CN103674870B - A kind of method measuring superoxide dismutase activity - Google Patents
A kind of method measuring superoxide dismutase activity Download PDFInfo
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- CN103674870B CN103674870B CN201210341599.6A CN201210341599A CN103674870B CN 103674870 B CN103674870 B CN 103674870B CN 201210341599 A CN201210341599 A CN 201210341599A CN 103674870 B CN103674870 B CN 103674870B
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Abstract
The invention provides a kind of method measuring superoxide dismutase activity.Solve the method measuring superoxide dismutase activity in prior art and there is detection difficult, accuracy is low, the technical matters of poor reproducibility.This method selects pyrogallol alkalescence autoxidation system, in conjunction with UV-VIS spectrophotometry, the scavenging action of superoxide dismutase to superoxide anion is evaluated, go out the clearance rate of superoxide dismutase to superoxide anion by the change calculations of the product water dissolubility formazan absorbance of trapping agent 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2 ˊ 4-disulfobenzene)-2H-tetrazole sodium salt after detection and superoxide anion effect, and then draw the activity of superoxide dismutase.This method has favorable reproducibility, and accuracy is high, advantage easy and simple to handle.<!--1-->
Description
Technical field
The invention belongs to analytical chemistry field, be specifically related to a kind of method measuring superoxide dismutase activity.
Background technology
Active oxygen comprises superoxide anion, hydroxyl radical free radical, hydrogen peroxide, singlet oxygen etc., and these particles are all very small.Owing to there is unpaired free electron, the chemical property of active oxygen is very active.A kind of secondary product of active oxygen often in biological biochemistry process, when it is excessive, the active oxygen in body can cause damage to cell and gene structure, causes various diseases, as cytotoxicity, aging, cancer etc.Superoxide anion is the source of other several active oxygens, superoxide dismutase can remove superoxide anion excessive in body, it can catalysis superoxide anion generation disproportionation reaction, produces oxygen and hydrogen peroxide, has anti-inflammatory, antiviral, the anti-ageing effect of waiting for a long time.Therefore, in recent years, the research of the method measuring superoxide dismutase activity is received much concern.
Method mainly Xanthine oxidase NBT method, epinephrine autoxidation method, Marklund pyrogallol method, the chemoluminescence method etc. of existing mensuration superoxide dismutase activity.But in Xanthine oxidase NBT method, NBT Chan Wu formazan poorly water-soluble, needing to add cosolvent increases water-soluble, and therefore the application of this method has certain restriction.Epinephrine autoxidation method and Marklund pyrogallol method are according to adrenaline and pyrogallol autoxidation in the basic conditions, produce superoxide anion and coloring matter, content according to coloring matter measures superoxide dismutase activity, but coloring matter is unstable, life period is short, therefore measure and have error, reappearance is poor.Chemoluminescence method is that the method is of short duration due to fluorescent lifetime, and measure difficult and easily produce error, reappearance is poor by using chemiluminescence agent to make system produce chemiluminescence, indirectly detecting the method for superoxide anion.
Summary of the invention
The present invention solves the method measuring superoxide dismutase activity in prior art to there is detection difficult, and accuracy is low, the technical matters of poor reproducibility, and provides a kind of method relating to the activity of UV-VIS spectrophotometry mensuration superoxide dismutase.
