A competitive binding method for assessing the interaction between trypsin and flavonoids

Abstract

The application discloses a competitive binding method for evaluating the interaction between trypsin and flavonoids, wherein the interaction between different flavonoids and trypsin is analyzed by means of electrospray ionization mass spectrometry (ESI-MS) in a competitive mode of mass spectrometry; the parameter R can be introduced to compare the relative strength of the affinity, to judge whether the different flavonoids compete with each other in the binding to trypsin, and to further infer whether the binding sites of the different flavonoids on trypsin are the same; the method is simple, fast and high in accuracy, and can provide a feasible scheme for rapidly screening trypsin inhibitors.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a competitive binding method for evaluating the interaction between trypsin and flavonoids. BACKGROUND

[0002] Trypsin is mainly secreted by the pancreas, as an important digestive enzyme, not only involved 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 immune physiological processes. However, 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 inhibitory effect on trypsin, and have certain curative effect on acute and chronic pancreatitis, emphysema, hemorrhagic shock and other diseases, which has attracted widespread attention in the medical field. At present, many natural product drugs have been reported to have inhibitory effect on trypsin, such as quercetin, genistein and other flavonoids. Therefore, the study of the interaction between flavonoids and trypsin can open up a new way for the design of flavonoid derivatives with better drug effect and smaller side effect, which has important 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 method, such as ultraviolet spectrometry, which can obtain the binding constant and binding stoichiometry of the two. However, the sensitivity and accuracy of this method are low. In addition, in the existing literature on competitive mass spectrometry experiments, the existence of competition relationship is usually judged by directly analyzing the change of the relative abundance of the binding peak or free protein peak, but this result is easily affected by the mass spectrometry state, and the accuracy and repeatability are not high. SUMMARY

[0003] To solve the problems raised in the background art, the present application provides a competitive binding method for evaluating the interaction between trypsin and flavonoids, which uses electrospray ionization mass spectrometry to explore the binding of trypsin and flavonoids, adopts a competitive binding mode, and can compare the affinity of multiple flavonoids to trypsin in one mass spectrum. At the same time, the introduction of parameter R can not only quantitatively compare the affinity of flavonoids to trypsin, but also judge whether there is a competition relationship between different flavonoids to trypsin, so as to infer whether the binding sites of different flavonoids on trypsin are the same. The competitive binding mass spectrometry method provided by the present application has fast analysis speed and high sensitivity, and can obtain the information related to the affinity and binding site of trypsin and flavonoids at the same time.

[0004] To achieve the above purpose, the present application provides the following technical scheme:

[0005] The application provides a competitive binding method for evaluating the interaction between trypsin and flavonoids, comprising the following steps:

[0006] S01, preparing a trypsin stock solution, dissolving trypsin dry powder in ultrapure water to prepare a trypsin stock solution; mixing the NH4OAc buffer solution and the trypsin stock solution, centrifuging at a speed of 12000 r / min for 20 min using a 10K ultrafiltration membrane, at a temperature of 4 DEG C, retaining the solution in the ultrafiltration membrane, diluting with the NH4OAc buffer solution, and repeating the centrifugation for 4-5 times; using a NanoDrop2000 ultramicro spectrophotometer to determine the concentration to obtain the trypsin stock solution, which is stored at -4 DEG C and kept away from light;

[0007] S02, preparing a flavonoid stock solution: dissolving flavonoid P1 powder and flavonoid P2 powder in methanol respectively to prepare 1 mmol / L flavonoid P1 stock solution and flavonoid P2 stock solution, which are stored at -4 DEG C and kept away from light;

[0008] S03, ESI-MS binding experiment: mixing a certain amount of trypsin stock solution, flavonoid P1 stock solution, NH4OAc buffer solution and ultrapure water uniformly, incubating at room temperature for 10 min. Adding a certain amount of methanol before detection, and performing mass spectrometry analysis, ESI-MS instrument parameters: positive ion mode, electrospray voltage is 3.0 kV, ion transmission tube temperature is 200 DEG C, sample flow rate is 2 muL / min, resolution is 12000, ion optical radio frequency voltage (RF) is 30%. Each sample is detected for 3 times.

[0009] S04, ESI-MS competition experiment: mixing a certain amount of trypsin stock solution, flavonoid P1 stock solution, flavonoid P2 stock solution, NH4OAc buffer solution and ultrapure water uniformly, incubating at room temperature for 10 min. Adding a certain amount of methanol before detection, and performing mass spectrometry analysis, ESI-MS instrument parameters: positive ion mode, electrospray voltage is 3.0 kV, ion transmission tube temperature is 200 DEG C, sample flow rate is 2 muL / min, resolution is 12000, ion optical radio frequency voltage (RF) is 30%. Each sample is detected for 3 times.

