CN113189069A - Pesticide-target protein screening method based on fluorescence analysis - Google Patents
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Abstract
The invention discloses a pesticide-target protein screening method based on fluorescence analysis. The method selects an environment-sensitive fluorescent probe to be pre-combined with protein to obtain a supramolecular system, and indicates the combination process of corresponding protein and pesticide according to the fluorescence change of the probe before and after the action of the protein and the pesticide; the method relates to flexible and controllable system composition, and can select a pre-combined fluorescent probe according to different proteins, and further add a specific pesticide for rapid screening to obtain a potential target protein with strong binding effect with the pesticide. The invention visually displays the effect of the pesticide and the target protein as spectral data by detecting the change of the fluorescent signal, provides a simple, universal and quick analysis method for the current pesticide target research field, and is particularly suitable for screening the target protein of new drugs and oriented pesticides, calculating the binding constant, Gibbs free energy change and the like.
Description
Technical Field
The invention belongs to the technical field of biochemistry, and particularly relates to a pesticide-target protein screening method based on fluorescence analysis.
Background
Traditional pesticideMost of the pesticide target proteins are combined with specific receptors (target proteins) to generate effects, and for the novel oriented pesticide with oriented transport, the target proteins of the pesticide are determined to help to clarify the oriented pesticide transport and enrichment processes, so that the establishment of a simple and effective analysis method for analyzing the interaction between the pesticide and the target proteins is important research content for researching and developing the novel oriented pesticide and clarifying the action mechanism of the pesticide. The prior art comprises isotope labeling (CN201810712528.X), mass spectrometry (CN201710103155.1), isothermal calorimetric titration and the like. The binding constant (K) of the protein and the small molecule can be obtained by the methoda) A series of physicochemical parameters such as Gibbs free energy change (delta G), combination proportion (n), entropy change (delta S), enthalpy change (delta H) and the like, but the existing method needs expensive instruments and complicated sample processing process, so that a simple and effective analysis method for pesticide-target protein mutual screening still needs to be developed.
Fluorescence assays have been used to analyze protein-small molecule interactions, such as the lantha screen eu kinase binding assay provided by ThermoFisher, and patent CN 111065926 a teaches methods for characterizing protein-small molecule interactions based on fluorescent dye-attached nucleic acids, among others. Although the method can provide the binding property of the protein and the specific small molecule, a more complicated sample preparation process is still needed, and a method for rapidly, universally and efficiently analyzing the interaction between different types of pesticides and potential target proteins is still lacked in the field of pesticide research.
Disclosure of Invention
The invention aims to provide a pesticide-target protein screening method based on fluorescence analysis and application thereof. The invention relates to protein-small molecule supramolecular action, and provides a protein-probe supramolecular system based on an environment-sensitive fluorescent probe, and application of the protein-probe supramolecular system in the aspects of pesticide and protein combination research, combination constant calculation and Gibbs free energy calculation.
The invention relates to a pesticide-target protein screening method based on fluorescence analysis, which comprises the following steps:
s1, selecting an environment-sensitive fluorescent probe capable of being combined with experimental protein, and respectively preparing a protein mother liquor a and a fluorescent probe mother liquor b; diluting the protein mother liquor a, adding a proper amount of fluorescent probe mother liquor b, mixing and dissolving to obtain a supramolecular system c, and testing the fluorescence spectrum of the system c;
s2, preparing a pesticide mother solution d to be tested, respectively dropwise adding pesticides to be tested with different concentrations into the system c, uniformly mixing, and testing fluorescence spectra under different titration concentrations;
s3, establishing a quantitative relation between the fluorescence signal measured in the steps S1 and S2 and the concentration of the pesticide to be measured, and calculating by using a Benesi-Hildebrand formula to obtain a dissociation constant KdFurther calculating to obtain the binding constant K of the pesticide to be detected and the experimental proteina、ΔG。
Preferably, the Benesi-Hildebrand formula in step S3 is as follows: y is Bmaxx/(Kd+ x), where x is the concentration of the pesticide, y is the fluorescence signal obtained experimentally at the concentration of the titration of x, BmaxIs a top asymptote, KdIs the dissociation constant; calculating the dissociation constant K by using a Benesi-Hildebrand formuladThen according to Ka＝1/KdCalculating to obtain a binding constant KaIs a reaction of KaSubstituting into the thermodynamic equation Δ G ═ RTln (K)a) Wherein R is a natural constant, T is temperature, KaΔ G was calculated as the binding constant.
