CN107796770A - A kind of isocitrate lyase Activity Assay Kit and its method - Google Patents
A kind of isocitrate lyase Activity Assay Kit and its method Download PDFInfo
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Abstract
The invention discloses a kind of isocitrate lyase Activity Assay Kit and its method, include in its kit by Tris HCl cushioning liquid, KCl, MnCl2, the solution that is mixed into of DTT and EDTA, the solution being mixed into by disodium hydrogen phosphate phosphate sodium dihydrogen buffer solution and water, by MgCl2·6H2O, the pulvis that DTT and NADH are mixed into, lactic dehydrogenase and isocitric acid.The measuring principle of this method is that ICL catalysis isocitric acids are degraded to glyoxalic acid and butanedioic acid, and glyoxalic acid and NADH generate ethanol and NAD, NADH in the presence of LDH characteristic absorption peak under 340nm, and the reduction speed of monitoring 340nm absorbances reflects ICL activity indirectly.The present invention is detected the height that NADH contents to reflect indirectly ICL activity under ultraviolet wavelength, can effectively be excluded the interference of coloring matter, substantially increase the degree of accuracy of detection by enzyme coupled action.
Description
Technical field
The invention belongs to life science field, and in particular to a kind of isocitrate lyase Activity Assay Kit and
Its method.
Background technology
Isocitrate lyase（ICL）Be primarily present in plant and microorganism, oil crop seeds in germination process,
Fat is transformed into by carbohydrate by glyoxalic acid circulation and other processes, isocitrate lyase is then that glyoxalic acid circulates
One of key enzyme.
Isocitrate lyase catalysis isocitric acid is degraded to glyoxalic acid and butanedioic acid, can be by determining the change of glyoxalic acid
The height of reflection isocitrate lyase activity indirectly.At present application compared with isocitrate lyase activity determination method be colorimetric
Method, i.e. glyoxalic acid and dinitrophenylhydrazine act on forming glyoxalic acid phenylhydrazine, show brown, the depth and acetaldehyde of color in the basic conditions
Acid content is proportional, calculates the activity of isocitrate lyase indirectly with this.Due to pyruvic acid also can with dinitrophenylhydrazine reaction,
The interference that this method is subject to is larger, false positive be present, and there is an urgent need to develop a kind of new method to detect different lemon in research work
Lemon lyase activity.
The content of the invention
The defects of in order to overcome prior art, the present invention is intended to provide a kind of isocitrate lyase Activity Assay Kit
And its method, it can efficiently and accurately determine the activity of isocitrate lyase.
To realize above-mentioned technical purpose and the technique effect, the present invention is achieved through the following technical solutions：
A kind of isocitrate lyase Activity Assay Kit, including following reagent：
Extract solution, liquid 100mL × 1 bottle, by Tris-HCl cushioning liquid, KCl, MnCl2, DTT and EDTA mix, and adjust
PH to 7.0, it is placed in 100mL volumetric flasks, 4 DEG C of preservations；
Reagent one, liquid 15mL × 1 bottle, mixed by disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution and water, be placed in 15mL examinations
In agent bottle, 4 DEG C of preservations；
Reagent two, pulvis × 1 bottle, by MgCl2·6H2O, DTT and NADH are mixed, and are placed in 1.5mL EP pipes, -20 DEG C of guarantors
Deposit；
Reagent three, μ L × 1 of liquid 360, is made up of lactic dehydrogenase, is placed in 1.5mL EP pipes, 4 DEG C of preservations；
Reagent four, pulvis × 1 bottle, is made up of isocitric acid, is placed in 5mL reagent bottles, 4 DEG C of preservations.
Further, the substance withdrawl syndrome of the Tris-HCl cushioning liquid contained in the extract solution is 100mmol/L
, pH 7.0, volume 100mL, KCl quality are 150mg, MnCl2Quality be 99mg, DTT quality is 31mg, EDTA
Quality be 3mg.
