CN114577745A - Method for detecting activity of methylenetetrahydrofolate dehydrogenase 2 - Google Patents
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Abstract
The application discloses a method for detecting activity of methylenetetrahydrofolate dehydrogenase 2. According to the activity detection method, methylene tetrahydrofolate dehydrogenase 2 to be detected and a catalyst NAD + cofactor are adopted to react with a reaction substrate 5, 10-methylene tetrahydrofolate, 340nM ultraviolet light absorption value detection is carried out on a reaction product in the reaction process, NADH content in the reaction product is calculated according to the 340nM ultraviolet light absorption value, and the activity of the methylene tetrahydrofolate dehydrogenase 2 is calculated according to the NADH content after different reaction times. According to the method, the NADH content in the reaction product can be obtained only by detecting the 340nM ultraviolet light absorption value in the reaction product according to the NADH standard curve, so that the activity of the methylenetetrahydrofolate dehydrogenase 2 is obtained by calculation. The method greatly shortens the detection time, and can be better suitable for the scene needing to quickly detect the activity of the methylenetetrahydrofolate dehydrogenase 2.
Description
Technical Field
The application relates to the technical field of polypeptide activity detection, in particular to a method for detecting activity of methylenetetrahydrofolate dehydrogenase 2.
Background
Methylenetetrahydrofolate dehydrogenase 2(MTHFD2), also known as methylenetetrahydrofolate cyclohydrolase, is a bifunctional enzyme with dual activities of methylenetetrahydrofolate dehydrogenase and cyclohydrolase, and exhibits high expression levels in mitochondrial folate metabolism. Studies have shown that the activity of MTHFD2 is closely related to the development and progression of various types of tumors; therefore, the detection of MTHFD2 activity is of great significance for tumor research.
The currently and commonly used MTHFD2 activity Detection method is that 5, 10-methylenetetrahydrofolic acid is dehydrogenated and catalyzed by using a catalyst NAD + cofactor and MTHFD2, NADH is finally generated after reacting for 30min at normal temperature, NADH-GLO Detection system and the generated NADH react for 60min at normal temperature, and then a chemiluminescence signal is detected by using a microplate reader; determining the amount of NADH and thus the activity of MTHFD2 based on the detected chemiluminescent signal. The method can effectively detect the activity of MTHFD 2; however, the reaction time is long, and at least 90min is needed; further, the reaction conditions are limited by the nature of the chemiluminescent reagent, and it is necessary to purchase a GLO Detection system Detection reagent or the like.
Therefore, how to develop a simpler, more convenient and faster detection method for MTHFD2 activity is a problem to be solved urgently.
Disclosure of Invention
The purpose of the application is to provide a novel method for detecting the activity of the methylenetetrahydrofolate dehydrogenase 2 antibody.
The following technical scheme is adopted in the application:
the application discloses a method for detecting activity of methylenetetrahydrofolate dehydrogenase 2, which comprises the steps of carrying out dehydrogenation catalytic reaction on a reaction substrate 5, 10-methylenetetrahydrofolate by adopting the methylenetetrahydrofolate dehydrogenase 2 to be detected and a catalyst NAD + cofactor, detecting an ultraviolet light absorption value of 340nM for a reaction product in the reaction process, calculating the NADH content in the reaction product according to the detected ultraviolet light absorption value of 340nM, and calculating the activity of the methylenetetrahydrofolate dehydrogenase 2 to be detected according to the NADH content in the reaction product after different reaction times.
It is noted that, the research of the application finds that the ultraviolet absorbance of NAD + is higher at 260nM, and the ultraviolet absorbance of NADH is higher at 340 nM; therefore, the activity of the methylenetetrahydrofolate dehydrogenase 2 can be calculated by directly utilizing the characteristics and detecting the ultraviolet absorbance value of the reaction product at 340nM so as to calculate the NADH content in the reaction product. It can be understood that the activity Detection method only needs to detect the ultraviolet absorbance value of 340nM, and does not need to additionally use Detection reagents such as GLO Detection system and the like, so that the Detection cost is saved, and the reaction time of NADH-GLO Detection system and NADH is also saved, so that the Detection method can be used for detecting the activity of MTHFD2 more simply and conveniently and rapidly.