The experimental protein is a known target protein or a candidate target protein of the pesticide to be detected, and the target protein is biological enzyme, receptor protein and serum protein.
Preferably, the test protein is acetylcholinesterase, GABA receptor, nicotinic acetylcholine receptor, ryanodine receptor or serum protein.
Preferably, the serum protein is human serum protein (HSA), Bovine Serum Albumin (BSA) or egg serum protein (ESA).
The solvent of the protein mother liquor a is common buffer solution, including but not limited to PBS, Tris and HEPES buffer solution, the pH range of the buffer solution is 4.9-8.9, and the concentration of the protein mother liquor is 1-80 mg/ml.
The fluorescent probe is a fluorescent molecule with an electron donor-acceptor structure (D-A) or a molecular rotor structure (molecular rotor). The solvent of the fluorescence probe mother liquor b is a common solvent, and includes but is not limited to tetrahydrofuran, dimethyl sulfoxide, acetonitrile, water and the like, and the concentration of the mother liquor is 1-10 mM.
Preferably, the fluorescent probe is a chalcone 4MC fluorescent probe.
Preferably, the step S1 specifically includes: selecting an environment-sensitive fluorescent probe capable of being combined with experimental protein, preparing a protein mother solution a with the concentration of 1-80 mg/ml by taking a PBS buffer solution as a solvent, and preparing a fluorescent probe mother solution b with the concentration of 1-10 mM by taking dimethyl sulfoxide (DMSO) as a solvent; diluting the protein mother liquor a to 10 mu M with PBS buffer solution, dripping the fluorescent probe mother liquor b under the oscillation condition of 2500rpm, and dripping the fluorescent probe mother liquor b into the system c, wherein the volume of the fluorescent probe mother liquor b is less than 5 percent of the total volume of the system c; and (5) standing for 2min after continuously oscillating for 1min to obtain a system c, and testing the fluorescence spectrum of the system c.
Preferably, the step S2 specifically includes: preparing a pesticide mother solution d to be detected, wherein the concentration of the pesticide mother solution d is 10-50 mM, and respectively dropwise adding pesticides to be detected with different concentrations into the system c, wherein the volume of the dropwise adding pesticides is less than 5% of the total volume of the system c; oscillating at 2500rpm for 1min, standing for 2min, and testing fluorescence spectra at different titration concentrations.
The pesticide to be detected can be a known pesticide and a new drug, and comprises but is not limited to carbamate pesticides, organophosphorus pesticides, benzopyrazoles pesticides, pyrethroid pesticides, neonicotinoids pesticides, anthranilamide pesticides, insect growth regulators, bactericides, herbicides, acaricides and the like.
Preferably, the pesticide to be detected is pyrazole spiro c-27, cyfluthrin, permethrin, cypermethrin or tetramethrin.
The solvent of the pesticide mother liquor d is a common solvent, and comprises but is not limited to dimethyl sulfoxide, tetrahydrofuran, N-dimethylformamide, methanol, water and the like, and the concentration of the mother liquor is 10-50 mM.
The titration spectra of the experimental protein and the pesticide to be detected are obtained according to the change of the fluorescence spectra after the pesticide to be detected with different concentrations is dripped into the system c, and the change of the fluorescence spectra comprises but is not limited to fluorescence intensity quenching, fluorescence intensity enhancement, fluorescence band red shift, blue shift and the like.
Preferably, the fluorescence signal is fluorescence intensity, ratio fluorescence intensity and luminescence wavelength.
The invention also provides application of the pesticide-target protein screening method based on fluorescence analysis in qualitative and quantitative analysis of interaction between pesticides and potential target proteins.
Has the advantages that:
the invention combines protein and fluorescent probe to form a composite system, and indicates the combination process of the protein and pesticide according to the fluorescent signal change before and after the pesticide replaces the fluorescent probe.
The invention can flexibly regulate and control the system composition according to the requirements, can screen the target protein for a single pesticide, and can analyze the binding force difference between the specific target protein and different pesticides.