In order to solve the problems of the technologies described above, the technical scheme of the method for mensuration superoxide dismutase activity of the present invention is specific as follows:
Measure a method for superoxide dismutase activity, the method comprises the following steps:
Step a, superoxide anion produce the preparation of system buffer solution
Secure ph is the ammonium bicarbonate buffer solution of 8.5 ~ 9.5 30 ~ 50 mM/ls;
The preparation of step b, standard items
With the ammonium bicarbonate buffer solution in step a, standard items are made into the standard solution that concentration is 50 micromoles per liter, described standard items are 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt;
The preparation of step c, reference substance, sample and blank sample
The preparation of reference substance: get 40 microlitre, 0.4 ~ 8 mM/l of disodium EDTA, add 40 microlitre physiological saline, add 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-the disulfobenzene)-2H-tetrazole sodium salt of 40 microlitre 50 micromoles per liter, finally add 40 microlitre, 1 mM/l of pyrogallol aqueous solution, 37 DEG C of reactions are after 8 ~ 20 minutes, product in contrast, the microplate reader for built-in ultraviolet-visible pectrophotometer measures;
The preparation of sample: get 40 microlitre, 0.4 ~ 8 mM/l of disodium EDTA, add 40 microlitre superoxide dismutase solution, add 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-the disulfobenzene)-2H-tetrazole sodium salt of 40 microlitre 50 micromoles per liter, finally add 40 microlitre, 1 mM/l of pyrogallol aqueous solution, 37 DEG C of reactions are after 8 ~ 20 minutes, as sample, the microplate reader for built-in ultraviolet-visible pectrophotometer measures;
The preparation of blank sample: get 40 microlitre, 0.4 ~ 8 mM/l of disodium EDTA, add 40 microlitre superoxide dismutase solution, add 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-the disulfobenzene)-2H-tetrazole sodium salt of 40 microlitre 50 micromoles per liter, finally add 40 microliters of water solution, 37 DEG C of reactions are after 8 ~ 20 minutes, as blank sample, the microplate reader for built-in ultraviolet-visible pectrophotometer measures;
The detection of steps d, reference substance, sample and blank sample
Setting ultraviolet-visible determined wavelength is 450 nanometers, in the microplate reader of built-in ultraviolet-visible pectrophotometer to step c in reference substance, sample and the blank sample prepared measure, obtain the absorbance A of reference substance, sample and blank sample
reference substance, A
sampleand A
blank sample;
The calculating of the activity of step e, superoxide dismutase
Superoxide dismutase to the clearance rate of superoxide anion according to following formulae discovery:
I
sample=[A
reference substance-(A
sample-A
blank sample)]/A
reference substance× 100%
In formula, I is the clearance rate of superoxide dismutase to superoxide anion;
A
reference substancefor the absorbance of the product water dissolubility formazan of 2-in reference substance (4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt, dimensionless;
A
samplefor the absorbance of the product water dissolubility formazan of 2-in sample (4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt, dimensionless;
A
blank samplefor the absorbance of the product water dissolubility formazan of 2-in blank sample (4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt, dimensionless;
The computing formula of superoxide dismutase activity is as follows:
Superoxide dismutase activity=I/ (1 – I)
In formula, I is the clearance rate of superoxide dismutase to superoxide anion; Superoxide dismutase activity unit is U.
In technique scheme, the buffer solution preferable ph described in step a is 9.0 ~ 9.5, and most preferably pH value is 9.3.
In technique scheme, the concentration of the disodium EDTA described in step c is preferably 0.4 ~ 2 mM/l, most preferably is 2 mM/ls.
The beneficial effect of the method for mensuration superoxide dismutase activity of the present invention is:
The method of mensuration superoxide dismutase activity of the present invention, select pyrogallol alkalescence autoxidation system, in conjunction with UV-VIS spectrophotometry, the clearance rate that superoxide dismutase removes superoxide anion is calculated, and then the activity of superoxide dismutase can be obtained, avoid Xanthine oxidase NBT method and produce thing formazan poorly water-soluble, application has the shortcoming of a definite limitation.Overcome adrenaline method, Marklund assay NBT photoreduction and chemoluminescence method unstable products, detection difficult also easily produces the problem of error simultaneously.
In addition, the method favorable reproducibility of mensuration superoxide dismutase activity of the present invention, accuracy is high, easy and simple to handle.
Embodiment
Invention thought of the present invention is: the present invention selects pyrogallol alkalescence autoxidation system, calculates, and then can obtain the activity of superoxide dismutase in conjunction with UV-VIS spectrophotometry to the clearance rate that superoxide dismutase removes superoxide anion.
The present invention selects pyrogallol alkalescence autoxidation system to produce superoxide anion, be superoxide anion trapping agent with 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt, add superoxide dismutase, product water dissolubility formazan has light absorption under 450 nanometers, go out the clearance rate of superoxide dismutase to superoxide anion by the absorbance change calculations of product water dissolubility formazan, and then the activity of superoxide dismutase can be obtained.
