CN107727589A - A kind of Activity Assay Kit of transhydrogenase 1 and its method - Google Patents
A kind of Activity Assay Kit of transhydrogenase 1 and its method Download PDFInfo
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Abstract
The invention discloses a kind of Activity Assay Kit of transhydrogenase 1 and its method, the solution being mixed into by Tris HCl cushioning liquid, sucrose and EDTA, Tris HCl cushioning liquid, NADH and AcPyADP are included in its kit.The measuring principle of the method for the present invention is the manually synthesis acetylpyridine adenine-dinucleotide phosphoric acid of substrate 3（APADP+）Substitute NADP+, APADP is catalyzed by determining TH 1+The APADPH of reduction generation is advanced the speed in 375nm light absorbs to calculate the activity of TH 1, can effectively exclude NAD interference, and detection selectivity is high.The present invention only need to add two kinds of reagents of sample and working solution in microcolorimetric ware or 96 orifice plates and can complete to determine by simply preparing working solution, detect and its conveniently.The present invention can efficiently separate plasmosin and mitochondrial protein, and Accurate Determining is located at the TH 1 of mitochondrial inner membrane.
Description
Technical field
The invention belongs to life science field, and in particular to a kind of Activity Assay Kit of transhydrogenase -1 and its side
Method.
Background technology
Transhydrogenase（TH）On the inner membrance of mitochondria, also known as respiratory electron transport chain complex six, NADH+ is catalyzed
NADP+And NAD++ NADPH is mutually converted.Catalysis forward reaction is referred to as transhydrogenase -1（TH-1）.During mitochondria NADH content increases
The H of mitochondrial membrane can be caused+Electrochemical gradient raises, thus promotes the generation of ROS on electron transport chain.Transhydrogenase -1（TH-
1）NADH is promoted to be converted to NADPH, so as to improve the oxidation resistance of mitochondria.
NADH and NADPH has feature light absorbs, therefore transhydrogenase under 340nm wavelength（TH）Catalysis turns hydrogen reaction not
340nm absorbances can be caused to change.Manually synthesize substrate 3- acetylpyridine adenine-dinucleotide phosphoric acid（APADP+）
Substitute NADP+, transhydrogenase -1（TH-1）It is catalyzed APADP+The APADPH of reduction generation has feature light absorbs under 375nm wavelength,
Therefore advanced the speed by determining 375nm light absorbs, transhydrogenase -1 can be calculated（TH-1）Activity.At present on the market also without one
The effective detection transhydrogenase -1 of kind（TH-1）The method of 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 Activity Assay Kit of transhydrogenase -1 and its side
Method, can effective detection transhydrogenase -1 activity.
To realize above-mentioned technical purpose and the technique effect, the present invention is achieved through the following technical solutions：
A kind of Activity Assay Kit of transhydrogenase -1, including following reagent：
Reagent one, liquid 100mL × 1 bottle, mixed by Tris-HCl cushioning liquid, sucrose and EDTA, be placed in 100mL capacity
In bottle, -20 DEG C of preservations；
Reagent two, liquid 50mL × 1 bottle, it is made up of, is placed in 50mL volumetric flasks Tris-HCl cushioning liquid, -20 DEG C of preservations；
Reagent three, liquid 18mL × 1 bottle, it is made up of, is placed in 20mL reagent bottles Tris-HCl cushioning liquid, 4 DEG C of preservations；
Reagent four, pulvis × 1, is made up of NADH, is placed in 1.5mL EP pipes, -20 DEG C of preservations；
Reagent five, pulvis × 1, is made up of AcPyADP, is placed in 1.5mL EP pipes, -20 DEG C of preservations.
Further, the substance withdrawl syndrome of the Tris-HCl cushioning liquid contained in the reagent one is 10mM, and pH is
7.5, volume 100mL, the quality of sucrose is 8.56mg, and EDTA quality is 0.44g.
Further, the substance withdrawl syndrome of the Tris-HCl cushioning liquid contained in the reagent two is 10mM, and pH is
7.5, volume 50mL.
Further, the substance withdrawl syndrome of the Tris-HCl cushioning liquid contained in the reagent three is 1mM, and pH is
7.5, volume 18mL.
Further, the NADH contained in the reagent four quality is 0.1mg.
Further, the AcPyADP contained in the reagent five quality is 0.1mg.
