A method for detecting the activity of phosphorylated enzymes based on nuclear magnetic resonance phosphorus spectroscopy

Abstract

A method for detecting the activity of a phosphorylated enzyme based on 31P NMR (nuclear magnetic resonance phosphorus spectroscopy) includes the following steps: (1) Prepare standard solutions of substrates or products with known different concentrations, conduct 31P NMR measurements respectively to obtain the peak area percentages of the standard solutions, and plot a standard curve with the concentration and the peak area percentage; (2) Measure the peak area percentage of the test solution by 31P NMR, substitute it into the linear equation to obtain the initial concentration of the substrate or product. Mix the test solution, buffer solution and phosphorylated enzyme for incubation to obtain a reaction solution, and detect by 31P NMR to obtain the concentration of the substrate or product in the reaction solution; (3) Compare the concentration of the substrate or product in the reaction solution with the initial concentration of the substrate or product to obtain the change in concentration, and calculate the enzyme activity using the change in concentration per unit time. The advantages are that it is fast, simple, does not rely on exogenous reagents, can recycle samples without pollution, has strong anti-interference ability, can conduct real-time monitoring, and is especially suitable for the biological field.

Description

Technical Field

[0001] The present invention relates to a method for detecting the activity of a phosphorylated enzyme, and more particularly to a method for detecting the activity of a phosphorylated enzyme based on phosphorus nuclear magnetic resonance spectroscopy 31 31P NMR. Background Art

[0002] Enzymes that reversibly bind to phosphate groups by covalent bonds, causing changes in enzyme structure and presenting different activities, are called phosphorylated enzymes. Common phosphorylated enzymes include phosphatases and kinases, etc. Phosphatases and kinases are widely distributed in the human body and are often used by cells as common signal transduction mechanisms to respond to external stimuli. A phosphatase is an enzyme that can dephosphorylate a corresponding substrate, that is, remove the phosphate group on the substrate molecule by hydrolyzing phosphomonoester to generate phosphate ions and free hydroxyl groups; the function of a kinase is the opposite of that of a phosphatase. A kinase is a phosphorylating enzyme that can use energy molecules, such as ATP, to add a phosphate group to the corresponding substrate molecule. Other phosphorylated enzymes important in the cell signal transduction pathway include cyclases that produce cyclic nucleotides (such as cyclic AMP (cAMP) and cyclic GMP (cGMP)) and phosphodiesterases that hydrolyze cyclic nucleotides to form corresponding acyclic monophosphonucleotides (i.e., AMP and GMP). Cyclic nucleotides cyclic AMP (cAMP) and cyclic GMP (cGMP) are important second messengers. Because of the important role of these phosphorylated enzymes in regulating cell functions, they are important targets for exploring and developing new drug therapies. Therefore, the detection of the activity of phosphorylated enzymes is particularly important.

[0003] Currently, common methods for detecting and analyzing the activity of phosphorylated enzymes include: fluorescence method, high performance liquid chromatography (HPLC), and capillary electrophoresis (CE), etc. The fluorescence method is a method for identifying substances and determining their contents based on the position and intensity of the fluorescence spectrum of the substances. The increase in fluorescence is proportional to the activity of the protease, and the enzyme activity is calculated based on the increase in fluorescence; the HPLC method is an analytical method that separates according to the different properties of the substances to be separated by selecting an appropriate chromatographic column (hydrophilic chromatography, hydrophobic chromatography, ion exchange chromatography, etc.) and then detecting with a corresponding detector, so as to detect the enzyme activity. Capillary electrophoresis is an analytical method that separates substances to be separated based on their different charges using a capillary as a separation channel under the action of an electric field, and then, with a suitable detector, the substances that reach the detector successively can be detected, so as to detect the enzyme activity.