Superoxide dismutase can catalysis superoxide anion generation disproportionation reaction, generate oxygen and hydrogen peroxide, thus cause the amount of the superoxide anion reacted with 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt to reduce, cause the amount of the reduzate water-soluble Xing formazan dyestuff of trapping agent 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt to reduce; So the activity of superoxide dismutase can be evaluated by the change of the product water dissolubility formazan dye content of trapping agent 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt.
Embodiment 1
A kind of method measuring superoxide dismutase activity provided by the invention, the method comprises the following steps:
Step a, superoxide anion produce the preparation of system buffer solution
Compound concentration is the ammonium bicarbonate buffer solution of 50 mM/ls, and adjust ph is 9.3;
The preparation of step b, standard items
With the ammonium bicarbonate buffer solution in step a, standard items are made into the standard solution that concentration is 50 micromoles per liter, described standard items are 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt, and energy and superoxide anion reaction produce water-soluble Xing formazan dyestuff;
The preparation of step c, reference substance, sample and blank sample
The preparation of reference substance: reaction cumulative volume 160 microlitre, first add 40 microlitre, 2 mM/ls of disodium EDTAs, add 40 microlitre physiological saline, add 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-the disulfobenzene)-2H-tetrazole sodium salt of 40 microlitre 50 micromoles per liter, finally add 40 microlitre, 1 mM/l of pyrogallol aqueous solution, 37 DEG C of reactions are after 12 minutes, product in contrast, the microplate reader for built-in ultraviolet-visible pectrophotometer measures;
The preparation of sample: reaction cumulative volume 160 microlitre, first add 40 microlitre, 2 mM/ls of disodium EDTAs, add 40 microlitre 3.12 mg/litre superoxide dismutase standard solution (physiological saline solution), add 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-the disulfobenzene)-2H-tetrazole sodium salt of 40 microlitre 50 micromoles per liter, finally add 40 microlitre, 1 mM/l of pyrogallol aqueous solution, 37 DEG C of reactions are after 12 minutes, as sample, the microplate reader for built-in ultraviolet-visible pectrophotometer measures;
The preparation of blank sample: reaction cumulative volume 160 microlitre, first add 40 microlitre, 2 mM/ls of disodium EDTAs, add 40 microlitre 3.12 mg/litre superoxide dismutase standard solution (physiological saline solution), add 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-the disulfobenzene)-2H-tetrazole sodium salt of 40 microlitre 50 micromoles per liter, finally add 40 microliters of water solution, 37 DEG C of reactions are after 12 minutes, as blank sample, the microplate reader for built-in ultraviolet-visible pectrophotometer measures;
The detection of steps d, reference substance, sample and blank sample
Reference substance detects: with 96 orifice plates for carrier, measure in the microplate reader of built-in ultraviolet-visible pectrophotometer, and setting ultraviolet-visible determined wavelength is 450 nanometers, measures, obtain the absorbance A of reference substance to reference substance prepared by step c
reference substance;
Sample detection: with 96 orifice plates for carrier, measures in the microplate reader of built-in ultraviolet-visible pectrophotometer, and setting ultraviolet-visible determined wavelength is 450 nanometers, measures, obtain the absorbance A of sample to the sample of step c preparation
sample;
Blank sample detects: with 96 orifice plates for carrier, measure in the microplate reader of built-in ultraviolet-visible pectrophotometer, and setting ultraviolet-visible determined wavelength is 450 nanometers, measures, obtain the absorbance A of blank sample to the blank sample of step c preparation
blank sample;
The calculating of the activity of step e, superoxide dismutase
Superoxide dismutase to the clearance rate of superoxide anion according to following formulae discovery:
I
sample=[A
reference substance-(A
sample-A
blank sample)]/A
reference substance× 100%
In formula, I is the clearance rate of superoxide dismutase to superoxide anion;
A
reference substancefor the absorbance of the product water dissolubility formazan of 2-in reference substance (4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt, dimensionless;
A
samplefor the absorbance of the product water dissolubility formazan of 2-in sample (4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt, dimensionless;
A
blank samplefor the absorbance of the product water dissolubility formazan of 2-in blank sample (4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt, dimensionless;
The computing formula of superoxide dismutase activity is as follows:
Superoxide dismutase activity=I/ (1 – I)
According to the absorbance A of the product water dissolubility formazan of 2-in the reference substance recorded, sample and blank sample (4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt
reference substance, A
sampleand A
blank sample, calculating 0.78 mg/litre superoxide dismutase standard items is 42% to the clearance rate of superoxide anion, and then show that the activity of superoxide dismutase standard items is 5.8U/ μ g.