Further, the substance withdrawl syndrome of the disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution contained in the reagent one
For 0.1mmol/L, pH 7.0, volume 3.85mL, the volume of water is 11.15mL.
Further, the MgCl contained in the reagent two2·6H2O quality is 20mg, and DTT quality is 3mg, NADH
Quality be 4.5mg.
Further, the enzyme concentration of the lactic dehydrogenase contained in the reagent three is 500U/mL, volume 360uL.
Further, the quality of the isocitric acid contained in the reagent four is 25.8mg.
A kind of isocitrate lyase activity determination method using mentioned reagent box, based on micromethod, its principle is different
Citrate lyase（ICL）Catalysis isocitric acid is degraded to glyoxalic acid and butanedioic acid, and glyoxalic acid and NADH are raw in the presence of LDH
Into ethanol and NAD, NADH has characteristic absorption peak under 340nm, and the reduction speed of monitoring 340nm absorbances can the different lemon of indirect reaction
The activity of lemon acid cleavage enzyme；
This method comprises the following steps that：
The preparation of step 1 instrument and articles for use；
Prepare ELIASA, desk centrifuge, water-bath, pipettor, micro quartz colorimetric utensil, mortar, ice and distilled water；
The pre-treatment of step 2 sample；
1）Pre-treatment for bacterium or culture cell, concrete operations are as follows：
Bacterium or cell are first collected in centrifuge tube, supernatant is abandoned after centrifugation；Then according to bacterium or cell quantity（104It is individual）：Carry
Liquid is taken to accumulate（mL）For 500 ~ 1000：1 ratio（It is recommended that 5,000,000 bacteriums or cell add 1mL extract solutions）, ultrasonic disruption is thin
Bacterium or cell（Ice bath, power 20% or 200W, ultrasonic 3s, 10s is spaced, repeated 30 times）；Finally using eccentricity 15000g, 4
10min is centrifuged at DEG C, takes supernatant, is put to be measured on ice；
2）Pre-treatment for tissue, concrete operations are as follows：
First according to liver mass（g）：Extracting liquid volume (mL) is 1：5 ~ 10 ratio（It is recommended that weighing about 0.1g tissues, 1mL is added
Extract solution）, ice bath homogenate is carried out, then using eccentricity 15000g, 10min is centrifuged at 4 DEG C, takes supernatant, put to be measured on ice；
ELIASA is preheated more than 30min, adjusting wavelength to 340nm, distilled water zeroing by step 3；
Step 4 sample measures；
1）The configuration of working solution, reagent two and reagent three are transferred in reagent one, are sufficiently mixed dissolving, be placed in 37 DEG C（Lactation
Animal）Or 25 DEG C（Other species）Water-bath 5min；- 20 DEG C of preservations after exhaustless reagent packing, forbid multigelation；
2）4mL distilled water is added in reagent four, is fully dissolved stand-by；- 20 DEG C of preservations after exhaustless reagent packing, forbid anti-
Multiple freeze thawing；
3）10 μ L samples are added in micro quartz colorimetric utensil, working solution and 40 μ L reagents four described in 150 μ L, mix, remember immediately
The light absorption value A2 after light absorption value A1 and 2min20s when recording 20s at 340nm, calculates Δ A=A1-A2；
Step 5 isocitrate lyase activity calculates；
1）Calculated by sample protein concentration：
ICL（nmol/min/mg prot）=[ΔA×VIt is anti-total÷（ε×d）×109]÷（VSample×Cpr）÷T = 3216×ΔA÷
Cpr；
ICL（nmol/min/mg prot）The definition of unit is the NADH definition per 1 nmol of consumption per minute in mg histones
For an enzyme activity unit；
2）Calculated by sample fresh weight：
ICL（Nmol/min/g fresh weights）=[ΔA×VIt is anti-total÷（ε×d）×109]÷（W×VSample÷VSample is total）÷T = 3216×ΔA
÷W；
ICL（Nmol/min/g fresh weights）The definition of unit：1 nmol of consumption per minute NADH is organized to be defined as an enzyme per g
Unit of activity；
3）Calculated by bacterium or cell density：
ICL（nmol/min/104cell）= [ΔA×VIt is anti-total÷（ε×d）×109]÷（500×VSample÷VSample is total）÷T = 6.25
×ΔA；
ICL（nmol/min/104cell）The definition of unit：1 nmol of every 10,000 bacteriums or cell consumption per minute NADH
It is defined as an enzyme activity unit；
Wherein, VIt is anti-totalRepresent reaction system cumulative volume, and VIt is anti-total=2×10-4 L；
ε represents NADH molar extinction coefficients, and ε=6.22 × 103L/mol/cm；
D represents micro quartz colorimetric utensil optical path, and d=0.5cm；
VSampleRepresent to add sample volume, and VSample=0.01mL；
VSample is totalRepresent the volume of addition extract solution, and VSample is total=1mL；
T represents the reaction time, the min of and T=2；
Cpr represents the concentration of sample protein, and its unit is mg/mL；
W represents the quality of sample, and its unit is g；
500 represent bacterium or TCS, represent 5,000,000.
