CN104165869B - A kind of method detecting hydrolase of proteolysis - Google Patents

Abstract

The invention provides a method for detecting the activity of proteolytic enzymes. The method comprises contacting a test sample containing proteolytic enzymes with a fluorescent probe of a coumarin derivative to obtain the contacted materials, and detecting that the contacted materials are excited. The intensity of fluorescence emitted under light, wherein the coumarin derivative fluorescent probe is a non-peptide coumarin derivative with a structure shown in formula (1) or formula (2); wherein, in formula (1) R ₁ is methyl or ethyl. Through the above technical scheme, the present invention provides a sensitive, simple and economical proteolytic enzyme activity detection method, a proteolytic enzyme activity detection kit, and a method for preparing and detecting proteolytic enzyme activity in samples with coumarin derivative fluorescent probes. Use in the kit. Formula (1) Formula (2).

Description

A method for detecting proteolytic enzyme activity

technical field

The invention relates to a method for detecting proteolytic enzyme activity.

Background technique

There are three main methods for the determination of proteolytic enzyme activity. The first is traditional high-performance liquid chromatography (HPLC), which can be directly used to monitor the hydrolysis of substrates by proteolytic enzymes. However, the experimental process of high performance liquid chromatography is cumbersome and the cycle is long, and it is difficult to screen inhibitors. The second is ultraviolet absorption spectroscopy, which is widely used because of its simplicity and rapidity, but ultraviolet absorption spectroscopy still has certain defects. First, this method suffers from large background interference and low sensitivity. In addition, the substrates involved in the analysis are all polypeptide derivatives, which are difficult to synthesize and lead to high preparation costs. The third is fluorescence emission spectrometry, which is a relatively novel method with high sensitivity, simple method, and can be used for high-throughput screening. Unfortunately, the currently used fluorescent probes are still polypeptides, most of which have poor stability under normal conditions, harsh storage and transportation conditions, and low atom utilization. Therefore, it is very urgent and necessary to design and develop sensitive, simple and economical detection methods based on non-peptide fluorescent probes. However, so far, there are few non-peptide fluorescent probes at home and abroad for the detection of elastase (EC number: 3.4.21.37), trypsin (EC number: 3.4.21.4), chymotrypsin (EC number: 3.4.21.1 ) and carboxypeptidase (EC number: 3.4.17.1) activities were reported.

Contents of the invention

The purpose of the present invention is to overcome the disadvantages of poor stability of polypeptide fluorescent probes in the prior art, harsh storage and transportation conditions, and low atom utilization, and provide a sensitive, simple, and economical detection method for proteolytic enzyme activity.

The invention provides a method for detecting the activity of proteolytic enzymes. The method comprises contacting a sample to be tested containing proteolytic enzymes with a coumarin derivative fluorescent probe to obtain contacted materials; The intensity of fluorescence emitted under excitation light; wherein, the fluorescent probe of coumarin derivatives is a non-peptide coumarin derivative with a structure shown in formula (1) or formula (2);

Formula (1) Formula (2)

Wherein, in formula (1), R ₁ is methyl or ethyl.

The present invention also includes the application of the fluorescent detection method for proteolytic enzyme activity of the present invention in the determination of proteolytic enzyme activity.

The present invention also provides a kit for detecting proteolytic enzyme activity in a sample, the kit comprising: a coumarin derivative fluorescent probe and a standard product of the proteolytic enzyme to be tested; the coumarin derivative fluorescent probe A non-peptide coumarin derivative with a structure shown in formula (1) or formula (2);

Formula (1) Formula (2)

Wherein, in formula (1), R ₁ is methyl or ethyl.

The present invention also provides the use of coumarin derivative fluorescent probes in the preparation of kits for detecting proteolytic enzyme activity in samples, and the coumarin derivative fluorescent probes are represented by formula (1) or formula (2). Non-peptidic coumarin derivatives showing the structure;

Formula (1) Formula (2)

Wherein, in formula (1), R ₁ is methyl or ethyl.