[0010] S05, evaluating the relative affinity of trypsin and flavonoids: the parameter R represents the ratio of the sum of the relative abundance of trypsin-flavonoid complex ions to the sum of the relative abundance of trypsin and its complex ions. The greater the R value, the stronger the affinity of the flavonoids to trypsin.

[0011]

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

[0013] wherein, the sum of relative abundance of peaks representing different charge states of the trypsin-flavonoid compound complex ions, the sum of relative abundance of peaks representing different charge states of the free state unbound flavonoid compound.

[0014] S06, judging whether there is a competitive relationship between different flavonoids and trypsin: ΔR / R(%) is the relative change of R value of flavonoid (P1) binding to trypsin before and after adding flavonoid (P2).

[0015]

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

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

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

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

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

[0021] Further, in step S03 or step S04, the content of methanol in the ESI-MS sample solution is 30%-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 two flavonoids P1 and 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 of the two is 1:1.

[0024] In step S05, the parameter R is introduced to evaluate the relative strength of the affinity of different flavonoids to trypsin. R represents the ratio of the sum of the relative abundances of different charge complex ions of trypsin-flavonoid to the sum of the relative abundances of different charge trypsin ions. 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 flavonoids to trypsin according to ΔR / R(%) in the competition mode, and further speculate whether the binding sites of different flavonoids to trypsin are the same.

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

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

[0027] The present application relates to a competitive binding method for evaluating the interaction between trypsin and flavonoids. Two (or more) flavonoids are mixed with trypsin and analyzed by mass spectrometry, so that the relative affinity strength and binding stoichiometry of different flavonoids to trypsin can be directly compared in one mass spectrum, and the existence of multi-component complexes can also be confirmed. In addition, the parameter R introduced in the present application can be used to determine whether there is a competitive relationship between different flavonoids to trypsin, and further speculate the binding sites. The method has fast analysis speed, high sensitivity and accuracy, and can simultaneously obtain relevant information such as the relative affinity strength and binding sites of different flavonoids. It is expected to be applied to rapid screening and mechanism analysis of trypsin inhibitors. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 The mass spectrum of the combination of trypsin and flavonoid Lut (P1) in Example 1 of the present application;

[0029] Figure 2Mass spectrum of the competition between Trypsin and flavonoids Lut (P1) and 3'-Lut (P2) in Example 2 of the present application;

[0030] Figure 3 R value change of Try and Lut (P1) binding after adding different flavonoids (P2) in Example 2 of the present application. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0032] Preparation of Trypsin stock solution: accurately weigh 2-5 mg of dry trypsin powder, dissolve 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 at a speed of 12000 r / min for 20 min at 4°C, retain the solution in the ultrafiltration membrane, dilute to 500 μL with 10 mmol / L NH4OAc, and repeat the centrifugation for 4-5 times. Use a NanoDrop 2000 ultramicro spectrophotometer to determine the concentration to obtain a trypsin stock solution, which is stored at -4°C in the dark.