[0004] However, there are certain limitations in these current methods. The fluorescence method for analyzing enzyme activity is affected by many external factors (such as temperature, solvent, acidity, fluorescence quenching agents), which can affect the fluorescence efficiency. The disadvantage of the HPLC method is the "extra-column effect". Between the injection point and the detector, in any dead space outside the column (injector, column connectors, connecting tubes, detection cell, etc.), if the flow pattern of the mobile phase changes, any diffusion and retention of the separated substances will significantly cause broadening of the chromatographic peak and a decrease in column efficiency. Capillary electrophoresis has the disadvantages of a short optical path due to the small diameter of the capillary, resulting in low sensitivity and poor separation reproducibility when using these detection methods. At the same time, these methods generally require complex sample pretreatment, with serious sample waste, a cumbersome operation process, low efficiency, often relying on exogenous reagents and being easily interfered with, etc. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a rapid, simple method for detecting the activity of phosphorylated enzymes based on nuclear magnetic resonance phosphorus spectrum 31 P NMR, which has simple sample pretreatment, can recycle samples without pollution, does not rely on exogenous reagents during detection, has strong anti-interference ability and can perform real-time monitoring.

[0006] The technical solution adopted by the present invention to solve the above technical problems is as follows:

[0007] A method for detecting the activity of phosphorylated enzymes based on nuclear magnetic resonance phosphorus spectrum 31 P NMR, comprising the following steps:

[0008] S1: Prepare standard solutions of substrates or products with known different concentrations, and perform nuclear magnetic resonance phosphorus spectrum 31 P NMR measurements respectively to obtain the peak area percentages of the standard solutions of the substrates or products with different concentrations, and plot a standard curve with the concentration and the peak area percentage to obtain a linear equation of the relationship between the concentration and the peak area percentage;

[0009] S2: Measure the peak area percentage of the test solution by nuclear magnetic resonance phosphorus spectrum 31 P NMR, substitute it into the linear equation of step S1 to obtain the initial concentration of the substrate or product, mix and incubate the test solution, buffer solution and phosphorylated enzyme, and after incubation, obtain a reaction solution. The reaction solution is detected by nuclear magnetic resonance phosphorus spectrum 31 P NMR to obtain the peak area percentage of the substrate or product in the reaction solution after incubation;

[0010] S3: Substitute the peak area percentage of the substrate or product in the reaction solution in step S2 into the linear equation obtained in step S1 to obtain the concentration of the substrate or product in the reaction solution;

[0011] S4: The change in concentration is obtained by comparing the concentration of the substrate or product in the reaction solution obtained in step S3 with the initial concentration of the substrate or product in step S2, and the enzyme activity of the phosphomodifying enzyme is calculated using the change in concentration per unit time.

[0012] The phosphomodifying enzyme in step S2 can catalyze the phosphorylation modification of the substrate to generate the product or catalyze the dephosphorylation modification of the substrate to generate the product.

[0013] The phosphomodifying enzyme in step S2 is a phosphatase, kinase, phosphodiesterase or cyclase.

[0014] The concentration of the phosphomodifying enzyme in step S2 is 0.2 - 1 mg / mL.

[0015] The buffer solution in step S2 is 40 - 50 mM Tris-HCl with a pH of 6.5 - 8.0 and contains 5 - 10 mM MgCl 2 .

[0016] The incubation time in step S2 is 10 min - 3 h.

[0017] The incubation temperature in step S2 is 37 °C.

[0018] Compared with the prior art, the advantages of the present invention are as follows:

[0019] (1) The present invention uses nuclear magnetic resonance phosphorus spectroscopy 31 P NMR technology to establish a standard curve of the concentration of the substrate or product of the enzyme-catalyzed reaction and the corresponding peak area percentage. By analyzing the decrease in the substrate or the increase in the product in the test sample, the content of the phosphorus-containing substance in the test substance is obtained, and thus the enzyme activity is calculated. The detection method of the present invention is rapid and simple, does not require the addition of exogenous reagents, does not require the debugging of complex test methods, has high sensitivity, and can analyze the enzyme activity of phosphomodifying enzymes in the biological field in real time.