Through the above technical scheme, the present invention provides a sensitive, simple and economical proteolytic enzyme activity assay method, a proteolytic enzyme activity detection kit and a coumarin derivative fluorescent probe for the preparation of proteolytic enzyme activity in samples Use in the kit.

Other features and advantages of the present invention will be described in detail in the following detailed description.

Description of drawings

The accompanying drawings are used to provide a further understanding of the present invention, and constitute a part of the description, together with the following specific embodiments, are used to explain the present invention, but do not constitute a limitation to the present invention. In the attached picture:

Figure 1 is a schematic diagram of the specificity of the fluorescent probe with the structure shown in formula (2) to elastase

detailed description

Specific embodiments of the present invention will be described in detail below. It should be understood that the specific embodiments described here are only used to illustrate and explain the present invention, and are not intended to limit the present invention.

The invention provides a method for detecting the activity of proteolytic enzymes. The method comprises contacting a sample to be tested containing proteolytic enzymes with a coumarin derivative fluorescent probe to obtain contacted materials; The intensity of fluorescence emitted under excitation light; wherein, the fluorescent probe of coumarin derivatives is a non-peptide coumarin derivative with a structure shown in formula (1) or formula (2);

Formula (1) Formula (2)

Wherein, in formula (1), R ₁ is methyl or ethyl, preferably, R ₁ is ethyl.

When the coumarin derivative has a structure represented by formula (1), the proteolytic enzyme is one or more of elastase, trypsin, chymotrypsin and carboxypeptidase.

When the coumarin derivative has the structure shown in formula (2), the proteolytic enzyme is elastase.

The detection method of proteolytic enzyme activity of the present invention also comprises:

The test sample containing proteolytic enzyme is contacted with the coumarin derivative of the present invention to obtain the contacted material; the intensity of the fluorescence emitted by the contacted material under excitation light is detected, and the intensity of the fluorescence indicates the The activity of the proteolytic enzyme in the sample to be tested; the wavelength of the excitation light is 360-405nm, and the emission wavelength of the fluorescence is 500-520nm.

In the detection method of proteolytic enzyme activity of the present invention, the consumption scope of coumarin derivative is relevant with the kind of proteolytic enzyme sample and coumarin derivative to be tested, usually, the coumarin derivative The amount can be used to characterize the activity of the proteolytic enzyme sample to be tested. Preferably, the amount of the coumarin derivative can be 1-100 μM for every 1 μM proteolytic enzyme in the sample to be tested. In order to achieve the best For a good detection effect, the dosage of the coumarin derivative is preferably 1-10 μM.

In the detection method of proteolytic enzyme activity provided by the present invention, the conditions for the contact of the proteolytic enzyme test sample with the coumarin derivative include: the temperature is 25-37°C, the pH value is 6-8, and the time is 5 -30min. Preferably, the conditions of the contacting include: the temperature is 25-30° C., the pH value is 6.5-8.0, and the time is 10-25 minutes.

In the detection method of the present invention, the contacting is carried out under the condition of avoiding light, and the contacting container is preheated to maintain the biological activity of the sample to be tested. Preferably, the contacted reaction vessel is a black flat-bottomed well plate, cuvette or microplate, and before contacting, each component and the reaction vessel that will participate in the contact are preheated to a suitable reaction temperature, and the preheating to be achieved The temperature is preferably 25-37°C.

The conditions of the method for detecting proteolytic enzyme activity of the present invention also include performing in 50-100mM Tris-HCl (containing 10mM CaCl ₂ , pH=7.0) buffer solution, and an appropriate amount of proteolytic enzymes can be hydrolyzed before the contact. Enzyme samples, 10 μM coumarin derivatives were mixed with 50-150 μL of the Tris-HCl buffer, respectively.