A kind of activity determination method of transhydrogenase -1 using mentioned reagent box, based on micromethod, its principle be NADH and
NADPH has feature light absorbs, therefore transhydrogenase under 340nm wavelength（TH）The hydrogen that turns of catalysis reacts and 340nm can not be caused to inhale
Luminosity changes；Using artificial synthesized substrate 3- acetylpyridines adenine-dinucleotide phosphoric acid（APADP+）Substitute NADP+, turn
Hydrogen enzyme -1（TH-1）It is catalyzed APADP+The APADPH of reduction generation has feature light absorbs under 375nm wavelength, therefore passes through measure
375nm light absorbs are advanced the speed, to calculate transhydrogenase -1（TH-1）Activity；
This method comprises the following steps that：
The preparation of step 1 instrument and articles for use；
Prepare visible spectrophotometer or ELIASA, desk centrifuge, water-bath, adjustable pipette, micro quartz colorimetric utensil
Or 96 orifice plates, mortar, ice and distilled water；
The pre-treatment of step 2 sample, the separation of plasmosin and mitochondrial protein in tissue, bacterium or cell；
1）Weigh 0.1g tissues or collect 5,000,000 cells, add 1mL reagents one, be homogenized with ice bathing homogenizer or mortar；
2）Homogenate is transferred in a centrifuge tube, 5min is centrifuged at eccentricity 600g, 4 DEG C；
3）Precipitation is abandoned, supernatant is moved in another centrifuge tube, 10min is centrifuged at eccentricity 11100g, 4 DEG C；
4）Supernatant is the plasmosin for removing mitochondria, the transhydrogenase -1 available for measure from mitochondria leakage（TH-1）
（This walks optional do）；
5）Precipitation in previous step is mitochondria, adds 500uL reagents two, ultrasonic disruption（Ice bath, power 20% or
200W, ultrasonic 3s, it is spaced 10 seconds, repeats 30 times）, for transhydrogenase -1（TH-1）Determination of activity；
ELIASA is preheated more than 30min, adjusting wavelength to 375nm, distilled water zeroing by step 3；
Step 4 sample measures；
1）The preparation of working solution, reagent four and reagent five are transferred to mixed dissolution in reagent three, are placed in 37 DEG C（Mammal）
Or 25 DEG C（Other species）Water-bath 5min；- 20 DEG C of preservations after exhaustless reagent packing, forbid multigelation；
2）Working solution described in 20 μ L samples and 180 μ L is added in micro quartz colorimetric utensil or 96 orifice plates, is mixed, immediate record
Light absorption value A2 at 375nm after initial light absorption value A1 and 10min, calculate Δ A=A2-A1；
The activity of step 5 transhydrogenase -1 calculates；
A. the calculation formula determined with micro quartz colorimetric utensil is as follows：
1）Calculated by sample protein concentration；
TH-1 activity（nmol/min/mg prot）= [ΔA×VIt is anti-total÷（ε×d）×109]÷（VSample×Cpr）÷T = 149×
ΔA÷Cpr；
TH-1 activity（nmol/min/mg prot）The definition of unit：Per 1 nmol APADPH of mg histones generation per minute
It is defined as a unit of enzyme activity；
2）Calculated by sample fresh weight；
TH-1 activity（Nmol/min/g fresh weights）= [ΔA×VIt is anti-total÷（ε×d）×109]÷（W×VSample÷VSample is total）÷T =
74.5×ΔA÷W；
TH-1 activity（Nmol/min/g fresh weights）The definition of unit：1 nmol APADPH of generation per minute are organized to be defined as per g
One unit of enzyme activity；
3）Calculated by bacterium or cell density
TH-1 activity（nmol/min/104cell）= [ΔA×VIt is anti-total÷（ε×d）×109]÷（500×VSample÷VSample is total）÷T =
0.149×ΔA；
TH-1 activity（nmol/min/104cell）The definition of unit：1 nmol of every 10,000 bacteriums or cell generation per minute
APADPH is defined as a unit of enzyme activity；
Wherein, VIt is anti-totalRepresent reaction system cumulative volume, and VIt is anti-total=2×10-4 L；
ε represents APADPH molar extinction coefficients, and ε=6.7 × 103L/mol/cm；
D represents micro quartz colorimetric utensil optical path, and d=1cm；
VSampleRepresent to add sample volume, and VSample=0.02 mL；