[0020] (2) The present invention qualitatively and quantitatively analyzes the activity of the phosphomodifying enzyme based on nuclear magnetic resonance phosphorus spectroscopy 31 P NMR method. Non-phosphorus-containing substances in the sample have no signal in the nuclear magnetic resonance phosphorus spectroscopy 31 P NMR spectrum, which has no influence on the test result. The test process will not be interfered by impurities, and the determination of phosphorus-containing substances has high specificity. As shown in Figure 5 , only the phosphorus-containing substrate and product have signals in the nuclear magnetic resonance phosphorus spectroscopy 31 P NMR spectrum of the reaction solution after enzyme catalysis; for phosphorus-containing impurities in the sample, as long as they are in the nuclear magnetic resonance phosphorus spectroscopy 31The chemical shift of the ³¹P NMR spectrum is different from that of the target phosphorus-containing product. As shown in Figure 8 the attached 31 ³¹P NMR spectrum of the reaction solution after enzymatic catalysis, although there are other phosphorus-containing impurities, since the chemical shifts of the impurities are inconsistent with those of the target phosphorus-containing product, the presence of the impurities still has no effect on the test results. Therefore, the present invention can quickly determine the concentration of phosphorus-containing substances, recover samples without pollution, and has strong anti-interference ability, and is particularly suitable for the detection and analysis of the activity of phosphorylated enzymes in the biological field. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the ³¹P NMR spectrum of cAMP standard solutions with different concentrations in Example 1 of the present invention; 31 ³¹P NMR chart;

[0022] Figure 2 is the linear relationship diagram between the concentrations of cAMP standard solutions with different concentrations in Example 1 of the present invention and the 31 ³¹P NMR peak area (converted to percentage);

[0023] Figure 3 is the ³¹P NMR spectrum of AMP standard solutions with different concentrations in Example 1 of the present invention; 31 ³¹P NMR chart;

[0024] Figure 4 is the linear relationship diagram between the concentrations of AMP standard solutions with different concentrations in Example 1 of the present invention and the 31 ³¹P NMR peak area (converted to percentage);

[0025] Figure 5 is the ³¹P NMR spectrum of the PDEII enzyme catalysis experiment in Example 1 of the present invention; 31 ³¹P-NMR detection chart;

[0026] Figure 6 is the ³¹P NMR spectrum of 6-phosphoglucose with different concentrations in Example 2 of the present invention; 31 ³¹P NMR chart;

[0027] Figure 7 is the linear relationship diagram between the concentration of 6-phosphoglucose in Example 2 of the present invention and the 31 ³¹P NMR peak area (converted to percentage);

[0028] Figure 8 is the ³¹P NMR spectrum of the HK enzyme catalysis experiment in Example 2 of the present invention; 31 ³¹P NMR detection chart. DETAILED DESCRIPTION OF THE INVENTION

[0029] The present invention will be further described in detail below in conjunction with the embodiments with reference to the accompanying drawings.

[0030] The present invention discloses a method for detecting the activity of a phosphoric acid-modified enzyme based on nuclear magnetic resonance phosphorus spectroscopy ( 31 31P NMR).

[0031] Important parameters of nuclear magnetic resonance (NMR) spectra include chemical shift, spectral line intensity (peak area), etc., which are also important bases for qualitative and quantitative analysis of compounds. Different phosphorus-containing substances have different chemical shifts in the 31 31P NMR spectrum, and the spectral line intensity (peak area) of the same substance is also different at different concentrations. Therefore, there is a linear relationship y = ax + b between the peak area and the substance concentration within a certain range. That is, a standard curve can be established for the corresponding NMR signals (peak areas) of a series of concentrations of a phosphorus-containing substance standard to quantify the unknown concentration of the phosphorus-containing substance. Enzyme activity analysis mainly depends on analyzing the decrease in the phosphorus-containing substrate or the increase in the product to judge. By establishing a standard curve y = ax + b of the peak area and the substance concentration for the substrate or product standard with known concentration gradients; after the actual substrate undergoes an enzyme-catalyzed reaction with an enzyme, the peak area of the phosphorus-containing substrate or phosphorus-containing product in the test solution is detected by nuclear magnetic resonance phosphorus spectroscopy 31 31PNMR, substituting it into the standard curve to obtain the corresponding concentration after the reaction, obtaining the change in concentration by comparing with the initial concentration, and then calculating the enzyme activity using a specific formula.