In an implementation manner of the application, the content of NADH in the reaction product is calculated according to the detected 340nM ultraviolet absorbance, specifically, the method includes the steps of detecting the 340nM ultraviolet absorbance by adopting a plurality of NADH with known concentrations, drawing a fitting curve of the NADH concentration and the 340nM ultraviolet absorbance according to a detection result, namely obtaining an NADH standard curve, and calculating the content of NADH in the reaction product according to the detected 340nM ultraviolet absorbance and the NADH standard curve of the reaction product.
In one implementation manner of the application, the NADH standard curve is obtained by diluting NADH of 2.5mmol/L in 2-fold gradient, obtaining 6 NADH with known concentration in total, detecting the ultraviolet absorbance value of 340nM, and drawing according to the detection results of 6 NADH.
In one implementation of the present application, the fitting formula of the NADH standard curve is Y0.9831X-0.0361, X is the NADH concentration, and Y is the ultraviolet absorbance of 340 nM.
In an implementation manner of the present application, the activity of the methylenetetrahydrofolate dehydrogenase 2 to be detected is calculated according to the content of NADH in the reaction product after different reaction times, and specifically includes:
1) detecting ultraviolet absorbance of 340nM, and calculating the NADH content generated by MTHFD2 enzyme reaction according to a fitting formula of the NADH standard curve;
2) fixing the concentration of a substrate and the concentration of NAD +, adding enzyme quantities with different concentrations, detecting the ultraviolet absorbance of 340nM once by a normal-temperature enzyme-labeling instrument in a 30s power cycle, subtracting a control group without MTHFD2 enzyme from the experimental group, calculating the NADH content according to a fitting formula of NADH standard curve, and taking the value within 4min to prepare a curve;
3) fitting the NADH content corresponding to different time within 4min, converting the slope of a fitting curve into a standard unit pmol/min as a reaction speed V, and preparing a curve chart of the amount of the reaction enzyme and the reaction speed;
4) fixing enzyme quantity and NAD + concentration, changing the concentration of a substrate 5, 10-methylenetetrahydrofolic acid, detecting the ultraviolet absorbance of 340nM once by a normal-temperature microplate reader for 30s power cycle, obtaining the relation between the reaction speed and the substrate concentration according to the calculation methods of the step 2) and the step 3), converting into a standard unit pmol/min/ug, making a curve chart, and calculating a Km value by using Prism 7 software; km is used for representing the affinity of the methylenetetrahydrofolate dehydrogenase 2 to be detected, the smaller Km is, the higher the affinity is, the stronger the enzyme activity is, the larger Km is, the lower the affinity is, and the smaller the enzyme activity is.
In an implementation manner of the present application, the following is specifically implemented:
(1) when the ultraviolet absorbance of 340nM is detected, the NADH content generated by MTHFD2 enzyme reaction can be deduced reversely, namely, according to the formula X, (Y + 0.0361)/0.9831; (2) according to a formula fitted by ultraviolet absorbance of 340nM corresponding to the NADH content of different concentrations, Y is 0.9831X-0.0361; (3) fixing the concentration of a substrate to be 500 mu mol/L and the concentration of NAD + to be 500 mu mol/L, adding enzyme amount with different concentrations, detecting the ultraviolet absorbance of 340nM once by a normal-temperature enzyme labeling instrument for 30s power circulation, subtracting a control group without MTHFD2 enzyme from an experimental group, calculating the content according to an NADH standard formula, taking the value within 4min to prepare a curve, and fitting the curve; (4) the slope of the reaction fitting curve at different time is the reaction speed V, then converted into the standard unit pmol/min, and a curve chart of the reaction enzyme amount and the reaction speed is made; (5) fixing enzyme amount is 40nmol/L, NAD + concentration is 500 mu mol/L, changing substrate concentration Folitixorin, starting from 500 mu mol/L, diluting by 2 times, carrying out 7 gradients, detecting ultraviolet absorbance of 340nM once by a room-temperature microplate reader in a 30s power cycle, obtaining the relation between reaction speed and substrate concentration according to the calculation method in the steps (2) and (4), converting into standard unit pmol/min/mu g, making a graph, and then calculating Km value by using Prism 7 software.