Superoxide dismutase standard items are to the clearance rate of superoxide anion, that superoxide dismutase can catalysis superoxide anion generation disproportionation reaction, generate oxygen and hydrogen peroxide, thus cause the amount of the superoxide anion reacted with 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt to reduce, cause the amount of the reduzate water-soluble Xing formazan dyestuff of trapping agent 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt to reduce; So the clearance rate of superoxide dismutase to superoxide anion is obtained by the change calculations of the product water dissolubility formazan dye content of trapping agent 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt, and then draw the activity of superoxide dismutase standard items.
Embodiment 2
A kind of method measuring superoxide dismutase activity provided by the invention, it comprises the following steps:
Step a, superoxide anion produce the preparation of system buffer solution
Compound concentration is the ammonium bicarbonate buffer solution of 30 mM/ls, and adjust ph is 9.5;
The preparation of step b, standard items
With the ammonium bicarbonate buffer solution in step a, standard items are made into the standard solution that concentration is 50 micromoles per liter, described standard items are 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt, and energy and superoxide anion reaction produce water-soluble Xing formazan dyestuff;
The preparation of step c, reference substance, sample and blank sample
The preparation of reference substance: reaction cumulative volume 160 microlitre, first add 40 microlitre, 4 mM/ls of disodium EDTAs, add 40 microlitre physiological saline, add 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-the disulfobenzene)-2H-tetrazole sodium salt of 40 microlitre 50 micromoles per liter, finally add 40 microlitre, 1 mM/l of pyrogallol aqueous solution, 37 DEG C of reactions are after 8 minutes, product in contrast, the microplate reader for built-in ultraviolet-visible pectrophotometer measures;
The preparation of sample: reaction cumulative volume 160 microlitre, first add 40 microlitre, 4 mM/ls of disodium EDTAs, add 40 microlitre rat liver homogenate liquid (physiological saline solution), add 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-the disulfobenzene)-2H-tetrazole sodium salt of 40 microlitre 50 micromoles per liter, finally add 40 microlitre, 1 mM/l of pyrogallol aqueous solution, 37 DEG C of reactions are after 8 minutes, as sample, the microplate reader for built-in ultraviolet-visible pectrophotometer measures;
The preparation of blank sample: reaction cumulative volume 160 microlitre, first add 40 microlitre, 4 mM/ls of disodium EDTAs, add 40 microlitre rat liver homogenate liquid (physiological saline solution), add 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-the disulfobenzene)-2H-tetrazole sodium salt of 40 microlitre 50 micromoles per liter, finally add 40 microliters of water solution, 37 DEG C of reactions are after 10 minutes, as blank sample, the microplate reader for built-in ultraviolet-visible pectrophotometer measures;
The detection of steps d, reference substance, sample and blank sample
Reference substance detects: with 96 orifice plates for carrier, measure in the microplate reader of built-in ultraviolet-visible pectrophotometer, and setting ultraviolet-visible determined wavelength is 450 nanometers, measures, obtain the absorbance A of reference substance to reference substance prepared by step c
reference substance;
Sample detection: with 96 orifice plates for carrier, measures in the microplate reader of built-in ultraviolet-visible pectrophotometer, and setting ultraviolet-visible determined wavelength is 450 nanometers, measures, obtain the absorbance A of sample to the sample of step c preparation
sample;
Blank sample detects: with 96 orifice plates for carrier, measure in the microplate reader of built-in ultraviolet-visible pectrophotometer, and setting ultraviolet-visible determined wavelength is 450 nanometers, measures, obtain the absorbance A of blank sample to the blank sample of step c preparation
blank sample;
Step e, superoxide dismutase are to the calculating of the clearance rate of superoxide anion
Superoxide dismutase to the clearance rate of superoxide anion according to following formulae discovery:
I
sample=[A
reference substance-(A
sample-A
blank sample)]/A
reference substance× 100%
In formula, I is the clearance rate of superoxide dismutase to superoxide anion;
A
