Competitive binding method for evaluating interaction between trypsin and flavonoid compounds

Abstract

The invention discloses a competitive binding method for evaluating interaction between trypsin and flavonoid compounds, which is characterized in that under the competitive mode of mass spectrum, the interaction between different flavonoid compounds and trypsin is analyzed through electrospray ionization mass spectrum (ESI-MS), and a parameter R is introduced, so that the relative strength of affinity can be compared; judging whether different flavonoid compounds have a competitive relationship with the combination of the trypsin or not, and further speculating whether the binding sites of the different flavonoid compounds on the trypsin are the same or not; the method is simple and rapid to operate and high in accuracy, and a feasible scheme can be provided for rapidly screening the trypsin inhibitor.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a competitive binding method for evaluating the interaction between trypsin and flavonoid compounds. Background Art

[0002] Trypsin is mainly secreted by the pancreas. As an important digestive enzyme, it not only participates in the digestion of protein in food, but also plays an important role in many physiological processes, such as hemostasis, apoptosis, signal transduction, reproduction and immunity. But at the same time, the occurrence of pancreatitis, emphysema, cancer and various cerebrovascular diseases is closely related to the excessive secretion of trypsin. Trypsin inhibitors are a class of drugs that have an inhibitory effect on trypsin. They have certain therapeutic effects on diseases such as acute and chronic pancreatitis, emphysema, and hemorrhagic shock, and have attracted widespread attention in the medical community. At present, many natural product drugs have been reported to have an inhibitory effect on trypsin, such as flavonoids such as quercetin and genistein. Therefore, studying the interaction between flavonoids and trypsin can open up new ways to design flavonoid derivatives with better drug effects and less side effects, which is of great significance for clinical medication, new drug development and design. However, the current research on the interaction between flavonoids and trypsin mainly relies on optical analysis methods, such as ultraviolet spectroscopy, which can obtain information such as the binding constant and binding stoichiometry between the two, but this method has low sensitivity and accuracy. In addition, in the existing literature on competitive mass spectrometry experiments, the change in the relative abundance of the binding peak or free protein peak is usually directly analyzed to determine the existence of a competitive relationship, but this result is easily affected by the mass spectrometry state, and the accuracy and repeatability are not high. Summary of the invention

[0003] In order to solve the problems raised in the above background technology, the present invention provides a competitive binding method for evaluating the interaction between trypsin and flavonoid compounds, using electrospray ionization mass spectrometry to explore the binding effect of trypsin and flavonoid compounds, using competitive binding mode, can simultaneously compare the affinity of multiple flavonoid compounds to trypsin in a mass spectrum, and introduce parameter R at the same time, not only can quantitatively compare the affinity of flavonoid compounds to trypsin, but also can judge whether there is a competitive relationship between different flavonoid compounds to trypsin, so as to infer whether the binding sites of different flavonoid compounds on trypsin are the same. The competitive binding mass spectrometry method provided by the present invention has fast analysis speed and high sensitivity, and can simultaneously obtain relevant information on the affinity and binding sites of trypsin and flavonoid compounds.

[0004] To achieve the above object, the present invention provides the following technical solutions:

[0005] The present invention provides a competitive binding method for evaluating the interaction between trypsin and flavonoid compounds, comprising the following steps:

[0006] S01, prepare a trypsin stock solution, dissolve the trypsin dry powder with ultrapure water to prepare a trypsin stock solution; mix the NH4OAc buffer solution and the trypsin stock solution, centrifuge with a 10K ultrafiltration membrane at a speed of 12000r / min for 20min at a temperature of 4°C, retain the solution in the ultrafiltration membrane, dilute with NH4OAc buffer solution, and repeat the centrifugation 4 to 5 times; use NanoDrop2000 ultra-micro spectrophotometer to determine the concentration to obtain a trypsin stock solution, and store it at -4°C in a dark place;

[0007] S02, preparing flavonoid compound stock solution: dissolving flavonoid compound P1 powder and flavonoid compound P2 powder in methanol respectively to prepare 1 mmol / L flavonoid compound P1 stock solution and flavonoid compound P2 stock solution, and storing them at -4°C in the dark;

[0008] S03, ESI-MS combination experiment: a certain amount of trypsin stock solution, flavonoid compound P1 stock solution, NH4OAc buffer solution and ultrapure water were mixed evenly and incubated at room temperature for 10 minutes. A certain amount of methanol was added before detection for mass spectrometry analysis. ESI-MS instrument parameters: positive ion mode, electrospray voltage of 3.0 kV, ion transfer tube temperature of 200 ° C, injection flow rate of 2 μL / min, resolution of 12000, ion optical radio frequency voltage (RF) of 30%. Each sample was tested 3 times.