It should be noted that Km is an important characteristic constant of MTFHD2 enzyme, and it is related to the nature of the enzyme and not to the concentration. The Km value can represent the affinity of the enzyme to the substrate, the smaller the Km value is, the greater the affinity is, and the larger the Km value is, the smaller the affinity is.
In an implementation manner of the application, the activity detection method further includes calculating the inhibition rate of the inhibitor LY345899 with different concentrations at different times on the activity of the methylenetetrahydrofolate dehydrogenase 2 to be detected according to an inhibition rate formula after calculating the content of NADH in the obtained reaction product, and calculating the IC50 of LY345899 by using Prism 7 software, so as to characterize the activity of the methylenetetrahydrofolate dehydrogenase 2 to be detected.
In one implementation of the present application, the concentration of the catalyst NAD + cofactor is 200-500. mu. mol/L.
In one implementation of the present application, the concentration of the reaction substrate 5, 10-methylenetetrahydrofolate is 100-500. mu. mol/L.
In one implementation manner of the present application, the concentration of the methylenetetrahydrofolate dehydrogenase 2 to be detected is 10-50 nmol/L.
The beneficial effect of this application lies in:
according to the method for detecting the activity of the methylenetetrahydrofolate dehydrogenase 2, the NADH content in the reaction product can be simply and conveniently obtained by detecting the 340nM ultraviolet absorption value of the 5, 10-methylenetetrahydrofolate dehydrogenation catalytic reaction product according to the NADH standard curve, so that the activity of the methylenetetrahydrofolate dehydrogenase 2 is obtained by calculation. The activity detection method greatly shortens the detection time, and can be better suitable for the scene needing to rapidly detect the activity of the methylenetetrahydrofolate dehydrogenase 2.
Drawings
FIG. 1 is a NADH standard curve plotted in the examples of the present application;
FIG. 2 is a graph of the concentration of MTHFD2 plotted for different examples of the present application;
FIG. 3 is a graph of the concentration of MTHFD2 versus the reaction rate for the different examples of the present application;
FIG. 4 is a graph of Folitixorin, a substrate, at various concentrations in the examples of the present application;
FIG. 5 is a plot of the concentrations of LY345899 inhibitor plotted against the examples of this application.
Detailed Description
The present application will be described in further detail with reference to specific examples. The following examples are intended to be illustrative of the present application only and should not be construed as limiting the present application. In the following description, numerous details are set forth in order to provide a better understanding of the present application. However, one skilled in the art will readily recognize that some of the features may be omitted in different instances or may be replaced by other kits, materials, methods. In some instances, certain operations related to the present application have not been shown or described in this specification in order not to obscure the core of the present application with unnecessary detail, but it is not necessary for those skilled in the art to describe these operations in detail, and the related operations will be fully understood from the description in the specification and the general knowledge in the art. In the following examples, the reagents or instruments used are not indicated by manufacturers, but are all conventional products available on the market.
Examples
First, main reagent and instrument
The main reagents and instruments used in this example are shown in table 1.
TABLE 1 Main reagents and instruments
| Product name | Specification and model | Manufacturer of the product | Genuine encoding |
| Recombinant Human MTHFD2 Protein | 0.5mg | novusbio | NBP2-51979-0.5mg |
| LY 345899 | 5mg | medchemexpress | HY-101943 |
| NAD+ | 500mg | medchemexpress | HY-B0445 |
| Folitixorin | 50mg | Shanghai Yifei creature | E044315 |
| 96-well transparent culture plate | NEST | 701001 | |
| Enzyme-linked immunosorbent assay (ELISA) instrument | Thermo | VARIOSKAN LUX | |
| NADH | 1g | Germany | 1.01E+10 |
| HEPES | 500g | Aladdin | H109406-500g |
The buffers used in this example included:
buffer mother liquor:
10mmol/L NADH: molecular weight is 709.4, 0.007094g is weighed, dissolved in sterile water to 1mL, split into 100 mu L/tube, and frozen in a refrigerator at-20 ℃ for storage.