reference substancefor the absorbance of the product water dissolubility formazan of 2-in reference substance (4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt, dimensionless;
A
samplefor the absorbance of the product water dissolubility formazan of 2-in sample (4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt, dimensionless;
A
blank samplefor the absorbance of the product water dissolubility formazan of 2-in blank sample (4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt, dimensionless;
The computing formula of superoxide dismutase activity is as follows:
Superoxide dismutase activity=I/ (1 – I)
According to the absorbance A of the product water dissolubility formazan of 2-in the reference substance recorded, sample and blank sample (4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt
reference substance, A
sampleand A
blank sample, calculate the clearance rate of healthy rat superoxide dismutase from liver to superoxide anion, then quality and extension rate per sample, the activity obtaining healthy rat superoxide dismutase from liver that converts is 19100U/g.
Healthy rat superoxide dismutase from liver is to the clearance rate of superoxide anion, that healthy rat superoxide dismutase from liver can catalysis superoxide anion generation disproportionation reaction, generate oxygen and hydrogen peroxide, thus cause the amount of the superoxide anion reacted with 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt to reduce, cause the amount of the reduzate water-soluble Xing formazan dyestuff of trapping agent 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt to reduce; So the clearance rate of healthy rat superoxide dismutase from liver to superoxide anion is obtained by the change calculations of the product water dissolubility formazan dye content of trapping agent 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt, and then draw the activity of healthy rat superoxide dismutase from liver.
Embodiment 3
A kind of method measuring superoxide dismutase activity provided by the invention, it comprises the following steps:
Step a, superoxide anion produce the preparation of system buffer solution
Compound concentration is the ammonium bicarbonate buffer solution of 45 mM/ls, and adjust ph is 9.0;
The preparation of step b, standard items
With the ammonium bicarbonate buffer solution in step a, standard items are made into the standard solution that concentration is 50 micromoles per liter, described standard items are 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt, and energy and superoxide anion reaction produce water-soluble Xing formazan dyestuff;
The preparation of step c, reference substance, sample and blank sample
The preparation of reference substance: reaction cumulative volume 160 microlitre, first add 40 microlitre, 0.4 mM/l of disodium EDTA, add 40 microlitre physiological saline, add 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-the disulfobenzene)-2H-tetrazole sodium salt of 40 microlitre 50 micromoles per liter, finally add 40 microlitre, 1 mM/l of pyrogallol aqueous solution, 37 DEG C of reactions are after 16 minutes, product in contrast, the microplate reader for built-in ultraviolet-visible pectrophotometer measures;
The preparation of sample: reaction cumulative volume 160 microlitre, first add 40 microlitre, 0.4 mM/l of disodium EDTA, add 40 microlitre rat kidney homogenates (physiological saline solution), add 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-the disulfobenzene)-2H-tetrazole sodium salt of 40 microlitre 50 micromoles per liter, finally add 40 microlitre, 1 mM/l of pyrogallol aqueous solution, 37 DEG C of reactions are after 16 minutes, as sample, the microplate reader for built-in ultraviolet-visible pectrophotometer measures;
The preparation of blank sample: reaction cumulative volume 160 microlitre, first add 40 microlitre, 0.4 mM/l of disodium EDTA, add 40 microlitre rat kidney homogenates (physiological saline solution), add 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-the disulfobenzene)-2H-tetrazole sodium salt of 40 microlitre 50 micromoles per liter, finally add 40 microliters of water solution, 37 DEG C of reactions are after 16 minutes, as blank sample, the microplate reader for built-in ultraviolet-visible pectrophotometer measures;
The detection of steps d, reference substance, sample and blank sample
Reference substance detects: with 96 orifice plates for carrier, measure in the microplate reader of built-in ultraviolet-visible pectrophotometer, and setting ultraviolet-visible determined wavelength is 450 nanometers, measures, obtain the absorbance A of reference substance to reference substance prepared by step c