The invention relies on a fluorescence analysis method, has the advantages of simple and convenient sample preparation, rapid sample measurement, high sensitivity and the like, and can be developed to utilize an enzyme-labeling instrument to rapidly screen various pesticides. The binding constant (K) can be calculated by fitting the fluorescent signal to the pesticide concentrationa) And the change of free energy (Δ G).
The invention visually displays the effect of the pesticide and the target protein as spectral data by detecting the change of the fluorescent signal, provides a simple, universal and quick analysis method for the current pesticide target research field, and is particularly suitable for screening the target protein of new drugs and oriented pesticides, calculating the binding constant, Gibbs free energy change and the like.
Drawings
FIG. 1 shows fluorescence spectra before and after the three protein-probe systems act on the pesticide pyrazole spiro ring c-27.
FIG. 2 shows the fluorescence titration spectra of the pesticide pyrazole spiro c-27 and egg serum protein and the fitting result of the fluorescence signal to the pesticide concentration.
FIG. 3 shows fluorescence spectra of human serum albumin before and after the action of four pyrethroid insecticides.
Figure 4 is structural characterization data for chalcone 4 MC.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
The following is described for the fluorescent probe chalcone 4MC referred to in the examples below:
chalcone 4MC (the structural formula is shown in formula I) is prepared by self,
the preparation of the compound of formula I is as follows:
dissolving 4-methoxy o-hydroxyacetophenone and p-dimethylaminobenzaldehyde in a mixed solvent of ethanol and water, wherein the water content is 30-50 wt%, adding 10ml of NaOH aqueous solution (10-20 wt%), stirring at 25-80 ℃ for 18-24 h, pouring the reaction solution into 1000ml of water, adding acid for neutralization, and recrystallizing to obtain 4 MC. Compound structure characterization data are shown in figure 4.
The invention provides a pesticide-target protein screening method based on fluorescence analysis.
Example 1:
this example discloses fluorescence spectra before and after the action of a new drug with three protein-probe systems, comprising the following steps:
A. 66.5mg of Human Serum Albumin (HSA), 66.4mg of Bovine Serum Albumin (BSA) and 44.7mg of Egg Serum Albumin (ESA) are respectively dissolved in 1ml of PBS buffer solution (0.1X, pH 7.4) to obtain protein mother solutions a1, a2 and a3, and 2.8mg of chalcone 4MC (structural formula I) fluorescent probe is dissolved in 5ml of DMSO to obtain a mother solution b.
B. 20 mu L of protein mother liquor a1, a2, a3 and 10 mu L of mother liquor b are respectively transferred to 2ml of PBS buffer solution, the solution is shaken at 2500rpm for 1min and then stands for 2min to respectively obtain solutions c1, c2 and c3, and the fluorescence spectra of the protein-probe systems of the solutions c1, c2 and c3 are tested.
C. Preparing a pyrazole spiro c-27 (the structural formula of the pyrazole spiro c-27 is shown in a formula II and disclosed in patent application CN 201810219338.4) mother solution d (40mM), respectively adding the pesticide pyrazole spiro c-27 mother solution d into solutions c1, c2 and c3 to an excessive concentration (0.2mM), oscillating at 2500rpm for 1min, standing for 2min, and testing the fluorescence spectrum of a sample.
Fluorescence spectra before and after the action of three protein-probe systems of solutions c1, c2 and c3 and the pesticide pyrazole spiro ring c-27 are shown in figure 1. When 20eq of the pesticide pyrazole spiro c-27 was added, the three serum protein-probe systems showed different spectral responses, in which the fluorescence of HSA-4MC decreased rapidly with increasing pyrazole spiro c-27 concentration, the fluorescence intensity of BSA-4MC hardly changed, and the fluorescence intensity of ESA-4MC increased progressively with increasing pyrazole spiro c-27 concentration. The result shows that the protein-probe system can analyze the binding effect of a plurality of proteins and the same pesticide, in the embodiment, the obvious binding effect of HSA, ESA and pyrazole spiro c-27 can be rapidly screened according to the spectrum response result, and the binding effect of BSA and pyrazole spiro c-27 in the method is weaker. The protein-probe system obtained by screening can be used for further analyzing the binding constant of the protein and the pesticide.