The detection of the fluorescence intensity of the material after the proteolytic enzyme to be tested is contacted with the coumarin derivative can be carried out in a microplate reader or a fluorescence spectrophotometer, and a time-dependent detection method can be used to obtain the proteolytic enzyme in the Fluorescence spectra of catalyzed hydrolysis of coumarin derivatives during exposure as a function of time, the increase in fluorescence intensity as a function of time was obtained. In addition, the reliability of the detection can be ensured by repeating the test multiple times and setting up several control groups.

When the coumarin derivative has the structure shown in formula (1), the method of the present invention can be used for one or more enzymes in elastase, trypsin, chymotrypsin and carboxypeptidase Determination of activity, wherein, when the sample to be tested contains one of elastase, trypsin, chymotrypsin and carboxypeptidase, the coumarin derivatives represented by the formula (1) provided by the present invention It can be used to determine the activity of known proteolytic enzymes contained in the sample; when the sample to be tested contains one of elastase, trypsin, chymotrypsin and carboxypeptidase but cannot determine which one When, the compound of formula (1) can be used for detection to obtain the Michaelis constant for the hydrolysis of the compound shown in formula (1) catalyzed by an unknown type of proteolytic enzyme, thereby judging the type of proteolytic enzyme contained in the sample; in addition, the present invention The provided coumarin derivatives represented by formula (1) can also be applied to simultaneously detect the total activity of various proteolytic enzymes contained in the sample, so as to analyze the total activity of proteolytic enzymes contained in the tested sample .

When the coumarin derivative has the structure shown in formula (2), the method of the present invention can be used for the determination of elastase activity in the sample, and can also be used to determine whether the proteolytic enzyme in the sample to be tested is elastase.

The method for detecting proteolytic enzyme activity of the present invention can be applied to any detection involving proteolytic enzyme activity in this field, for example, detection of proteolytic enzyme activity, screening of proteolytic enzyme inhibitors or accelerators, proteolytic enzyme inhibition Kinetic characterization of agents or accelerators, etc., while the method described in the present invention can also be used in the diagnosis and treatment of related diseases, but the present invention is not limited thereto.

When the method for detecting proteolytic enzyme activity of the present invention is used for the screening of proteolytic enzyme inhibitors, proteolytic enzymes containing specific concentrations, various inhibitors or organic compound solutions of a certain concentration and 50-100mM Tris-HCl (containing 10 mM CaCl ₂ , pH=7.0) was preheated in a constant temperature water tank at 30°C; at the same time, a black flat-bottom 96-well plate was preheated in a 30°C incubator. Subsequently, an appropriate amount of preheated Tris-HCl (containing 10 mM CaCl ₂ , pH=7.0) buffer solution, the same volume of various proteolytic enzyme inhibitors and an appropriate amount of fluorescent probes were contacted with the proteolytic enzyme. And use a microplate reader or a full-featured fluorescence spectrophotometer to monitor the fluorescence intensity of the sample in a time-dependent manner.

The present invention also provides a kit for detecting proteolytic enzyme activity in a sample, the kit comprising: a coumarin derivative fluorescent probe and a standard product of the proteolytic enzyme to be tested; the coumarin derivative fluorescent probe A non-peptide coumarin derivative with a structure shown in formula (1) or formula (2);

Formula (1) Formula (2)

Wherein, in formula (1), R ₁ is methyl or ethyl, preferably, R ₁ is ethyl.

The kit provided by the present invention can be applied to detect the activity of proteolytic enzymes in the sample to be tested, screen proteolytic enzyme inhibitors or promoters and other related proteolytic enzyme activity detection or proteolytic enzyme inhibitors or promoter functions In addition, the kit can also be used in the field of medical detection and treatment related to the detection of proteolytic enzyme activity, but the present invention is not limited thereto.