reference substance;
Sample detection: with 96 orifice plates for carrier, measures in the microplate reader of built-in ultraviolet-visible pectrophotometer, and setting ultraviolet-visible determined wavelength is 450 nanometers, measures, obtain the absorbance A of sample to the sample of step c preparation
sample;
Blank sample detects: with 96 orifice plates for carrier, measure in the microplate reader of built-in ultraviolet-visible pectrophotometer, and setting ultraviolet-visible determined wavelength is 450 nanometers, measures, obtain the absorbance A of blank sample to the blank sample of step c preparation
blank sample;
Step e, superoxide dismutase are to the calculating of the clearance rate of superoxide anion
Superoxide dismutase to the clearance rate of superoxide anion according to following formulae discovery:
I
sample=[A
reference substance-(A
sample-A
blank sample)]/A
reference substance× 100%
In formula, I is the clearance rate of superoxide dismutase to superoxide anion;
A
reference substancefor the absorbance of the product water dissolubility formazan of 2-in reference substance (4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt, dimensionless;
A
samplefor the absorbance of the product water dissolubility formazan of 2-in sample (4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt, dimensionless;
A
blank samplefor the absorbance of the product water dissolubility formazan of 2-in blank sample (4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt, dimensionless;
The computing formula of superoxide dismutase activity is as follows:
Superoxide dismutase activity=I/ (1 – I)
According to the absorbance A of the product water dissolubility formazan of 2-in the reference substance recorded, sample and blank sample (4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt
reference substance, A
sampleand A
blank sample, to calculate in healthy rat kidney superoxide dismutase to the clearance rate of superoxide anion, then quality and extension rate per sample, the activity obtaining superoxide dismutase in healthy rat kidney that converts is 4900U/g.
In healthy rat kidney, superoxide dismutase is to the clearance rate of superoxide anion, that in healthy rat kidney, superoxide dismutase can catalysis superoxide anion generation disproportionation reaction, generate oxygen and hydrogen peroxide, thus cause the amount of the superoxide anion reacted with 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt to reduce, cause the amount of the reduzate water-soluble Xing formazan dyestuff of trapping agent 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt to reduce; So in healthy rat kidney, the clearance rate of superoxide dismutase to superoxide anion is obtained by the change calculations of the product water dissolubility formazan dye content of trapping agent 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt, and then draw the activity of superoxide dismutase in healthy rat kidney.
Embodiment 4
A kind of method measuring superoxide dismutase activity provided by the invention, it comprises the following steps:
Step a, superoxide anion produce the preparation of system buffer solution
Compound concentration is the ammonium bicarbonate buffer solution of 40 mM/ls, and adjust ph is 8.5;
The preparation of step b, standard items
With the ammonium bicarbonate buffer solution in step a, standard items are made into the standard solution that concentration is 50 micromoles per liter, described standard items are 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt, and energy and superoxide anion reaction produce water-soluble Xing formazan dyestuff;
The preparation of step c, reference substance, sample and blank sample
The preparation of reference substance: reaction cumulative volume 160 microlitre, first add 40 microlitre, 8 mM/ls of disodium EDTAs, add 40 microlitre physiological saline, add 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-the disulfobenzene)-2H-tetrazole sodium salt of 40 microlitre 50 micromoles per liter, finally add 40 microlitre, 1 mM/l of pyrogallol aqueous solution, 37 DEG C of reactions are after 20 minutes, product in contrast, the microplate reader for built-in ultraviolet-visible pectrophotometer measures;