20mmol/L NAD +: molecular weight 663, weigh 0.01326g, dissolve in sterile water to 1mL, subpackage 50 μ L/tube, freeze in-20 degree refrigerator and store.
1mol/L KCl: molecular weight 74.55, weighing 3.7275g, dissolved in sterile water to 50 mL.
1mol/L MgCl2: molecular weight 203.3, weigh 2.03g, dissolve in 10mL of sterile water.
250mmol/L HEPES: molecular weight 238.30, weighing 1.19g, dissolved in 10mL of sterile water, pH8.0 was adjusted.
0.2mol/L NaH2PO4 2H2O: molecular weight 156.01, weigh 0.312g, dissolve in 10mL of sterile water.
0.2mol/L of Na2HPO4 12H2O: molecular weight 358.14, weighing 3.5814g, dissolved in sterile water to 50 mL.
10ml of 200mmol/L phosphate buffer was prepared: taking 0.2mol/L NaH2PO4 2H2O buffer 530. mu.L, 0.2mol/L Na2HPO4 12H29.47mL of O buffer, pH 8.0.
Configure 2 × MTFHD2 Assay buffer 10 mL: 4mL of 250mmol/L HEPES, 2mL of 1mol/L KCl, and 1mol/L MgCl20.01mL of 200mmol/L phosphorusAcid buffer 2.5mL, ddH2O1.49mL。
Second, enzyme activity detection experiment
1. Preparation of NADH Standard Curve
Adopting an NADH standard substance with the concentration of 2.5mmol/L, diluting for five times by 2-time gradient to obtain 6 gradient NADH standard substances; and (3) taking 90 mu L of each gradient NADH standard substance, detecting the ultraviolet absorbance value of 340nM by using a microplate reader, and drawing an NADH standard curve.
The NADH standard curve is shown in FIG. 1, and in FIG. 1, the abscissa represents the concentration of NADH in units; the ordinate is the UV absorbance at 340 nM. The results in FIG. 1 show that NADH concentration has a good linear relationship with 340nM UV absorbance, the fitting formula is Y0.9831X-0.0361, X is NADH concentration, Y is 340nM UV absorbance, and the correlation coefficient of the two is R2＝0.9986。
Plotting the different enzyme amounts of MTHFD2
Preparing a reaction solution, wherein the concentration of a substrate Folitixorin in the reaction solution is 500 mu mol/L, NAD +, the concentration is 500 mu mol/L, and 50 mu L of 2 XMTFHD 2 Assay buffer is added into the reaction solution; the amount of MTHFD2 added was controlled so that the concentrations of MTHFD2 in the reaction mixture were 50nmol/L, 40nmol/L, 30nmol/L, 20nmol/L, and 10nmol/L, respectively, and the total volume of the reaction mixture was 100. mu.L. Namely, five reaction solution systems with the concentrations of MTHFD2 in the reaction solution of 50nmol/L, 40nmol/L, 30nmol/L, 20nmol/L and 10nmol/L are respectively prepared and used for drawing reaction curves of different enzyme quantities; the reaction system without MTHFD2 was designed as a control, i.e., MTHFD2 was 0 nmol/L.