[0032] The phosphoric acid-modified enzymes detected in the present invention include kinases and phosphatases, phosphodiesterases, cyclases, etc. that phosphorylate and dephosphorylate polypeptide molecules respectively. The term "substrate" refers to the molecule on which the enzyme catalyzing phosphoric acid modification acts. The term "product" refers to the molecule modified by phosphoric acid catalyzed by the enzyme.

[0033] Main reagents: cAMP, AMP, phosphodiesterase II (PDEII), hexokinase, glucose 6-phosphate, Tris-HCl, MgCl 2 , BSA

[0034] Main instrument: Bruker AVANCE 500 MHz nuclear magnetic resonance spectrometer

[0035] Instrument parameters: BBFO intelligent probe, constant experimental temperature 298K; controlled by Bruker VT-200 temperature controller, temperature control accuracy ±0.1

[0036] Example 1 31 Determination of the enzyme activity of phosphodiesterase (PDEII) by 31P NMR

[0037] To determine the enzyme activity, it is necessary to first determine the standard curve of the substrate or product, and then after the enzyme-catalyzed experiment, substitute the experimental data into the standard curve to calculate and obtain the enzyme activity.

[0038] 1.1. 31 Determination of the concentration of cyclic adenosine monophosphate (cAMP, the specific substrate of PDEII) by \(^{31}\)P NMR

[0039] Prepare five standard solutions of cAMP with known concentrations of 25 μM, 50 μM, 100 μM, 250 μM, and 500 μM, and perform 31 the determination of \(^{31}\)P NMR signals respectively to obtain the 31 \(^{31}\)P NMR spectra of cAMP standard solutions with different concentrations, as shown in the appendix Figure 1 shown. Obtain the peak areas (converted to percentages) of the cAMP standard solutions at different concentrations, and use the concentration as the abscissa \(x\) and the peak area (converted to percentage) as the ordinate \(y\) to plot a standard curve, obtaining the corresponding linear relationship diagram, as Figure 2 shown, with the equation \(y = 4.9373x + 0.9454\). \(R\) 2 \(= 0.9986\), indicating a good linear correlation between the peak area and the concentration.

[0040] Prepare a cAMP solution with a certain concentration, perform 31 \(^{31}\)P NMR detection to obtain the peak area value. After converting it to a percentage and substituting it into the above equation \(y = 4.9373x + 0.9454\), obtain the concentration value (\(c_1\)), and compare it with the true concentration value (\(c_2\)), as shown in Table 1 specifically.

[0041] Table 1 Comparison of the true values and experimental values of cAMP solutions with different concentrations

[0042]

[0043] Note: \(\Delta\) is the difference between \(c_1\) and \(c_2\), \(\Delta=(c_1 - c_2) / c_2\times100\%\).

[0044] Experimental results: From the above experimental results, it can be seen that using 31 the \(^{31}\)P NMR technique, the concentration of cAMP can be obtained quickly and accurately.

[0045] 1.2. 31 Determination of the concentration of adenosine monophosphate (AMP, the product of PDEII) by \(^{31}\)P NMR

[0046] Prepare five standard solutions of AMP with known concentrations of 25 μM, 50 μM, 100 μM, 250 μM, and 500 μM, and perform 31 the determination of \(^{31}\)P NMR signals respectively, as shown in the appendix Figure 3As shown, the peak areas (converted to percentages) of the standard solutions of AMP at different concentrations were obtained. With the concentration as the abscissa x and the peak area (converted to percentage) as the ordinate y, a standard curve was plotted to obtain the corresponding linear relationship diagram, as Figure 4 shown, and the equation was y = 4.9196x - 1.4554. R 2 = 0.9969, indicating a good linear correlation between the peak area and the concentration.

[0047] An AMP solution with a certain concentration was prepared and subjected to 31 31P NMR detection to obtain the peak area value. After converting it to a percentage and substituting it into the above equation y = 4.9196x - 1.4554, the concentration value (c1) was obtained and compared with the true concentration value (c2). The specific results are shown in Table 2.