[0009] S04, ESI-MS competition experiment: a certain amount of trypsin stock solution, flavonoid compound P1 stock solution, flavonoid compound P2 stock solution, NH4OAc buffer solution and ultrapure water were mixed evenly and incubated at room temperature for 10 minutes. A certain amount of methanol was added before detection, and mass spectrometry analysis was performed. ESI-MS instrument parameters: positive ion mode, electrospray voltage of 3.0 kV, ion transfer tube temperature of 200 ° C, injection flow rate of 2 μL / min, resolution of 12000, ion optical radio frequency voltage (RF) of 30%. Each sample was tested 3 times.

[0010] S05. Evaluate the relative affinity between trypsin and flavonoids: Parameter R represents the ratio of the sum of the relative abundances of trypsin-flavonoid complex ions to the sum of the relative abundances of trypsin and its complex ions. The larger the R value, the stronger the affinity of flavonoids to trypsin.

[0011]

[0012] N = 1-4; m = 8-10;

[0013] in, The sum of the relative abundances of the peaks representing trypsin-flavonoid complex ions of different charges, The relative abundance of the peaks representing the free, unbound flavonoids from trypsin of different charges is summed.

[0014] S06. Determine whether there is a competitive relationship between the binding of different flavonoid compounds and trypsin: ΔR / R (%) is the relative change in the R value of the binding of the flavonoid compound (P1) and trypsin before and after the addition of the flavonoid compound (P2).

[0015]

[0016] Wherein, R represents the R value of flavonoid compound (P1) and trypsin obtained in the competitive mode, and R0 represents the R value of flavonoid compound (P1) and trypsin obtained in the binding mode. If ΔR / R(%)>0, it means that the addition of P2 is conducive to the binding of P1 and trypsin, and P1 and P2 may have a synergistic effect; if ΔR / R(%)<0, it means that the addition of P2 is not conducive to the binding of P1 and trypsin, and P1 and P2 may have a competitive effect; if ΔR / R(%)=0, it means that the addition of P2 has no significant effect on the binding of P1 and trypsin, and there is no competitive relationship between the two.

[0017] Furthermore, in step S02, the flavonoid compound is one or more of chrysin (Chr), galangin (Gal), baicalin (Bai), luteolin (Lut), luteolin-3'-glucuronide (3'-Lut), and luteolin-7-glucuronide (7-Lut).

[0018] Furthermore, in step S03 or step S04, the concentration of trypsin in the ESI-MS sample solution is 5-10 μmol / L.

[0019] Furthermore, in step S03 or step S04, the concentration of the single flavonoid compound in the ESI-MS sample solution is 40-100 μmol / L.

[0020] Furthermore, in step S03 or step S04, the concentration ratio of trypsin to the single flavonoid compound in the ESI-MS sample solution is 1:8 to 1:10.

[0021] Furthermore, in step S03 or step S04, the content of methanol in the ESI-MS sample solution is 30% to 60%.

[0022] Further, in step S03 or step S04, the concentration of the NH4OAc buffer solution in the ESI-MS sample solution is 10-30 mmol / L pH=8.6.

[0023] Further, in step S04, the flavonoid compound P1 and the flavonoid compound P2 in the ESI-MS sample solution are two of chrysin (Chr), galangin (Gal), baicalein (Bai), luteolin (Lut), luteolin-3'-glucuronide (3'-Lut), and luteolin-7-glucuronide (7-Lut), and the concentration ratio between the two is 1:1.