And (3) placing the reaction solution at the normal temperature of 20-30 ℃ for reaction for 30min, detecting the ultraviolet light absorption value at 340nM by means of kinetic cycle of a microplate reader every 30s, collecting data, and drawing a curve. The curves plotted for the different concentrations of MTHFD2 are shown in figure 2. In FIG. 2, the abscissa represents time (min) and the ordinate represents the concentration of NADH in the product (nmol/L). The results of figure 2 show that it is,
0nmol/L y＝(-7E-15)x+36.721 R2＝-1E-16
10nmol/L y＝0.9392x+49.026 R2＝0.9942
20nmol/L y＝2.326x+49.485 R2＝0.9949
30nmol/L y＝3.0109x+52.354 R2＝0.9948
40nmol/L y＝5.3369x+84.976 R2＝0.9976
50nmol/L y＝2.6989x+61.409 R2＝0.9862
from the results of FIG. 2, the slope values of the respective curves of FIG. 2 were taken and the reaction rate was plotted against the amount of the enzyme, and the results are shown in FIG. 3. In FIG. 3, the abscissa represents the amount of enzyme, i.e., the concentration (nmol/L) of MTHFD2, and the ordinate represents the reaction rate V (pM/min). The results in FIG. 3 show that the reaction rate is the fastest at a concentration of MTHFD2 of 40 nmol/L.
Thirdly, calculating the Michaelis constant Km of the MTFHD2 enzyme
Preparing reaction liquid of Folitixorin with different concentrations, starting from the concentration of Folitixorin in the reaction liquid of 500 mu mol/L, carrying out gradient dilution by 2 times, and diluting for six times in total to obtain reaction liquid of 7 substrates with gradient concentrations, wherein the concentration of MTHFD2 in the reaction liquid is 40nmol/L, the concentration of NAD + is 500 mu mol/L, and the total volume of the reaction liquid is 100 mu L. And detecting the ultraviolet light absorption value at 340nM by means of dynamic circulation of a microplate reader once every 30s, collecting data for 30min, drawing a curve, and calculating the substrate concentration Km corresponding to half of the maximum reaction rate. The results are shown in FIG. 4.
The results in fig. 4 show that Km ═ 160 μmol/L. Km is an important characteristic constant of MTFHD2 enzyme, and it is related to the nature of the enzyme, and not to the concentration. The Km value can represent the affinity of the enzyme to the substrate, the smaller the Km value is, the greater the affinity is, and the larger the Km value is, the smaller the affinity is.
IC50 of LY345899 for inhibiting MTFHD2 and Folitixorin action
Reaction solutions with different concentrations of LY345899 were prepared, and from the reaction solution in which the concentration of LY345899 was 10. mu. mol/L, 2-fold gradient dilution was performed, and five dilutions were performed in total to obtain 6 reaction solutions with gradient concentrations of LY 345899. The concentration of MTHFD2 in the reaction mixture was 40nmol/L, and the concentration of NAD + was 250. mu. mol/L. Firstly, LY345899 and MTFHD2 react with the reaction solution of NAD + for 10min, then substrate Folitixorin with the final concentration of 250 mu mol/L is added, the ultraviolet absorbance value is detected at 340nM by the dynamic cycle of a microplate reader, the detection is carried out every 2min, 20min data are collected, a curve is drawn, and the result is shown in figure 5. In FIG. 5, the respective curves are curves of reaction times of 2 minutes, 4 minutes, 6 minutes, and 8 minutes, respectively; the abscissa is the logarithm of the concentration of LY345899, and the ordinate is the inhibition (%).
The results in FIG. 5 show that within 8min, the repeatability and consistency of the experiment are relatively good, and the reaction speed is relatively fast. Therefore, the activity detection method of the embodiment can characterize the activity of the MTFHD2 to be detected by detecting the IC50 within 8min, that is, the detection method of the embodiment can complete the detection in only 8 minutes.
IC50 was calculated within 8min using Prism 7 software, and the results are shown in Table 2.
Statistical results of IC50 within 28 min
| Time (min) | 2 | 4 | 6 | 8 | Mean value of |
| IC50(μmol/L) | 0.6555 | 0.6443 | 0.7108 | 0.6504 | 0.66525 |
| R2 | 0.9943 | 0.9993 | 0.9998 | 0.9999 |
Inhibition rate calculation formula (100%; positive control-experimental well)/(positive control-negative control) ]
Positive control: contains MTFHD2 Assay buffer, 250 mu mol/L NAD +, 250 mu mol/L substrate Folitixorin, 40nmol/L MTHFD2 and water.