VSample is totalRepresent the volume of addition extract solution, and VSample is total=0.5 mL；
T represents the reaction time, the min of and T=10；
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；
B. the calculation formula determined with 96 orifice plates is as follows：
1）Calculated by sample protein concentration；
TH-1 activity（nmol/min/mg prot）= [ΔA×VIt is anti-total÷（ε×d）×109]÷（VSample×Cpr）÷T = 298×
ΔA÷Cpr；
TH-1 activity（nmol/min/mg prot）The definition of unit：Per 1 nmol APADPH of mg histones generation per minute
It is defined as a unit of enzyme activity；
2）Calculated by sample fresh weight；
TH-1 activity（Nmol/min/g fresh weights）= [ΔA×VIt is anti-total÷（ε×d）×109]÷（W×VSample÷VSample is total）÷T = 149
×ΔA÷W；
TH-1 activity（Nmol/min/g fresh weights）The definition of unit：1 nmol APADPH of generation per minute are organized to be defined as per g
One unit of enzyme activity；
3）Calculated by bacterium or cell density；
TH-1 activity（nmol/min/104cell）= [ΔA×VIt is anti-total÷（ε×d）×109]÷（500×VSample÷VSample is total）÷T =
0.298×ΔA；
TH-1 activity（nmol/min/104cell）The definition of unit：Every 10,000 bacteriums or cell generation 1nmol per minute
APADPH is defined as a unit of enzyme activity；
Wherein, VIt is anti-totalRepresent reaction system cumulative volume, and VIt is anti-total=2×10-4 L；
ε represents APADPH molar extinction coefficients, and ε=6.7 × 103L/mol/cm；
D represents 96 orifice plate optical paths, and d=0.5cm；
VSampleRepresent to add sample volume, and VSample=0.02 mL；
VSample is totalRepresent the volume of addition extract solution, and VSample is total=0.5 mL；
T represents the reaction time, the min of and T=10；
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 manually synthesis substrate 3- acetylpyridine adenine-dinucleotide phosphoric acid（APADP+）Substitute NADP+, APADP is catalyzed by determining TH-1+The APADPH of reduction generation is advanced the speed in 375nm light absorbs to calculate
TH-1 activity, can effectively exclude NAD interference, and detection selectivity is high.
2nd, method of the invention can efficiently separate plasmosin and mitochondrial protein, and Accurate Determining is located at mitochondrial inner membrane
TH-1.
3rd, method of the invention is by simply preparing working solution, need to only be added in microcolorimetric ware or 96 orifice plates sample with
Two kinds of reagents of working solution can be completed to determine, and detect and its conveniently.
4th, 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.
5th, 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 Activity Assay Kit of transhydrogenase -1, including following reagent：
Reagent one, liquid 100mL × 1 bottle, mixed by Tris-HCl cushioning liquid, sucrose and EDTA, be placed in 100mL capacity
In bottle, -20 DEG C of preservations；
Reagent two, liquid 50mL × 1 bottle, it is made up of, is placed in 50mL volumetric flasks Tris-HCl cushioning liquid, -20 DEG C of preservations；
Reagent three, liquid 18mL × 1 bottle, it is made up of, is placed in 20mL reagent bottles Tris-HCl cushioning liquid, 4 DEG C of preservations；
Reagent four, pulvis × 1, is made up of NADH, is placed in 1.5mL EP pipes, -20 DEG C of preservations；
Reagent five, pulvis × 1, is made up of AcPyADP, is placed in 1.5mL EP pipes, -20 DEG C of preservations.
Further, the substance withdrawl syndrome of the Tris-HCl cushioning liquid contained in the reagent one is 10mM, and pH is
7.5, volume 100mL, the quality of sucrose is 8.56mg, and EDTA quality is 0.44g.
Further, the substance withdrawl syndrome of the Tris-HCl cushioning liquid contained in the reagent two is 10mM, and pH is
7.5, volume 50mL.
Further, the substance withdrawl syndrome of the Tris-HCl cushioning liquid contained in the reagent three is 1mM, and pH is
7.5, volume 18mL.
Further, the NADH contained in the reagent four quality is 0.1mg.
Further, the AcPyADP contained in the reagent five quality is 0.1mg.