[0048] Table 2 Comparison of the true values and experimental values of AMP solutions at different concentrations

[0049]

[0050]

[0051] Note: Δ is the difference between c1 and c2, and Δ = (c1 - c2) / c2 * 100%.

[0052] Experimental results: From the above experimental results, it can be seen that using 31 31P NMR technology, the concentration of AMP can be obtained quickly and accurately.

[0053] 1.3. Determination of PDEII enzyme activity

[0054] The method for detecting the activity of PDEII enzyme is to add the substrate cAMP (100 μM) and the enzyme PDEII (0.2 mg / mL) to the corresponding buffer solution (40 mM Tris-HCl pH 6.5, 10 mM MgCl, 0.1 mg / mL BSA), and then place the sample in an environment at 37°C for reaction for 10 min. Then, through 31 31P NMR detection, the 31P NMR detection map of the PDEII enzyme-catalyzed experiment was obtained, as 31 shown in Figure 5 Figure.

[0055] The enzyme-catalyzed reaction consumed the substrate cAMP and produced the product AMP.

[0056] Experimental results: In the enzyme-catalyzed experiment 31The detection diagram of ³¹P NMR can obtain the peak areas of substrate cAMP or AMP in the test solution. After percentage conversion through the corresponding standard curve, it is found that 15.62% of substrate cAMP remains (product AMP increased by 4.73%). Substituting into the standard curve for calculation, it is obtained that approximately 78.07 μM of substrate cAMP remains (product AMP increased by 21.81 μM). Enzyme activity, also known as enzyme vigor, refers to the ability of an enzyme to catalyze a certain chemical reaction. The unit of enzyme activity is U, and 1 U is the amount of enzyme required to convert 1 micromole of substrate or generate 1 micromole of product in 1 minute. Therefore, the enzyme activity of this PDEII enzyme calculated using substrate cAMP is 2.19 U (the enzyme activity calculated using product AMP is 2.18 U). After the substrate in the test solution undergoes an enzyme-catalyzed reaction using PDEII enzyme, 31 ³¹P NMR is used to detect the content changes of the substrate or product in the test solution. The peak area of the substrate or the peak area of the product is obtained and substituted into the standard curve to obtain the corresponding concentration after the reaction. The results of calculating the enzyme activity through the content changes of the substrate or product are almost the same.

[0057] Example 2 31 Determination of the enzyme activity of hexokinase (HK) by ³¹P NMR

[0058] 2.1. 31 Determination of the concentration of glucose 6-phosphate (the product of HK) by ³¹P NMR

[0059] Five glucose 6-phosphate solutions with known concentrations of 1 mM, 4 mM, 6 mM, 8 mM, and 10 mM are prepared for 31 the determination of ³¹P NMR signals. As shown in the appendix Figure 6 shown, the peak areas (converted to percentages) of the glucose 6-phosphate standard solution at different concentrations are obtained. With the concentration as the abscissa x and the peak area (converted to percentage) as the ordinate y, a standard curve is plotted to obtain the corresponding linear relationship diagram, as Figure 7 shown. The equation is y = 0.1008x + 0.1264. R 2 ² = 0.9984, indicating a good linear correlation between the peak area and the concentration.

[0060] A glucose 6-phosphate solution with a certain concentration is prepared for 31 ³¹P NMR detection. The peak area value is obtained, converted to a percentage, and substituted into the above equation y = 0.1008x + 0.1264 to obtain the concentration value (c1), which is compared with the true concentration value (c2). See Table 3 for details.

[0061] Table 3 Comparison of the true values and experimental values of glucose 6-phosphate solutions with different concentrations

[0062]

[0063] Note: Δ is the difference between c1 and c2, Δ = (c1-c2) / c2*100%.

[0064] Experimental results: From the above experimental results, we can see that using 31 P NMR technology can quickly and accurately determine the concentration of glucose 6-phosphate.

[0065] 2.2. HK enzyme activity assay

[0066] HK enzyme activity was detected by adding substrate glucose (2 mM) and ATP (1 mM) and HK enzyme (1 mg / mL) into the corresponding buffer (50 mM Tris-HCl pH 8.0, 5 mM MgCl 2 ) and then placed the sample in a 37°C environment for 3 h. 31 P NMR detection, HK enzyme catalysis experiment 31 P NMR detection diagram, such as Figure 8 shown.