Example 2:
the embodiment discloses data for calculating a protein and pesticide binding constant by using fluorescence titration spectrum, which comprises the following steps:
A. solution c3 (egg serum albumin (ESA) with chalcone 4MC fluorescent probe) prepared as in example 1 was titrated using pesticide mother liquor d (40mM pyrazole spiro c-27) formulated at different concentrations (0, 0.04, 0.08, 0.12, 0.16, 0.2, 0.24, 0.28mM) from example 1 and tested for fluorescence spectra at the different titrated concentrations.
B. Substituting the fluorescence intensity I corresponding to the fluorescence peak and the pesticide addition concentration C into the Benesi-Hildebrand formula y ═ Bmaxx/(Kd+ x), where x is the pesticide concentration in the system after addition, y is the fluorescence signal obtained by the experiment under the titration concentration of x, BmaxIs a top asymptote, KdFor the dissociation constant, K is calculateddThen according to Ka＝1/KdFrom K bydCalculating to obtain KaAnd substituting into the thermodynamic formula Δ G ═ RTln (K)a) Obtaining Δ G, where R is a natural constant, T is temperature, KaIs the binding constant.
The fluorescence titration spectra of pyrazole spiro c-27 and egg serum protein are shown in FIG. 2a, and the fitting result of fluorescence intensity I corresponding to fluorescence peak to concentration of pesticide pyrazole spiro c-27 is shown in FIG. 2b, according to the fitting curve (y ═ 1.233 × 10)-8x+7.383×10-5R of which20.9973), K is calculatedd＝1.6×10-4，ΔG＝-21.7kJ/mol。
Example 3:
this example shows fluorescence titration spectra of Human Serum Albumin (HSA) and four pyrethroid insecticides (permethrin, cypermethrin, tetramethrin).
The method specifically comprises the following steps:
1) and (3) preparing four pyrethroid pesticides namely cyfluthrin, permethrin, cypermethrin and tetramethrin according to the step C in the example 1, wherein the concentration of the mother liquor is 40mM, and d 1-4.
2) Human Serum Albumin (HSA) and chalcone 4MC fluorescent probe solutions were prepared according to step A, B in example 1, specifically:
A. 66.5mg Human Serum Albumin (HSA) was dissolved in 1ml PBS buffer (0.1X, pH 7.4) to give protein stock a1, and 2.8mg chalcone 4MC fluorescent probe was dissolved in 5ml DMSO to give stock b.
B. Transferring 20 mu L of the protein mother liquor a1 and 10 mu L of the mother liquor b to 2ml of PBS buffer solution, shaking at 2500rpm for 1min, standing for 2min to obtain a solution c1, and testing the fluorescence spectrum of the solution c1 protein-probe system.
3) The fluorescence titration spectra of four pesticides on the HSA-fluorescent probe are respectively tested, and specifically comprise the following steps:
pesticide with different concentrations (40mM of cyhalothrin, permethrin, cypermethrin or tetramethrin mother liquor is respectively diluted to prepare different concentration gradients, the final concentration range of the pesticide in the system is 0-500 mu M), the mixture is oscillated at 2500rpm for 1min and then is kept stand for 2min, and the fluorescence spectrum of each sample is tested.
FIG. 3 shows fluorescence spectra of HSA-4MC before and after the action with the four pyrethroid pesticides, and as shown in the figure, the fluorescence spectrum of HSA-4MC has good response performance to the four pyrethroid pesticides. The result shows that the protein-probe system can analyze and compare the combination effect of the same protein and various pesticides with the same type and similar structures.
In conclusion, the method provided by the invention can judge whether the pesticide and the protein have the binding effect through the change of the fluorescence spectrum, and then the binding constant (K) can be calculated by fitting the concentration by using the fluorescence signala) And Gibbs free energy change (delta G). The method can quickly screen the protein combined with the existing pesticide, can also investigate the combination effect of a certain selected protein and different pesticides, and ensures the universality of the method by the flexible controllability of the system composition.