A kind of activity determination method of transhydrogenase -1 using mentioned reagent box, based on micromethod, its principle be NADH and
NADPH has feature light absorbs, therefore transhydrogenase under 340nm wavelength（TH）The hydrogen that turns of catalysis reacts and 340nm can not be caused to inhale
Luminosity changes；Using artificial synthesized substrate 3- acetylpyridines adenine-dinucleotide phosphoric acid（APADP+）Substitute NADP+, turn
Hydrogen enzyme -1（TH-1）It is catalyzed APADP+The APADPH of reduction generation has feature light absorbs under 375nm wavelength, therefore passes through measure
375nm light absorbs are advanced the speed, to calculate transhydrogenase -1（TH-1）Activity；
This method comprises the following steps that：
The preparation of step 1 instrument and articles for use；
Prepare visible spectrophotometer or ELIASA, desk centrifuge, water-bath, adjustable pipette, micro quartz colorimetric utensil
Or 96 orifice plates, mortar, ice and distilled water；
The pre-treatment of step 2 sample, the separation of plasmosin and mitochondrial protein in tissue, bacterium or cell；
1）Weigh 0.1g tissues or collect 5,000,000 cells, add 1mL reagents one, be homogenized with ice bathing homogenizer or mortar；
2）Homogenate is transferred in a centrifuge tube, 5min is centrifuged at eccentricity 600g, 4 DEG C；
3）Precipitation is abandoned, supernatant is moved in another centrifuge tube, 10min is centrifuged at eccentricity 11100g, 4 DEG C；
4）Supernatant is the plasmosin for removing mitochondria, the transhydrogenase -1 available for measure from mitochondria leakage（TH-1）
（This walks optional do）；
5）Precipitation in previous step is mitochondria, adds 500uL reagents two, ultrasonic disruption（Ice bath, power 20% or
200W, ultrasonic 3s, it is spaced 10 seconds, repeats 30 times）, for transhydrogenase -1（TH-1）Determination of activity；
ELIASA is preheated more than 30min, adjusting wavelength to 375nm, distilled water zeroing by step 3；
Step 4 sample measures；
1）The preparation of working solution, reagent four and reagent five are transferred to mixed dissolution in reagent three, are placed in 37 DEG C（Mammal）
Or 25 DEG C（Other species）Water-bath 5min；- 20 DEG C of preservations after exhaustless reagent packing, forbid multigelation；
2）Working solution described in 20 μ L samples and 180 μ L is added in micro quartz colorimetric utensil or 96 orifice plates, is mixed, immediate record
Light absorption value A2 at 375nm after initial light absorption value A1 and 10min, calculate Δ A=A2-A1；
The activity of step 5 transhydrogenase -1 calculates；
A. the calculation formula determined with micro quartz colorimetric utensil is as follows：
1）Calculated by sample protein concentration；
TH-1 activity（nmol/min/mg prot）= [ΔA×VIt is anti-total÷（ε×d）×109]÷（VSample×Cpr）÷T = 149×
ΔA÷Cpr；
TH-1 activity（nmol/min/mg prot）The definition of unit：Per 1 nmol APADPH of mg histones generation per minute
It is defined as a unit of enzyme activity；
2）Calculated by sample fresh weight；
TH-1 activity（Nmol/min/g fresh weights）= [ΔA×VIt is anti-total÷（ε×d）×109]÷（W×VSample÷VSample is total）÷T =
74.5×ΔA÷W；
TH-1 activity（Nmol/min/g fresh weights）The definition of unit：1 nmol APADPH of generation per minute are organized to be defined as per g
One unit of enzyme activity；
3）Calculated by bacterium or cell density
TH-1 activity（nmol/min/104cell）= [ΔA×VIt is anti-total÷（ε×d）×109]÷（500×VSample÷VSample is total）÷T =
0.149×ΔA；
TH-1 activity（nmol/min/104cell）The definition of unit：1 nmol of every 10,000 bacteriums or cell generation per minute
APADPH is defined as a unit of enzyme activity；
Wherein, VIt is anti-totalRepresent reaction system cumulative volume, and VIt is anti-total=2×10-4 L；