Experiment hole: contains MTFHD2 Assay buffer, 250 mu mol/L NAD +, 250 mu mol/L substrate Folitixorin, 40nmol/L MTHFD2, water and different concentrations of LY 345899.
Negative control: contains MTFHD2 Assay buffer, 250 mu mol/L NAD +, 250 mu mol/L substrate Folitixorin and water.
The results in table 2 show that the average IC50 is 665nmol/L, which is very close to the literature reported IC50 is 663nmol/L, since the deviation is about 2% normal for each experimental condition operation, so that LY345899 can be considered as consistent with the inhibition IC50 of MTFHD2, thus proving that the activity detection method of the present example is feasible.
Fifth, sample detection
5 MTHFD2 samples were assayed for activity using the assay of this example; meanwhile, the same 5 samples of MTHFD2 were tested using GLO Detection system Detection reagent according to the conventional method, and the test results are shown in Table 3.
The specific detection method of this example is as follows:
(1) in a 96-well plate, the experimental group was added MTFHD2 Assay buffer, 250. mu. mol/L NAD +, 40nmol/L MTHFD2, water, 10. mu. mol/L LY345899, volume 100. mu.L, 2 fold dilution, 7 gradients, 2 replicates. Positive control: contains MTFHD2 Assay buffer, 250 mu mol/L NAD +, 40nmol/LMTHFD2 and water. Negative control: contains MTFHD2 Assay buffer, 250. mu. mol/L NAD +, water. The total volume of the reaction solution was 100. mu.L, and the reaction was carried out at room temperature for 10 min.
(2) The substrate Folitixorin was added at 100. mu. mol/L and the UV absorbance was measured at 340nM once in a dynamic cycle of the microplate reader for 30 s.
(3) And (3) calculating the activity of the methylenetetrahydrofolate dehydrogenase 2 to be detected according to the content of NADH in the reaction product.
The specific Detection method by using the GLO Detection system Detection reagent is as follows:
(1) in a 96-well plate, the experimental group was added MTFHD2 Assay buffer, 250. mu. mol/L NAD +, 40nmol/L MTHFD2, water, 10. mu. mol/L LY345899, volume 100. mu.L, 2 fold dilution, 7 gradients, 2 replicates. Positive control: contains MTFHD2 Assay buffer, 250 mu mol/L NAD +, 40nmol/LMTHFD2 and water. Negative control: contains MTFHD2 Assay buffer, 250. mu. mol/L NAD +, water. The total volume is 100 mu L, and the reaction is carried out for 10min at normal temperature.
(2) Adding 100 mu mol/L substrate Folitixorin, and reacting for 30min at normal temperature.
(3) Addition of NAD (P) H-GloTMThe Detection System dispenses the reagent and reacts for 60min at normal temperature.
(4) Chemiluminescence was detected with a microplate reader.
(5) And (3) calculating the activity of the methylenetetrahydrofolate dehydrogenase 2 to be detected according to the content of NADH in the reaction product.
TABLE 3 IC50 Activity results in nmol/L of MTHFD2 obtained with different assays
| Sample numbering | The results of the activity test in this example | Results of Activity detection by |
| 1 | 675.7 | 665.5 |
| 2 | 670.7 | 667.2 |
| 3 | 668.5 | 664.3 |
| 4 | 669.2 | 668.6 |
| 5 | 668.5 | 669.3 |
The results in Table 3 show that the assay results of MTHFD2 activity in this example are consistent with those of conventional GLO Detection system reagents; the activity detection method of the present example is demonstrated that MTHFD2 activity can be accurately and effectively detected by detecting the ultraviolet absorbance at 340nM in the reaction system. The method for detecting the activity of MTHFD2 omits a GLO Detection system reagent reaction step and time, and is simpler, more convenient and quicker.
The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. It will be apparent to those skilled in the art from this disclosure that many more simple derivations or substitutions can be made without departing from the spirit of the disclosure.