[0024] In step S05, the parameter R is introduced to evaluate the relative strength of the affinity of different flavonoid compounds to trypsin, and R represents the ratio of the relative abundance of complex ions of different charges formed by trypsin-flavonoid compounds to the relative abundance of trypsin ions of different charges. The parameter R can not only compare the relative strength of the binding force, but also determine whether there is a competitive relationship between different flavonoid compounds and trypsin according to ΔR / R (%) in the competition mode, and then infer whether the binding sites of different flavonoid compounds on trypsin are the same.

[0025] According to the mass spectra obtained by S03 and S04, the trypsin-flavonoid complex ion, trypsin and its complex ion can be determined according to the relative molecular masses of trypsin and flavonoid.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention relates to a competitive binding method for evaluating the interaction between trypsin and flavonoid compounds. By using an electrospray ionization mass spectrometry method, two (or more) flavonoid compounds are mixed with trypsin and analyzed by mass spectrometry. The relative affinity strength and binding stoichiometry of different flavonoid compounds to trypsin can be intuitively compared in a mass spectrum, and the existence of a multi-component complex can be confirmed at the same time. In addition, the parameter R introduced by the present invention can be used to determine whether there is a competitive relationship between the binding of different flavonoid compounds to trypsin, and then the binding site can be inferred. The method has a fast analysis speed, high sensitivity and accuracy, and can simultaneously obtain relevant information such as the relative affinity strength and binding site of different flavonoid compounds, and is expected to be applied to the rapid screening and mechanism analysis of trypsin inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the mass spectrum of the combination of trypsin and flavonoid compound Lut (P1) in Example 1 of the present invention;

[0029] Figure 2This is a mass spectrum of competitive binding between trypsin and flavonoid compounds Lut (P1) and 3'-Lut (P2) in Example 2 of the present invention;

[0030] Figure 3 The R value change of the combination of Try and Lut (P1) after adding different flavonoid compounds (P2) in Example 2 of the present invention. DETAILED DESCRIPTION

[0031] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Preparation of trypsin stock solution: Accurately weigh 2-5 mg of trypsin dry powder and dissolve it in 1 mL of ultrapure water to prepare a trypsin stock solution of a certain concentration. Mix 300 μL of 10 mmol / L NH4OAc and 200 μL of trypsin stock solution, centrifuge with a 10K ultrafiltration membrane: speed 12000 r / min, centrifugation time 20 min, temperature 4°C, retain the solution in the ultrafiltration membrane, dilute to 500 μL with 10 mmol / L NH4OAc, and repeat centrifugation 4-5 times. Use NanoDrop 2000 ultra-micro spectrophotometer to determine the concentration to obtain trypsin stock solution, refrigerate at -4°C and store away from light.

[0033] 2) Preparation of flavonoid stock solution: accurately weigh a certain amount of flavonoid powder, dissolve it in 1 mL of methanol to prepare a 1 mmol / L flavonoid stock solution, and store it at -4°C in the dark.

[0034] Example 1

[0035] A certain amount of trypsin stock solution, Lut stock solution, NH4OAc buffer solution and ultrapure water were mixed evenly, and incubated at room temperature for 10 minutes to obtain a trypsin and Lut mixed solution (containing 5 μmol / L trypsin, 50 μmol / L Lut, 20 mmol / L NH4OAc, pH = 8.6). 50% methanol was added to mix and then mass spectrometry analysis was performed. Figure 1 This is the mass spectrum of trypsin combined with Lut. From the figure, we can observe the ion peaks with Try charges of 10+ to 8+ ([Try] 10+ 、[Try] 9+ and [Try] 8+, m / z were 2333.69, 2592.88, and 2916.86, and the relative abundances were 15.5%, 34.0%, and 8.3%, respectively), and a complex ion peak of Try-Lut with a 1:1 binding ([Try+P1] 10+ 、[Try+P1] 9+ and [Try+P1] 8+ , m / z were 2362.29, 2624.76, and 2952.61, with relative abundances of 6.2%, 13.2%, and 3.0%, respectively), and the 1:2 binding complex ion peak ([Try+2P1] 10+ 、[Try+2P1] 9+ and [Try+2P1] 8+ , m / z were 2390.99, 2656.65, 2988.61, and the relative abundances were 3.5%, 6.2%, and 1.6%, respectively), and the 1:3 binding complex ion peak ([Try+3P1] 10+ 、[Try+3P1] 9+ and [Try+3P1] 8+ , m / z were 2419.49, 2688.32, 3024.37, and the relative abundances were 2.3%, 3.0%, and 1.1%, respectively). The R of Try-Lut was calculated to be 0.41. The mixed solution of trypsin and Chr was prepared in the same way, and mass spectrometry was performed to obtain the R of Try-Chr to be 0.24. By comparing the R values ​​of different flavonoid compounds, the results showed that the relative affinity of Lut to trypsin is stronger than that of Chr.