The beneficial effects of the invention are as follows：
1st, the measuring principle of the inventive method is that ICL catalysis isocitric acids are degraded to glyoxalic acid and butanedioic acid, glyoxalic acid and NADH
Ethanol and NAD, NADH are generated in the presence of LDH characteristic absorption peak under 340nm, monitors the reduction speed of 340nm absorbances
Rate reflects ICL activity indirectly.At present on the market also without a kind of enzyme micro ICL detection kits of coupling ultraviolet spectrophotometry and
Its detection method, this method detect the height that NADH contents to reflect indirectly ICL activity by enzyme coupled action under ultraviolet wavelength
It is low, the interference of coloring matter can be effectively excluded, substantially increases the degree of accuracy of detection.
2nd, method of the invention can complete the survey of a sample by simply preparing working solution and reagent 4 in 3 minutes
It is fixed, substantially increase detection efficiency.
3rd, reaction system of the invention is 200 μ L, compared to the 3mL reaction systems of AAS, greatly reduces examination
Agent dosage, cost are extremely low.
4th, preferably, the coefficient of variation (CV values) is within 2% for method of the invention repeatability.
Described above is only the general introduction of technical solution of the present invention, in order to better understand the technological means of the present invention,
And can be practiced according to the content of specification, described in detail below with presently preferred embodiments of the present invention.The specific reality of the present invention
Mode is applied to be shown in detail by following examples.
Embodiment
Below in conjunction with embodiment, to describe the present invention in detail.
A kind of isocitrate lyase Activity Assay Kit, including following reagent：
Extract solution, liquid 100mL × 1 bottle, by Tris-HCl cushioning liquid, KCl, MnCl2, DTT and EDTA mix, and adjust
PH to 7.0, it is placed in 100mL volumetric flasks, 4 DEG C of preservations；
Reagent one, liquid 15mL × 1 bottle, mixed by disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution and water, be placed in 15mL examinations
In agent bottle, 4 DEG C of preservations；
Reagent two, pulvis × 1 bottle, by MgCl2·6H2O, DTT and NADH are mixed, and are placed in 1.5mL EP pipes, -20 DEG C of guarantors
Deposit；
Reagent three, μ L × 1 of liquid 360, is made up of lactic dehydrogenase, is placed in 1.5mL EP pipes, 4 DEG C of preservations；
Reagent four, pulvis × 1 bottle, is made up of isocitric acid, is placed in 5mL reagent bottles, 4 DEG C of preservations.
Further, the substance withdrawl syndrome of the Tris-HCl cushioning liquid contained in the extract solution is 100mmol/L
, pH 7.0, volume 100mL, KCl quality are 150mg, MnCl2Quality be 99mg, DTT quality is 31mg, EDTA
Quality be 3mg.