ε represents APADPH molar extinction coefficients, and ε=6.7 × 103L/mol/cm；
D represents micro quartz colorimetric utensil optical path, and d=1cm；
VSampleRepresent to add sample volume, and VSample=0.02 mL；
VSample is totalRepresent the volume of addition extract solution, and VSample is total=0.5 mL；
T represents the reaction time, the min of and T=10；
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；
B. the calculation formula determined with 96 orifice plates is as follows：
1）Calculated by sample protein concentration；
TH-1 activity（nmol/min/mg prot）= [ΔA×VIt is anti-total÷（ε×d）×109]÷（VSample×Cpr）÷T = 298×
ΔA÷Cpr；
TH-1 activity（nmol/min/mg prot）The definition of unit：Per 1 nmol APADPH of mg histones generation per minute
It is defined as a unit of enzyme activity；
2）Calculated by sample fresh weight；
TH-1 activity（Nmol/min/g fresh weights）= [ΔA×VIt is anti-total÷（ε×d）×109]÷（W×VSample÷VSample is total）÷T = 149
×ΔA÷W；
TH-1 activity（Nmol/min/g fresh weights）The definition of unit：1 nmol APADPH of generation per minute are organized to be defined as per g
One unit of enzyme activity；
3）Calculated by bacterium or cell density；
TH-1 activity（nmol/min/104cell）= [ΔA×VIt is anti-total÷（ε×d）×109]÷（500×VSample÷VSample is total）÷T =
0.298×ΔA；
TH-1 activity（nmol/min/104cell）The definition of unit：Every 10,000 bacteriums or cell generation 1nmol per minute
APADPH is defined as a unit of enzyme activity；
Wherein, VIt is anti-totalRepresent reaction system cumulative volume, and VIt is anti-total=2×10-4 L；
ε represents APADPH molar extinction coefficients, and ε=6.7 × 103L/mol/cm；
D represents 96 orifice plate optical paths, and d=0.5cm；
VSampleRepresent to add sample volume, and VSample=0.02 mL；
VSample is totalRepresent the volume of addition extract solution, and VSample is total=0.5 mL；
T represents the reaction time, the min of and T=10；
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 Activity Assay Kit of transhydrogenase -1, it is characterised in that including following reagent：
Reagent one, liquid 100mL × 1 bottle, mixed by Tris-HCl cushioning liquid, sucrose and EDTA, be placed in 100mL capacity
In bottle, -20 DEG C of preservations；
Reagent two, liquid 50mL × 1 bottle, it is made up of, is placed in 50mL volumetric flasks Tris-HCl cushioning liquid, -20 DEG C of preservations；
Reagent three, liquid 18mL × 1 bottle, it is made up of, is placed in 20mL reagent bottles Tris-HCl cushioning liquid, 4 DEG C of preservations；
Reagent four, pulvis × 1, is made up of NADH, is placed in 1.5mL EP pipes, -20 DEG C of preservations；
Reagent five, pulvis × 1, is made up of AcPyADP, is placed in 1.5mL EP pipes, -20 DEG C of preservations.
2. the Activity Assay Kit of transhydrogenase -1 according to claim 1, it is characterised in that：Contain in the reagent one
The substance withdrawl syndrome of Tris-HCl cushioning liquid is 10mM, pH 7.5, volume 100mL, and the quality of sucrose is 8.56mg,
EDTA quality is 0.44g.
3. the Activity Assay Kit of transhydrogenase -1 according to claim 1, it is characterised in that：Contain in the reagent two
The substance withdrawl syndrome of Tris-HCl cushioning liquid is 10mM, pH 7.5, volume 50mL.
4. the Activity Assay Kit of transhydrogenase -1 according to claim 1, it is characterised in that：Contain in the reagent three
The substance withdrawl syndrome of Tris-HCl cushioning liquid is 1mM, pH 7.5, volume 18mL.
5. the Activity Assay Kit of transhydrogenase -1 according to claim 1, it is characterised in that：Contain in the reagent four
NADH quality is 0.1mg.
6. the Activity Assay Kit of transhydrogenase -1 according to claim 1, it is characterised in that：Contain in the reagent five
AcPyADP quality is 0.1mg.