[0036] Example 2

[0037] A certain amount of trypsin stock solution, Lut stock solution (P1), NH4OAc buffer solution and ultrapure water were mixed evenly, incubated at room temperature for 10 minutes, and a mixed solution of trypsin and Lut (containing 5μmol / L trypsin, 50μmol / LLut, 20mmol / LNH4OAc, pH=8.6) was obtained as a control group (control) for mass spectrometry analysis. The remaining five flavonoid compound stock solutions (P2, the same concentration as Lut) were added to a certain amount of trypsin stock solution, Lut stock solution, NH4OAc buffer solution and ultrapure water mixed solution, mixed evenly, incubated at room temperature for 10 minutes, and a mixed solution of trypsin and P1 and P2 (containing 5μmol / L trypsin, 50μmol / LP1, 50μmol / LP2, 20mmol / LNH4OAc, pH=8.6) was obtained for mass spectrometry analysis. Among them, the mass spectrum results of competitive binding between trypsin and flavonoid compounds Lut (P1) and 3'-Lut (P2) are shown as follows: Figure 2As shown in the figure, the complex ion peaks of Try-Lut(P1) with a binding ratio of 1:1 to 1:2 can be observed ([Try+P1] 9+ and [Try+2P1] 9+ , m / z 2624.75 and 2656.54, with relative abundances of 22.3% and 7.8%, respectively), and the complex ion peak of Try-3'-Lut(P2) with a ratio of 1:1 to 1:2 ([Try+P2] 9+ and [Try+2P2] 9+ , m / z 2643.97 and 2695.64, with relative abundances of 21.8% and 9.0%, respectively), and the ternary complex ion peak of Try combined with P1 and P2 ([Try+P1+P2] 9+ , m / z is 2676.08, and the relative abundance is 14.0%).

[0038] The R values ​​obtained in the experiment are plotted into a bar graph, such as Figure 3 As shown, the addition of Gal has no significant effect on the R value of Lut and Try, while the addition of Chr, 3'-Lut, and 7-Lut leads to a decrease in the R of Try-Lut by 21.4%, 20.6%, and 18.3%, respectively. This indicates that the three flavonoids Chr, 3'-Lut, and 7-Lut are in a competitive relationship with Lut. It is speculated that the flavonoids Chr, 3'-Lut, 7-Lut, and Lut have the same binding site on Try, while the binding site of Gal and Try is different from that of Lut. In addition, it was found that the addition of Bai led to an increase of 16.7% in the R value of Try-Lut, indicating that the addition of Bai is conducive to the binding of Try and Lut, and it is speculated that Bai and Lut have a synergistic effect.

[0039] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0040] It should be noted that the above content only illustrates the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications all fall within the protection scope of the claims of the present invention.