Further, the substance withdrawl syndrome of the disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution contained in the reagent one
For 0.1mmol/L, pH 7.0, volume 3.85mL, the volume of water is 11.15mL.
Further, the MgCl contained in the reagent two2·6H2O quality is 20mg, and DTT quality is 3mg, NADH
Quality be 4.5mg.
Further, the enzyme concentration of the lactic dehydrogenase contained in the reagent three is 500U/mL, volume 360uL.
Further, the quality of the isocitric acid contained in the reagent four is 25.8mg.
A kind of isocitrate lyase activity determination method using mentioned reagent box, based on micromethod, its principle is different
Citrate lyase（ICL）Catalysis isocitric acid is degraded to glyoxalic acid and butanedioic acid, and glyoxalic acid and NADH are raw in the presence of LDH
Into ethanol and NAD, NADH has characteristic absorption peak under 340nm, and the reduction speed of monitoring 340nm absorbances can the different lemon of indirect reaction
The activity of lemon acid cleavage enzyme；
This method comprises the following steps that：
The preparation of step 1 instrument and articles for use；
Prepare ELIASA, desk centrifuge, water-bath, pipettor, micro quartz colorimetric utensil, mortar, ice and distilled water；
The pre-treatment of step 2 sample；
1）Pre-treatment for bacterium or culture cell, concrete operations are as follows：
Bacterium or cell are first collected in centrifuge tube, supernatant is abandoned after centrifugation；Then according to bacterium or cell quantity（104It is individual）：Carry
Liquid is taken to accumulate（mL）For 500 ~ 1000：1 ratio（It is recommended that 5,000,000 bacteriums or cell add 1mL extract solutions）, ultrasonic disruption is thin
Bacterium or cell（Ice bath, power 20% or 200W, ultrasonic 3s, 10s is spaced, repeated 30 times）；Finally using eccentricity 15000g, 4
10min is centrifuged at DEG C, takes supernatant, is put to be measured on ice；
2）Pre-treatment for tissue, concrete operations are as follows：
First according to liver mass（g）：Extracting liquid volume (mL) is 1：5 ~ 10 ratio（It is recommended that weighing about 0.1g tissues, 1mL is added
Extract solution）, ice bath homogenate is carried out, then using eccentricity 15000g, 10min is centrifuged at 4 DEG C, takes supernatant, put to be measured on ice；
ELIASA is preheated more than 30min, adjusting wavelength to 340nm, distilled water zeroing by step 3；
Step 4 sample measures；
1）The configuration of working solution, reagent two and reagent three are transferred in reagent one, are sufficiently mixed dissolving, be placed in 37 DEG C（Lactation
Animal）Or 25 DEG C（Other species）Water-bath 5min；- 20 DEG C of preservations after exhaustless reagent packing, forbid multigelation；
2）4mL distilled water is added in reagent four, is fully dissolved stand-by；- 20 DEG C of preservations after exhaustless reagent packing, forbid anti-
Multiple freeze thawing；
3）10 μ L samples are added in micro quartz colorimetric utensil, working solution and 40 μ L reagents four described in 150 μ L, mix, remember immediately
The light absorption value A2 after light absorption value A1 and 2min20s when recording 20s at 340nm, calculates Δ A=A1-A2；
Step 5 isocitrate lyase activity calculates；
1）Calculated by sample protein concentration：
ICL（nmol/min/mg prot）=[ΔA×VIt is anti-total÷（ε×d）×109]÷（VSample×Cpr）÷T = 3216×ΔA÷
Cpr；
ICL（nmol/min/mg prot）The definition of unit is the NADH definition per 1 nmol of consumption per minute in mg histones
For an enzyme activity unit；
2）Calculated by sample fresh weight：
ICL（Nmol/min/g fresh weights）=[ΔA×VIt is anti-total÷（ε×d）×109]÷（W×VSample÷VSample is total）÷T = 3216×ΔA
÷W；
ICL（Nmol/min/g fresh weights）The definition of unit：1 nmol of consumption per minute NADH is organized to be defined as an enzyme per g
Unit of activity；
3）Calculated by bacterium or cell density：
ICL（nmol/min/104cell）= [ΔA×VIt is anti-total÷（ε×d）×109]÷（500×VSample÷VSample is total）÷T = 6.25
×ΔA；
ICL（nmol/min/104cell）The definition of unit：1 nmol of every 10,000 bacteriums or cell consumption per minute NADH
It is defined as an enzyme activity unit；
Wherein, VIt is anti-totalRepresent reaction system cumulative volume, and VIt is anti-total=2×10-4 L；
ε represents NADH molar extinction coefficients, and ε=6.22 × 103L/mol/cm；
D represents 96 orifice plate optical paths, and d=0.5cm；
VSampleRepresent to add sample volume, and VSample=0.01mL；
VSample is totalRepresent the volume of addition extract solution, and VSample is total=1mL；
T represents the reaction time, the min of and T=2；
Cpr represents the concentration of sample protein, and its unit is mg/mL；
W represents the quality of sample, and its unit is g；
500 represent bacterium or TCS, represent 5,000,000.