7. a kind of activity determination method of transhydrogenase -1 using kit as claimed in claim 1, it is characterised in that based on micro-
Amount method, is comprised the following steps that：
The preparation of step 1 instrument and articles for use；
Prepare visible spectrophotometer or ELIASA, desk centrifuge, water-bath, adjustable pipette, micro quartz colorimetric utensil
Or 96 orifice plates, mortar, ice and distilled water；
The pre-treatment of step 2 sample, the separation of plasmosin and mitochondrial protein in tissue, bacterium or cell；
1）Weigh 0.1g tissues or collect 5,000,000 cells, add 1mL reagents one, be homogenized with ice bathing homogenizer or mortar；
2）Homogenate is transferred in a centrifuge tube, 5min is centrifuged at eccentricity 600g, 4 DEG C；
3）Precipitation is abandoned, supernatant is moved in another centrifuge tube, 10min is centrifuged at eccentricity 11100g, 4 DEG C；
4）Supernatant is the plasmosin for removing mitochondria, the transhydrogenase -1 available for measure from mitochondria leakage；
5）Precipitation in previous step is mitochondria, adds 500uL reagents two, ultrasonic disruption, is surveyed for the activity of transhydrogenase -1
It is fixed；
ELIASA is preheated more than 30min, adjusting wavelength to 375nm, distilled water zeroing by step 3；
Step 4 sample measures；
The preparation of working solution, reagent four and reagent five are transferred to mixed dissolution in reagent three, are placed in 37 DEG C or 25 DEG C of water-baths
5min；
2）Working solution described in 20 μ L samples and 180 μ L is added in micro quartz colorimetric utensil or 96 orifice plates, is mixed, immediate record
Light absorption value A2 at 375nm after initial light absorption value A1 and 10min, calculate Δ A=A2-A1；
The activity of step 5 transhydrogenase -1 calculates；
A. the calculation formula determined with micro quartz colorimetric utensil is as follows：
1）Calculated by sample protein concentration；
TH-1 activity（nmol/min/mg prot）= [ΔA×VIt is anti-total÷（ε×d）×109]÷（VSample×Cpr）÷T = 149×
ΔA÷Cpr；
2）Calculated by sample fresh weight；
TH-1 activity（Nmol/min/g fresh weights）= [ΔA×VIt is anti-total÷（ε×d）×109]÷（W×VSample÷VSample is total）÷T = 74.5
×ΔA÷W；
3）Calculated by bacterium or cell density
TH-1 activity（nmol/min/104cell）= [ΔA×VIt is anti-total÷（ε×d）×109]÷（500×VSample÷VSample is total）÷T =
0.149×ΔA；
Wherein, VIt is anti-totalRepresent reaction system cumulative volume, and VIt is anti-total=2×10-4 L；
ε represents APADPH molar extinction coefficients, and ε=6.7 × 103L/mol/cm；
D represents micro quartz colorimetric utensil optical path, and d=1cm；
VSampleRepresent to add sample volume, and VSample=0.02 mL；
VSample is totalRepresent the volume of addition extract solution, and VSample is total=0.5 mL；
T represents the reaction time, the min of and T=10；
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；
B. the calculation formula determined with 96 orifice plates is as follows：
1）Calculated by sample protein concentration；
TH-1 activity（nmol/min/mg prot）= [ΔA×VIt is anti-total÷（ε×d）×109]÷（VSample×Cpr）÷T = 298×
ΔA÷Cpr；
2）Calculated by sample fresh weight；
TH-1 activity（Nmol/min/g fresh weights）= [ΔA×VIt is anti-total÷（ε×d）×109]÷（W×VSample÷VSample is total）÷T = 149
×ΔA÷W；
3）Calculated by bacterium or cell density；
TH-1 activity（nmol/min/104cell）= [ΔA×VIt is anti-total÷（ε×d）×109]÷（500×VSample÷VSample is total）÷T =
0.298×ΔA；
Wherein, VIt is anti-totalRepresent reaction system cumulative volume, and VIt is anti-total=2×10-4 L；
ε represents APADPH molar extinction coefficients, and ε=6.7 × 103L/mol/cm；
D represents 96 orifice plate optical paths, and d=0.5cm；
VSampleRepresent to add sample volume, and VSample=0.02 mL；
VSample is totalRepresent the volume of addition extract solution, and VSample is total=0.5 mL；
T represents the reaction time, the min of and T=10；
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. the activity determination method of transhydrogenase -1 according to claim 1, it is characterised in that：The 5 of step 2）In, described is super
The method of sonication bacterium or cell is first ice bath, then using power 20% or 200W, ultrasonic 3s, is spaced 10s, repeats 30
It is secondary.
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| 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 |
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