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

[0034] Example 1

[0035] Mix a certain amount of trypsin stock solution, Lut stock solution, NH4OAc buffer solution and ultrapure water uniformly, incubate at room temperature for 10 min to obtain a trypsin and Lut mixed solution (containing 5 μmol / L trypsin, 50 μmol / L Lut, 20 mmol / L NH4OAc, pH = 8.6). Mix with 50% methanol and perform mass spectrometry analysis. Figure 1 Mass spectrum of Trypsin and Lut binding, from which it can be observed that the ion peaks of Try charge are 10+~8+ ([Try] 10+ , [Try] 9+ and [Try] 8+, m / z 2333.69, 2592.88, 2916.86, relative abundance 15.5%, 34.0%, 8.3%, respectively), the complex ion peak of Try-Lut combined in 1:1 ([Try+P1] 10+ , [Try+P1] 9+ and [Try+P1] 8+ , m / z 2362.29, 2624.76, 2952.61, relative abundance 6.2%, 13.2%, 3.0%, respectively), the complex ion peak of Try-Lut combined in 1:2 ([Try+2P1] 10+ , [Try+2P1] 9+ and [Try+2P1] 8+ , m / z 2390.99, 2656.65, 2988.61, relative abundance 3.5%, 6.2%, 1.6%, respectively), the complex ion peak of Try-Lut combined in 1:3 ([Try+3P1] 10+ , [Try+3P1] 9+ and [Try+3P1] 8+ , m / z 2419.49, 2688.32, 3024.37, relative abundance 2.3%, 3.0%, 1.1%, respectively), and the R of Try-Lut was calculated as 0.41. The trypsin and Chr mixed solution was prepared in the same way, and the mass spectrum analysis was performed, and the R of Try-Chr was 0.24. By comparing the R values of different flavonoid compounds, it was found that the relative affinity of Lut to trypsin was 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 uniformly, incubated at room temperature for 10 min, and a trypsin and Lut mixed solution (containing 5 μmol / L trypsin, 50 μmol / L Lut, 20 mmol / L NH4OAc, pH = 8.6) was obtained, which was used as a control group for mass spectrum analysis. To a certain amount of trypsin stock solution, Lut stock solution, NH4OAc buffer solution and ultrapure water mixed solution, five kinds of flavonoid compound stock solutions (P2, the same concentration as Lut) were added respectively, mixed uniformly, incubated at room temperature for 10 min, and a trypsin and P1, P2 mixed solution (containing 5 μmol / L trypsin, 50 μmol / L P1, 50 μmol / L P2, 20 mmol / L NH4OAc, pH = 8.6) was obtained, which was used for mass spectrum analysis. Among them, the mass spectrum analysis results of the competition of trypsin and flavonoid compounds Lut (P1) and 3'-Lut (P2) for binding are as follows Figure 2As shown, the complex ion peak of Try-Lut (P1) combined at 1:1~1:2 ([Try+P1] 9+ and [Try+2P1] 9+ , m / z were 2624.75, 2656.54, respectively, and relative abundance were 22.3%, 7.8%, respectively), and the complex ion peak of Try-3'-Lut (P2) combined at 1:1~1:2 ([Try+P2] 9+ and [Try+2P2] 9+ , m / z were 2643.97, 2695.64, respectively, and relative abundance were 21.8%, 9.0%, respectively), and the ternary complex ion peak of Try combined with P1 and P2 ([Try+P1+P2] 9+ , m / z was 2676.08, and relative abundance was 14.0%).

[0038] The R values obtained in the experiment were plotted into a column chart, as shown in Figure 3 It can be seen that 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 of 21.4%, 20.6% and 18.3% in the R value of Try-Lut, respectively, which indicates that the combination of Chr, 3'-Lut and 7-Lut all has a competitive relationship with Lut, and it is inferred that Chr, 3'-Lut, 7-Lut and Lut have the same binding site on Try, while the binding site of Gal on Try is different from that of Lut. In addition, it is found that the addition of Bai leads to an increase of 16.7% in the R value of Try-Lut, which indicates that the addition of Bai is beneficial to the combination of Try and Lut, and it is inferred that Bai and Lut have a synergistic effect.

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

[0040] It should be noted that the above only illustrates the technical idea of the present application, and cannot limit the protection scope of the present application. For those skilled in the art, without departing from the principle of the present application, a number of improvements and refinements can be made, which all fall within the protection scope of the claims of the present application.

Claims

1. A competitive binding method for evaluating the interaction between trypsin and flavonoids, characterized in that: The following steps are involved: S01. Prepare a trypsin stock solution by dissolving trypsin dry powder in ultrapure water to prepare a trypsin stock solution; mix the NH4OAc buffer solution and the trypsin stock solution, centrifuge at 12,000 rpm using a 10K ultrafiltration membrane for 20 minutes at 4°C, retain the solution in the ultrafiltration membrane, dilute with NH4OAc buffer solution, and repeat the centrifugation 4 to 5 times; determine the concentration using a NanoDrop 2000 ultramicro spectrophotometer to obtain the trypsin stock solution, and store it at -4°C in the dark. S02. Prepare flavonoid stock solutions: Dissolve flavonoid P1 powder and flavonoid P2 powder in methanol to prepare 1 mmol / L flavonoid P1 stock solutions and flavonoid P2 stock solutions, respectively. Store 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; a larger R value indicates a stronger affinity of the flavonoids for 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 flavonoid-containing trypsin with different charges; n = 1-4, m = 8-10; S06, determine whether there is a competitive relationship between different flavonoids and trypsin binding: ΔR / R (%) is the relative change in the R value of flavonoid P1 binding to trypsin before and after the addition of flavonoid P2; Among them, 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 beneficial 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 flavonoids according to claim 1, characterized in that: In step S02, the flavonoid compound is one or more of chrysin, galangin, baicalein, 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 flavonoids 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 and the pH is 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 between 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 as follows: 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, and ion optical radio frequency voltage (RF) of 30%; each sample is detected 3 times.

Images