The preparation of sample: reaction cumulative volume 160 microlitre, first add 40 microlitre, 4 mM/ls of disodium EDTAs, add 40 microlitre rat blood serums (normal saline dilution), add 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-the disulfobenzene)-2H-tetrazole sodium salt of 40 microlitre 50 micromoles per liter, finally add 40 microlitre, 1 mM/l of pyrogallol aqueous solution, 37 DEG C of reactions are after 20 minutes, as sample, the microplate reader for built-in ultraviolet-visible pectrophotometer measures;
The preparation of blank sample: reaction cumulative volume 160 microlitre, first add 40 microlitre, 4 mM/ls of disodium EDTAs, add 40 microlitre rat blood serums (normal saline dilution), add 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-the disulfobenzene)-2H-tetrazole sodium salt of 40 microlitre 50 micromoles per liter, finally add 40 microliters of water solution, 37 DEG C of reactions are after 20 minutes, as blank sample, the microplate reader for built-in ultraviolet-visible pectrophotometer measures;
The detection of steps d, reference substance, sample and blank sample
Reference substance detects: with 96 orifice plates for carrier, measure in the microplate reader of built-in ultraviolet-visible pectrophotometer, and setting ultraviolet-visible determined wavelength is 450 nanometers, measures, obtain the absorbance A of reference substance to reference substance prepared by step c
reference substance;
Sample detection: with 96 orifice plates for carrier, measures in the microplate reader of built-in ultraviolet-visible pectrophotometer, and setting ultraviolet-visible determined wavelength is 450 nanometers, measures, obtain the absorbance A of sample to the sample of step c preparation
sample;
Blank sample detects: with 96 orifice plates for carrier, measure in the microplate reader of built-in ultraviolet-visible pectrophotometer, and setting ultraviolet-visible determined wavelength is 450 nanometers, measures, obtain the absorbance A of blank sample to the blank sample of step c preparation
blank sample;
Step e, superoxide dismutase are to the calculating of the clearance rate of superoxide anion
Superoxide dismutase to the clearance rate of superoxide anion according to following formulae discovery:
I
sample=[A
reference substance-(A
sample-A
blank sample)]/A
reference substance× 100%
In formula, I is the clearance rate of superoxide dismutase to superoxide anion;
A
reference substancefor the absorbance of the product water dissolubility formazan of 2-in reference substance (4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt, dimensionless;
A
samplefor the absorbance of the product water dissolubility formazan of 2-in sample (4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt, dimensionless;
A
blank samplefor the absorbance of the product water dissolubility formazan of 2-in blank sample (4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt, dimensionless;
The computing formula of superoxide dismutase activity is as follows:
Superoxide dismutase activity=I/ (1 – I)
According to the absorbance A of the product water dissolubility formazan of 2-in the reference substance recorded, sample and blank sample (4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt
reference substance, A
sampleand A
blank sample, to calculate in rat blood serum superoxide dismutase to the clearance rate of superoxide anion, then volume and extension rate per sample, the activity obtaining superoxide dismutase in rat blood serum that converts is 1100U/mL.
In rat blood serum, superoxide dismutase is to the clearance rate of superoxide anion, that in rat blood serum, superoxide dismutase can catalysis superoxide anion generation disproportionation reaction, generate oxygen and hydrogen peroxide, thus cause the amount of the superoxide anion reacted with 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt to reduce, cause the amount of the reduzate water-soluble Xing formazan dyestuff of trapping agent 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt to reduce; So in rat blood serum, the clearance rate of superoxide dismutase to superoxide anion is obtained by the change calculations of the product water dissolubility formazan dye content of trapping agent 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt, and then draw the activity of superoxide dismutase in rat blood serum.
Obviously, above-described embodiment is only for clearly example being described, and the restriction not to embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here exhaustive without the need to also giving all embodiments.And thus the apparent change of extending out or variation be still among the protection domain of the invention.