Above-described embodiment is in the art the purpose is to be to allow simply to illustrate that the technical concepts and features of the present invention
Those of ordinary skill can understand present disclosure and implement according to this, and it is not intended to limit the scope of the present invention.It is all
It is the equivalent change or modification according to made by the essence of present invention, should all covers within the scope of the present invention.
Claims (8)
1. a kind of isocitrate lyase Activity Assay Kit, it is characterised in that including following reagent：
Extract solution, liquid 100mL × 1 bottle, by Tris-HCl cushioning liquid, KCl, MnCl2, DTT and EDTA mix, and adjust
PH to 7.0, it is placed in 100mL volumetric flasks, 4 DEG C of preservations；
Reagent one, liquid 15mL × 1 bottle, mixed by disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution and water, be placed in 15mL examinations
In agent bottle, 4 DEG C of preservations；
Reagent two, pulvis × 1 bottle, by MgCl2·6H2O, DTT and NADH are mixed, and are placed in 1.5mL EP pipes, -20 DEG C of guarantors
Deposit；
Reagent three, μ L × 1 of liquid 360, is made up of lactic dehydrogenase, is placed in 1.5mL EP pipes, 4 DEG C of preservations；
Reagent four, pulvis × 1 bottle, is made up of isocitric acid, is placed in 5mL reagent bottles, 4 DEG C of preservations.
2. isocitrate lyase Activity Assay Kit according to claim 1, it is characterised in that：In the extract solution
The substance withdrawl syndrome of the Tris-HCl cushioning liquid contained is 100mmol/L, pH 7.0, volume 100mL, KCl matter
Measure as 150mg, MnCl2Quality be 99mg, DTT quality is 31mg, and EDTA quality is 3mg.
3. isocitrate lyase Activity Assay Kit according to claim 1, it is characterised in that：In the reagent one
The substance withdrawl syndrome of the disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution contained is 0.1mmol/L, pH 7.0, and volume is
3.85mL, the volume of water is 11.15mL.
4. isocitrate lyase Activity Assay Kit according to claim 1, it is characterised in that：In the reagent two
The MgCl contained2·6H2O quality is 20mg, and DTT quality is 3mg, and NADH quality is 4.5mg.
5. isocitrate lyase Activity Assay Kit according to claim 1, it is characterised in that：In the reagent three
The enzyme concentration of the lactic dehydrogenase contained is 500U/mL, volume 360uL.
6. isocitrate lyase Activity Assay Kit according to claim 1, it is characterised in that：In the reagent four
The quality of the isocitric acid contained is 25.8mg.