Claims

1. A competitive binding method for evaluating the interaction between trypsin and flavonoid compounds, characterized in that: The following steps are involved: S01, prepare a trypsin stock solution, dissolve the trypsin dry powder with ultrapure water to prepare a trypsin stock solution; mix the NH4OAc buffer solution and the trypsin stock solution, centrifuge with a 10K ultrafiltration membrane at a speed of 12000r / min for 20min at a temperature of 4°C, retain the solution in the ultrafiltration membrane, dilute with NH4OAc buffer solution, and repeat the centrifugation 4 to 5 times; use NanoDrop2000 ultra-micro spectrophotometer to determine the concentration to obtain a trypsin stock solution, and store it at -4°C in a dark place; S02, preparing flavonoid compound stock solution: dissolving flavonoid compound P1 powder and flavonoid compound P2 powder in methanol respectively to prepare 1 mmol / L flavonoid compound P1 stock solution and flavonoid compound P2 stock solution, and storing them at -4°C in the dark; S03, ESI-MS combined experiment, trypsin stock solution, flavonoid compound P1 stock solution, NH4OAc buffer solution and ultrapure water were mixed evenly and incubated at room temperature for 10 minutes; methanol was added before detection to obtain ESI-MS sample solution for mass spectrometry analysis; S04, ESI-MS competition experiment, trypsin stock solution, flavonoid compound P1 stock solution, flavonoid compound P2 stock solution, NH4OAc buffer solution and ultrapure water were mixed evenly and incubated at room temperature for 10 minutes; methanol was added before detection to obtain ESI-MS sample solution for mass spectrometry analysis; S05. Evaluate the relative affinity between trypsin and flavonoids: The parameter R represents the ratio of the sum of the relative abundances of trypsin-flavonoid complex ions to the sum of the relative abundances of trypsin and its complex ions, as shown in the following formula; the larger the R value, the stronger the affinity of flavonoids to trypsin. in, The sum of the relative abundances of the peaks representing trypsin-flavonoid complex ions of different charges, The sum of the relative abundances of the peaks representing the free unbound flavonoids trypsin with different charges; n = 1-4, m = 8-10; S06. Determine whether there is a competitive relationship between the binding of different flavonoid compounds and trypsin: ΔR / R (%) is the relative change in the R value of the binding of flavonoid compound P1 and trypsin before and after the addition of flavonoid compound P2. Wherein, R represents the R value of flavonoid compound P1 and trypsin obtained in the competition mode, and R0 represents the R value of flavonoid compound P1 and trypsin obtained in the binding mode. If ΔR / R(%)>0, it means that the addition of P2 is conducive to the binding of P1 and trypsin, and P1 and P2 may have a synergistic effect; if ΔR / R(%)<0, it means that the addition of P2 is not conducive to the binding of P1 and trypsin, and P1 and P2 may have a competitive effect; if ΔR / R(%)=0, it means that the addition of P2 has no significant effect on the binding of P1 and trypsin, and there is no competitive relationship between the two.

2. A competitive binding method for evaluating the interaction between trypsin and flavonoid compounds according to claim 1, characterized in that: In step S02, the flavonoid compound is one or more of chrysin, galangin, baicalin, luteolin, luteolin-3'-glucuronide, and luteolin-7-glucuronide.

3. A competitive binding method for evaluating the interaction between trypsin and flavonoids according to claim 1, characterized in that: In step S03 or step S04, the concentration of trypsin in the ESI-MS sample solution is 5 to 10 μmol / L.

4. A competitive binding method for evaluating the interaction between trypsin and flavonoid compounds according to claim 1, characterized in that: In step S03 or step S04, the concentration of the single flavonoid compound in the ESI-MS sample solution is 40-100 μmol / L.

5. A competitive binding method for evaluating the interaction between trypsin and flavonoids according to claim 1, characterized in that: In step S03 or step S04, the concentration ratio of trypsin to the single flavonoid compound in the ESI-MS sample solution is 1:8 to 1:

10.

6. A competitive binding method for evaluating the interaction between trypsin and flavonoids according to claim 1, characterized in that: In step S03 or step S04, the content of methanol in the ESI-MS sample solution is 30% to 60%.

7. A competitive binding method for evaluating the interaction between trypsin and flavonoids according to claim 1, characterized in that: In step S03 or step S04, the concentration of the NH4OAc buffer solution in the ESI-MS sample solution is 10-30 mmol / L pH=8.

6.

8. A competitive binding method for evaluating the interaction between trypsin and flavonoids according to claim 1, characterized in that: In step S04, the flavonoid compound P1 and the flavonoid compound P2 in the ESI-MS sample solution are two of chrysin, galangin, baicalein, luteolin, luteolin-3'-glucuronide, and luteolin-7-glucuronide, and the concentration ratio of the two is 1:

1.

9. A competitive binding method for evaluating the interaction between trypsin and flavonoids according to claim 1, characterized in that: In step S03 or step S04, the ESI-MS instrument parameters are: positive ion mode, electrospray voltage of 3.0 kV, ion transfer tube temperature of 200°C, injection flow rate of 2 μL / min, resolution of 12000, ion optical radio frequency voltage (RF) of 30%; each sample is detected 3 times.

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