Claims (5)
1. measure a method for superoxide dismutase activity, the method comprising the steps of a, b, be specially:
Step a, superoxide anion produce the preparation of system buffer solution
Secure ph is the ammonium bicarbonate buffer solution of 8.5 ~ 9.5 30 ~ 50 mM/ls;
The preparation of step b, standard items
With the ammonium bicarbonate buffer solution in step a, standard items are made into the standard solution that concentration is 50 micromoles per liter, described standard items are 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt;
It is characterized in that, the method also comprises step c, d and e, is specially:
The preparation of step c, reference substance, sample and blank sample
The preparation of reference substance: get 40 microlitre, 0.4 ~ 8 mM/l of disodium EDTA, add 40 microlitre physiological saline, add 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-the disulfobenzene)-2H-tetrazole sodium salt of 40 microlitre 50 micromoles per liter, finally add 40 microlitre, 1 mM/l of pyrogallol aqueous solution, 37 DEG C of reactions are after 8 ~ 20 minutes, product in contrast, the microplate reader for built-in ultraviolet-visible pectrophotometer measures;
The preparation of sample: get 40 microlitre, 0.4 ~ 8 mM/l of disodium EDTA, add 40 microlitre superoxide dismutase solution, add 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-the disulfobenzene)-2H-tetrazole sodium salt of 40 microlitre 50 micromoles per liter, finally add 40 microlitre, 1 mM/l of pyrogallol aqueous solution, 37 DEG C of reactions are after 8 ~ 20 minutes, as sample, the microplate reader for built-in ultraviolet-visible pectrophotometer measures;
The preparation of blank sample: get 40 microlitre, 0.4 ~ 8 mM/l of disodium EDTA, add 40 microlitre superoxide dismutase solution, add 2-(4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-the disulfobenzene)-2H-tetrazole sodium salt of 40 microlitre 50 micromoles per liter, finally add 40 microliters of water solution, 37 DEG C of reactions are after 8 ~ 20 minutes, as blank sample, the microplate reader for built-in ultraviolet-visible pectrophotometer measures;
The detection of steps d, reference substance, sample and blank sample
Setting ultraviolet-visible determined wavelength is 450 nanometers, in the microplate reader of built-in ultraviolet-visible pectrophotometer to step c in reference substance, sample and the blank sample prepared measure, obtain the absorbance A of reference substance, sample and blank sample
reference substance, A
sampleand A
blank sample;
The calculating of the activity of step e, superoxide dismutase
Superoxide dismutase to the clearance rate of superoxide anion according to following formulae discovery:
I
sample=[A
reference substance-(A
sample-A
blank sample)]/A
reference substance× 100%
In formula, I
samplefor superoxide dismutase is to the clearance rate of superoxide anion;
A
reference substancefor the absorbance of the product water dissolubility formazan of 2-in reference substance (4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt, dimensionless;
A
samplefor the absorbance of the product water dissolubility formazan of 2-in sample (4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt, dimensionless;
A
blank samplefor the absorbance of the product water dissolubility formazan of 2-in blank sample (4-iodobenzene)-3-(4-nitrobenzene)-5-(2'4-disulfobenzene)-2H-tetrazole sodium salt, dimensionless;
The computing formula of superoxide dismutase activity is as follows:
Superoxide dismutase activity=I
sample/ (1 – I
sample)
In formula, superoxide dismutase activity unit is U.
2. the method measuring superoxide dismutase activity as claimed in claim 1, it is characterized in that, the pH value of buffer solution described in step a is 9.0 ~ 9.5.
3. the method measuring superoxide dismutase activity as claimed in claim 2, it is characterized in that, the pH value of buffer solution described in step a is 9.3.
4. the method measuring superoxide dismutase activity as claimed in claim 1, it is characterized in that, the concentration of the disodium EDTA described in step c is 0.4 ~ 2 mM/l.
5. the method measuring superoxide dismutase activity as claimed in claim 4, it is characterized in that, the concentration of the disodium EDTA described in step c is 2 mM/ls.
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| CN106290346A (en) * | 2016-09-29 | 2017-01-04 | 浙江达美生物技术有限公司 | Mensuration reagent of superoxide dismutase and preparation method thereof |
| CN106770209A (en) * | 2017-01-12 | 2017-05-31 | 南京欣迪生物药业工程有限责任公司 | A kind of refining superoxide dismutase detection kit and its application |
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