A kind of 7. isocitrate lyase activity determination method using kit as claimed in claim 1, it is characterised in that
Based on micromethod, comprise the following steps that：
The preparation of step 1 instrument and articles for use；
Prepare ELIASA, desk centrifuge, water-bath, pipettor, micro quartz colorimetric utensil, mortar, ice and distilled water；
The pre-treatment of step 2 sample；
Pre-treatment for bacterium or culture cell, concrete operations are as follows：
Bacterium or cell are first collected in centrifuge tube, supernatant is abandoned after centrifugation；Then according to bacterium or cell quantity（104It is individual）：Extraction
Liquid accumulates（mL）For 500 ~ 1000：1 ratio, ultrasonic disruption bacterium or cell；Finally using eccentricity 15000g, at 4 DEG C
10min is centrifuged, supernatant is taken, puts to be measured on ice；
Pre-treatment for tissue, concrete operations are as follows：
First according to liver mass（g）：Extracting liquid volume (mL) is 1：5 ~ 10 ratio, ice bath homogenate is carried out, then using centrifugation
Rate 15000g, 10min is centrifuged at 4 DEG C, supernatant is taken, puts to be measured on ice；
ELIASA is preheated more than 30min, adjusting wavelength to 340nm, distilled water zeroing by step 3；
Step 4 sample measures；
1）The configuration of working solution, reagent two and reagent three are transferred in reagent one, are sufficiently mixed dissolving, be placed in 37 DEG C or 25 DEG C
Water-bath 5min；
2）4mL distilled water is added in reagent four, is fully dissolved stand-by；3）Added in micro quartz colorimetric utensil 10 μ L samples,
Working solution described in 150 μ L and 40 μ L reagents four, mix, after the light absorption value A1 and 2min20s at immediate record 340nm during 20s
Light absorption value A2, calculate Δ A=A1-A2；
Step 5 isocitrate lyase activity calculates；
1）Calculated by sample protein concentration：
ICL（nmol/min/mg prot）=[ΔA×VIt is anti-total÷（ε×d）×109]÷（VSample×Cpr）÷T = 3216×ΔA÷
Cpr；
2）Calculated by sample fresh weight：
ICL（Nmol/min/g fresh weights）=[ΔA×VIt is anti-total÷（ε×d）×109]÷（W×VSample÷VSample is total）÷T = 3216×ΔA
÷W；
3）Calculated by bacterium or cell density：
ICL（nmol/min/104cell）= [ΔA×VIt is anti-total÷（ε×d）×109]÷（500×VSample÷VSample is total）÷T = 6.25
×ΔA；
Wherein, VIt is anti-totalRepresent reaction system cumulative volume, and VIt is anti-total=2×10-4 L；
ε represents NADH molar extinction coefficients, and ε=6.22 × 103L/mol/cm；
D represents micro quartz colorimetric utensil optical path, and d=0.5cm；
VSampleRepresent to add sample volume, and VSample=0.01mL；
VSample is totalRepresent the volume of addition extract solution, and VSample is total=1mL；
T represents the reaction time, the min of and T=2；
Cpr represents the concentration of sample protein, and its unit is mg/mL；
W represents the quality of sample, and its unit is g；
500 represent bacterium or TCS, represent 5,000,000.
8. isocitrate lyase activity determination method according to claim 7, it is characterised in that：The 1 of step 2）In, institute
The ultrasonic disruption bacterium stated or the method for cell are first ice bath, then using power 20% or 200W, ultrasonic 3s, are spaced 10s,
Repeat 30 times.
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| CN109211817A (en) * | 2018-09-06 | 2019-01-15 | 苏州科铭生物技术有限公司 | A kind of CoA contents assay kit and its method based on micromethod |
| CN109324006A (en) * | 2018-11-12 | 2019-02-12 | 苏州科铭生物技术有限公司 | 1,3 diphospho glycerate acid content assay kit and its method based on micromethod |
| CN109541052A (en) * | 2018-11-12 | 2019-03-29 | 苏州科铭生物技术有限公司 | A kind of spermidine synthase activity assay kit and its method |
| CN110779887A (en) * | 2019-09-30 | 2020-02-11 | 浙江大学 | Method for determining phosphoglycerate kinase activity |
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