Claims (10)
1. A pesticide-target protein screening method based on fluorescence analysis is characterized by comprising the following steps:
s1, selecting an environment-sensitive fluorescent probe capable of being combined with experimental protein, and respectively preparing a protein mother liquor a and a fluorescent probe mother liquor b; diluting the protein mother liquor a, adding a proper amount of fluorescent probe mother liquor b, mixing and dissolving to obtain a supramolecular system c, and testing the fluorescence spectrum of the system c;
s2, preparing a pesticide mother solution d to be tested, respectively dropwise adding pesticides to be tested with different concentrations into the system c, uniformly mixing, and testing fluorescence spectra under different titration concentrations;
s3, establishing a quantitative relation between the fluorescence signal measured in the steps S1 and S2 and the concentration of the pesticide to be measured, and calculating by using a Benesi-Hildebrand formula to obtain a dissociation constant KdFurther calculating to obtain the binding constant K of the pesticide to be detected and the experimental proteina、ΔG。
2. The fluorescence analysis-based screening method for pesticide-target protein as claimed in claim 1, wherein the Benesi-Hildebrand formula in step S3 is as follows: y is Bmaxx/(Kd+ x), where x is the concentration of the pesticide, y is the fluorescence signal obtained experimentally at the concentration of the titration of x, BmaxIs a top asymptote, KdIs the dissociation constant; calculating the dissociation constant K by using a Benesi-Hildebrand formuladThen according to Ka＝1/KdCalculating to obtain a binding constant KaIs a reaction of KaSubstituting into the thermodynamic equation Δ G ═ RTln (K)a) Wherein R is a natural constant, T is temperature, KaΔ G was calculated as the binding constant.
3. The method of claim 1, wherein the test protein is acetylcholinesterase, GABA receptor, nicotinic acetylcholine receptor, ryanodine receptor or serum protein.
4. The method for screening a pesticide-target protein based on fluorescence analysis of claim 3, wherein the serum protein is human serum protein, bovine serum albumin or egg serum protein.
5. The method for screening pesticide-target protein based on fluorescence analysis of claim 1, wherein the fluorescent probe is chalcone 4MC fluorescent probe.
6. The method for screening a pesticide-target protein based on fluorescence analysis according to claim 1, wherein the step S1 specifically comprises: selecting an environment-sensitive fluorescent probe capable of being combined with experimental protein, preparing a protein mother solution a with the concentration of 1-80 mg/ml by using a PBS buffer solution as a solvent, and preparing a fluorescent probe mother solution b with the concentration of 1-10 mM by using dimethyl sulfoxide as a solvent; diluting the protein mother liquor a to 10 mu M with PBS buffer solution, dripping the fluorescent probe mother liquor b under the oscillation condition of 2500rpm, and dripping the fluorescent probe mother liquor b into the system c, wherein the volume of the fluorescent probe mother liquor b is less than 5 percent of the total volume of the system c; and (5) standing for 2min after continuously oscillating for 1min to obtain a system c, and testing the fluorescence spectrum of the system c.
7. The method for screening a pesticide-target protein based on fluorescence analysis according to claim 1, wherein the step S2 specifically comprises: preparing a pesticide mother solution d to be detected, wherein the concentration of the pesticide mother solution d is 10-50 mM, and respectively dropwise adding pesticides to be detected with different concentrations into the system c, wherein the volume of the dropwise adding pesticides is less than 5% of the total volume of the system c; oscillating at 2500rpm for 1min, standing for 2min, and testing fluorescence spectra at different titration concentrations.
8. The method for screening pesticide-target protein based on fluorescence analysis of claim 1, wherein the pesticide to be detected is pyrazole spiro c-27, cyfluthrin, permethrin, cypermethrin or tetramethrin.
9. The method for screening a pesticide-target protein based on fluorescence analysis of claim 1, wherein the fluorescence signal is fluorescence intensity, ratio fluorescence intensity and luminescence wavelength.
10. Use of the fluorescence analysis based pesticide-target protein screening method of any one of claims 1 to 9 for qualitative and quantitative analysis of pesticide interaction with potential target proteins.
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| US5900944A (en) * | 1997-03-03 | 1999-05-04 | Day-Glo Color Corp. | Method and device for the analysis of pesticides |
| CN101293131A (en) * | 2007-04-29 | 2008-10-29 | 上海普天欣生物技术有限公司 | Complex enzyme preparation for eliminating residual pesticides and application thereof |
| CN110092771A (en) * | 2019-04-19 | 2019-08-06 | 深圳大学 | A kind of fluorescence probe and preparation method thereof for human serum albumins detection, Fluorescence kit |
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