In the kit provided by the present invention, the reaction needs to be carried out in a reaction buffer, preferably, the reaction buffer is 50-100mM Tris-HCl (containing 10mM CaCl ₂ , pH=7.0), and the storage condition of the kit is at Store at minus 20 to 4°C in the dark. The specific method of use of the kit can be carried out according to the methods listed below:

(1) Prepare a 10mM DMSO solution of coumarin derivatives, and store it in the dark at minus 20°C to 4°C.

(2) Dissolve the proteolytic enzyme standard product in 0.1M Tris-HCl (containing 10mM CaCl ₂ , pH=7.0) buffer solution, prepare a 100U/ml proteolytic enzyme standard product solution, and dilute it by 10 times Perform serial dilutions.

(3) Dissolve the test sample containing proteolytic enzyme with 100 μL of 100 mM Tris-HCl (containing 10 mM CaCl ₂ , pH=7.0) buffer solution.

(4) Add 0.2mL of Tris-HCl (containing 10mM CaCl ₂ , pH=7.0) buffer solution, coumarin derivative, proteolytic enzyme sample to be tested and proteolytic enzyme standard to the black microtiter plate . The activity of the proteolytic enzyme in the sample to be tested and the proteolytic enzyme standard product is detected by using the method for detecting proteolytic enzyme activity of the present invention.

(5) Draw a standard curve with the fluorescence intensity change values of the detected serially diluted proteolytic enzyme standards, and determine the proteolytic enzyme activity in the detected samples by comparison.

When the kit of the present invention is used for screening proteolytic enzyme inhibitors or accelerators, the inhibitors can be added to the reaction mixture at an appropriate ratio or viscosity before detection. Then add an appropriate amount of proteolytic enzyme standard product to start the reaction, the reaction condition is 25-37°C, and the reaction time is 5-30min.

In addition, the present invention also provides the use of coumarin derivative fluorescent probes in the preparation of kits for detecting proteolytic enzyme activity in samples, and the coumarin derivative fluorescent probes are of formula (1) or formula (2 ) non-peptidic coumarin derivatives of the structure shown;

Formula (1) Formula (2)

Wherein, in formula (1), R ₁ is methyl or ethyl, preferably, R ₁ is ethyl.

The present invention will be described in detail below by way of examples. The polypeptide substrate Suc-Ala-Ala-Ala-pNA used in the following examples is a commercial product purchased from Sigma-alderich Reagent Company with product number 70967-97-4. The polypeptide substrate N-α-Benzoyl-DL-Arg-pNA (CAS: 911-77-3) was a commercial product purchased from Sigma-Aldrich Reagent Company, and the soybean trypsin inhibitor was purchased from Maya Company with the number 10063. The silica gel chromatography column is a product of Shinwell Company. A microplate reader was purchased from Molecular Devices, model SpectraMax M5. The coumarin derivatives with the structures shown in formula (1), formula (2), formula (3) and formula (4) are all according to the literature (Journal of East China Institute of Chemical Technology, 1991, 17, 536-539; Tetrahedron, 2011, 67, 7348-7353) prepared by the method recorded.

The source and parameters of the enzyme used in the following examples are as follows:

Acetylcholinesterase was purchased from a-Aldrich (C1682) with specific activity ≥1,500U/mg

Butyrylcholinesterase was purchased from Sigma-Aldrich (C1057) specific activity ≥ 900U/mg

Bovine serum albumin was purchased from Bailingwei Technology Co., Ltd. (109636) with a purity of 98%.

Porcine pancreatic elastase was purchased from Maya Company (10095) with specific activity ≥30U/mg

Trypsin was purchased from Maya Company (10020) with specific activity ≥250U/mg

Human neutrophil elastase was purchased from Maya Company (E8140) with specific activity ≥50U/mg

Chymotrypsin hydrolase was purchased from Maya Company (10001) with specific activity ≥1500U/mg

Carboxypeptidase was purchased from Worthington (CLS005304) specific activity ≥ 170U/mg

In the following examples, the fluorescence intensity change rate (ΔF/Δt) is calculated according to the following formula:

$\frac{\mathrm{<span\; class="notranslate">\; \&Delta;F</span>}}{\mathrm{<span\; class="notranslate">\; \&Delta;t</span>}}<span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; f</span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; f</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}}{{\mathrm{<span\; class="notranslate">\; t</span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; t</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}}$

Among them, F2 represents the fluorescence intensity _of the sample at time _t2 , and _F1 represents the fluorescence intensity of the sample at time _t1 .