[0067] The enzyme-catalyzed reaction consumes the substrate glucose and ATP and produces the product glucose-6-phosphate.

[0068] Experimental results: In the enzyme catalysis experiment 31 The peak area of ​​the product 6-phosphoglucose can be obtained from the detection graph of P NMR. After conversion to the percentage of the standard curve, 23.9% of 6-phosphoglucose was produced (assuming 10mM as 100%). Substituting it into the standard curve, the product produced 2.54mM. Enzyme activity is also called enzyme activity, which refers to the ability of an enzyme to catalyze a certain chemical reaction. The unit of enzyme activity is U, 1U = the amount of enzyme required to convert 1 micromole of substrate in 1 minute. Therefore, the actual enzyme activity of HK is 14.1U.

[0069] In summary, using 31 P NMR technology can be used for quantitative analysis of phosphorus-containing substances and rapid and accurate analysis of the activity of phosphate-modifying enzymes.

[0070] The above is only a preferred embodiment of the present invention. It should be pointed out that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for detecting the activity of a phospho-modifying enzyme based on 31P NMR of nuclear magnetic resonance phosphorus spectrum, characterized in that it includes the following steps: S1: Prepare standard solutions of substrates or products with known different concentrations, and perform nuclear magnetic resonance phosphorus spectroscopy (PNMR) measurements respectively. Obtain the peak area percentages of the standard solutions of the substrates or products with different concentrations, and plot a standard curve of the concentration versus the peak area percentage to obtain a linear equation for the relationship between the concentration and the peak area percentage; 31 ​ S2: By phosphorus nuclear magnetic resonance 31 The peak area percentage of the test solution is measured by PNMR, and the initial concentration of the substrate or product is obtained by substituting it into the linear equation of step S1. The test solution, buffer solution, and phosphoric acid-modifying enzyme are mixed and incubated, and a reaction solution is obtained after incubation. The reaction solution is analyzed by phosphorus nuclear magnetic resonance 31 PNMR detection to obtain the peak area percentage of the substrate or product in the reaction solution after incubation; the phosphoric acid-modifying enzyme is phosphatase, kinase, phosphodiesterase, or cyclase; S3: Substitute the peak area percentage of the substrate or product of the reaction solution in step S2 into the linear equation obtained in step S1 to obtain the concentration of the substrate or product of the reaction solution; S4: By comparing the concentration of the substrate or product of the reaction solution obtained in step S3 with the initial concentration of the substrate or product in step S2 to obtain the change in concentration, and calculate the enzyme activity of the phospho-modifying enzyme using the change in concentration per unit time.

2. A method for detecting the activity of a phosphoric acid-modified enzyme based on nuclear magnetic resonance phosphorus spectrum 31 PNMR characterized in that the phospho-modifying enzyme in step S2 can catalyze the phosphorylation modification of the substrate to generate the product or catalyze the dephosphorylation modification of the substrate to generate the product.

3. A method for detecting the activity of a phosphoric acid-modified enzyme based on nuclear magnetic resonance phosphorus spectrum 31 PNMR characterized in that the concentration of the phospho-modifying enzyme in step S2 is 0.2 - 1 mg / mL.

4. A method for detecting the activity of a phosphoric acid-modified enzyme based on phosphorus nuclear magnetic resonance 31 PNMR characterized in that The buffer solution described in step S2 is 40-50 mM Tris HCl with a pH of 6.5-8.0 and contains MgCl with a concentration of 5-10 mM 2 .

5. A method for detecting the activity of a phosphoric acid-modified enzyme based on nuclear magnetic resonance phosphorus spectrum 31 PNMR characterized in that the incubation time in step S2 is 10 min - 3 h.

6. A method for detecting the activity of a phosphoric acid-modified enzyme based on phosphorus nuclear magnetic resonance 31 PNMR characterized in that the incubation temperature in step S2 is 37 °C.

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