Claims (9)
1. A method for detecting the activity of methylenetetrahydrofolate dehydrogenase 2 is characterized in that: the method comprises the steps of carrying out dehydrogenation catalytic reaction on a reaction substrate 5, 10-methylenetetrahydrofolate by adopting methylenetetrahydrofolate dehydrogenase 2 to be detected and a catalyst NAD + cofactor, detecting an ultraviolet absorbance value of 340nM on a reaction product in the reaction process, calculating the content of NADH in the reaction product according to the detected ultraviolet absorbance value of 340nM, and calculating the activity of the methylenetetrahydrofolate dehydrogenase 2 to be detected according to the content of NADH in the reaction product after different reaction times.
2. The method of claim 1, wherein: and calculating the content of NADH in the reaction product according to the detected 340nM ultraviolet absorbance value, specifically, detecting the 340nM ultraviolet absorbance value by adopting a plurality of NADH with known concentration, drawing a fitting curve of the NADH concentration and the 340nM ultraviolet absorbance value according to the detection result to obtain an NADH standard curve, and calculating the content of NADH in the reaction product according to the detected 340nM ultraviolet absorbance value of the reaction product and the NADH standard curve.
3. The method of claim 2, wherein: and diluting the NADH standard curve by 2.5mmol/L NADH in a 2-fold gradient, obtaining 6 NADH with known concentration in total, detecting the ultraviolet absorbance value of 340nM, and drawing according to the detection results of the 6 NADH.
4. The method of claim 3, wherein: the fitting formula of the NADH standard curve is that Y is 0.9831X-0.0361, X is NADH concentration, and Y is ultraviolet absorbance value of 340 nM.
5. The method of claim 2, wherein: the activity of the methylenetetrahydrofolate dehydrogenase 2 to be detected is calculated according to the NADH content in the reaction products after different reaction times, which specifically comprises,
1) detecting ultraviolet absorbance of 340nM, and calculating the NADH content generated by the MTHFD2 enzyme reaction according to the fitting formula of the NADH standard curve;
2) fixing the concentration of a substrate and the concentration of NAD +, adding enzyme quantities with different concentrations, detecting the ultraviolet absorbance of 340nM once by a normal-temperature enzyme-labeling instrument for 30s power cycle, subtracting a control group without MTHFD2 enzyme from an experimental group, calculating the NADH content according to a fitting formula of NADH standard curve, and preparing a curve by taking the value within 4 min;
3) fitting the NADH content corresponding to different time within 4min, converting the slope of a fitting curve into a standard unit pmol/min as a reaction speed V, and preparing a curve chart of the amount of the reaction enzyme and the reaction speed;
4) fixing enzyme quantity and NAD + concentration, changing the concentration of a substrate 5, 10-methylenetetrahydrofolic acid, detecting the ultraviolet absorbance of 340nM once in a 30s power cycle by a normal-temperature enzyme-labeling instrument, obtaining the relation between the reaction speed and the substrate concentration according to the calculation methods of the step 2) and the step 3), converting into a standard unit pmol/min/ug, making a curve chart, and calculating a Km value by using Prism 7 software; km is used for representing the affinity of the methylenetetrahydrofolate dehydrogenase 2 to be detected, the smaller Km is, the higher the affinity is, the stronger the enzyme activity is, the larger Km is, the lower the affinity is, and the smaller the enzyme activity is.
6. The method according to any one of claims 1 to 5, wherein: after the NADH content in the reaction product is obtained through calculation, the inhibition rate of the inhibitor LY345899 with different time and different concentration to the activity of the methylenetetrahydrofolate dehydrogenase 2 to be detected is calculated according to an inhibition rate formula, and IC50 of the inhibitor LY345899 is calculated by Prism 7 software, so that the activity of the methylenetetrahydrofolate dehydrogenase 2 to be detected is represented.
7. The method according to any one of claims 1 to 5, wherein: the concentration of the catalyst NAD + cofactor is 200-500. mu. mol/L.
8. The method according to any one of claims 1 to 5, wherein: the concentration of the reaction substrate 5, 10-methylenetetrahydrofolate is 500 mu mol/L.
9. The method according to any one of claims 1 to 5, wherein: the concentration of the methylenetetrahydrofolate dehydrogenase 2 to be detected is 10-50 nmol/L.
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