorbance determination in constant-temperature catalytic reaction process |
| CN111620441A (en) * | 2020-06-11 | 2020-09-04 | 南京中洲环保科技有限公司 | Synchronous sewage nitrification and denitrification control method and device |
| CN112553294A (en) * | 2020-11-25 | 2021-03-26 | 浙江工业大学 | Method for detecting enzyme activity of steroid C1, 2-dehydrogenase |
| CN112684160A (en) * | 2019-10-17 | 2021-04-20 | 潍坊峡山荆卫生物科技有限公司 | Efficient blood plasma, erythrocyte and lymphocyte NAD+Detection method |
| CN112760359A (en) * | 2019-11-04 | 2021-05-07 | 南京盛德生物科技研究院有限公司 | GDH inhibitor high-flux screening method based on human mutant protein |
| CN113670911A (en) * | 2021-08-30 | 2021-11-19 | 武汉纺织大学 | Method for determining total number of viable bacteria based on WST-8 chromogenic reaction |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1378077A (en) * | 2002-05-10 | 2002-11-06 | 肖洪武 | Method for detecting total bile acid and detecting reagent |
| CN101717814A (en) * | 2009-12-18 | 2010-06-02 | 北京九强生物技术有限公司 | Liquid double reagent diagnostic reagent kit for determining content of potassium ions in serum and blood plasma |
| CN105418526A (en) * | 2015-11-09 | 2016-03-23 | 杭州伽玛生物科技有限公司 | Compound with mono-sulfophenyl tetrazole structure and application of compound with mono-sulfophenyl tetrazole structure |
-
2016
- 2016-05-11 CN CN201610312284.7A patent/CN107365830A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1378077A (en) * | 2002-05-10 | 2002-11-06 | 肖洪武 | Method for detecting total bile acid and detecting reagent |
| CN101717814A (en) * | 2009-12-18 | 2010-06-02 | 北京九强生物技术有限公司 | Liquid double reagent diagnostic reagent kit for determining content of potassium ions in serum and blood plasma |
| CN105418526A (en) * | 2015-11-09 | 2016-03-23 | 杭州伽玛生物科技有限公司 | Compound with mono-sulfophenyl tetrazole structure and application of compound with mono-sulfophenyl tetrazole structure |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112684160A (en) * | 2019-10-17 | 2021-04-20 | 潍坊峡山荆卫生物科技有限公司 | Efficient blood plasma, erythrocyte and lymphocyte NAD+Detection method |
| CN112760359A (en) * | 2019-11-04 | 2021-05-07 | 南京盛德生物科技研究院有限公司 | GDH inhibitor high-flux screening method based on human mutant protein |
| CN110887933A (en) * | 2019-11-28 | 2020-03-17 | 中国科学院天津工业生物技术研究所 | Enzyme activity determination method based on real-time absorbance determination in constant-temperature catalytic reaction process |
| CN111620441A (en) * | 2020-06-11 | 2020-09-04 | 南京中洲环保科技有限公司 | Synchronous sewage nitrification and denitrification control method and device |
| CN112553294A (en) * | 2020-11-25 | 2021-03-26 | 浙江工业大学 | Method for detecting enzyme activity of steroid C1, 2-dehydrogenase |
| CN113670911A (en) * | 2021-08-30 | 2021-11-19 | 武汉纺织大学 | Method for determining total number of viable bacteria based on WST-8 chromogenic reaction |
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