The change rate (ΔA/Δt) of the UV-visible absorption value is calculated according to the following formula:

$\frac{\mathrm{<span\; class="notranslate">\; \&Delta;A</span>}}{\mathrm{<span\; class="notranslate">\; \&Delta;t</span>}}<span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; A</span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; A</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}}{{\mathrm{<span\; class="notranslate">\; t</span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; t</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}}$

Among them, A ₂ represents the UV-visible absorption value of the sample at 405 nm at t ₂ time, and A ₁ represents the UV-visible absorption value of the sample at 405 nm at t ₁ time.

Example 1

This example is used to illustrate the method for detecting elastase activity of the compound represented by formula (2) provided by the present invention.

Dissolve 1mg of elastase sample in 100mM Tris-HCl (containing 10mM CaCl ₂ , pH=7.0) buffer solution and perform gradient dilution, prepare different concentrations of elastase standard solution according to the concentration given in Table 1, and obtain the configuration The elastase standard solution was preheated in a 30°C constant temperature water tank; at the same time, the black flat-bottomed 96-well plate was preheated in a 30°C incubator. Subsequently, 40 μL of the preheated elastase standard solution was placed in a preheated black flat-bottomed 96-well plate, and then 120 μL of 100 mM Tris-HCl (containing 10 mM CaCl ₂ , pH=7.0) buffer solution was added, and a negative control group ( Only add 160 μl of buffer), under dark conditions, 40 μL of non-peptide coumarin derivatives with the structure shown in formula (2) were contacted with elastase standard and control buffer respectively, and each The fluorescence intensity of the elastase standard solution and the control group solution under the excitation light of 395nm was scanned kinetically, and the change rate of the fluorescence intensity in the first 5-10 minutes was obtained. The results are listed in Table 1.

Comparative example 1

This comparative example is used to illustrate the detection method of elastase activity in the prior art.

The same method as in Example 1 was used to detect the hydrolysis of the substrate of the series of concentration gradients catalyzed by the elastase standard substance, except that the substrate probe used was Suc-Ala-Ala-Ala-pNA (purchased from Sigma-Aldrich company, the product number is S7388), at a wavelength of 405nm, a kinetic scan was performed on the UV-visible absorption value after contact, and the change rate of the UV-visible absorption value within 5-10 minutes was obtained. The results are listed in Table 1.

Table 1

Example 2

This example is used to illustrate the method for measuring the activities of elastase, chymotrypsin, trypsin and carboxypeptidase using the non-peptide fluorescent probe represented by formula (3) provided by the present invention.

Formula (3)

Each proteolytic enzyme sample was dissolved in 100mM Tris-HCl (containing 10mM CaCl ₂ , pH=7.0) buffer and diluted to prepare an enzyme solution with a concentration of 0.01mg/L; at the same time, place a black flat-bottomed 96-well plate Preheat in a 30°C incubator. Subsequently, 40 μL of the preheated enzyme solution was put into a preheated black flat-bottomed 96-well plate, and then 120 μL of 100 mM Tris-HCl (containing 10 mM CaCl ₂ , pH=7.0) buffer solution was added, and a negative control group (only 160 μl of Buffer), under light-shielding conditions, 40 μL of non-peptide coumarin derivatives with the structure shown in formula (3) were contacted with the enzyme solution and the control group respectively, and the enzyme standard solution and the control group solution were tested with a microplate reader. The post-contact fluorescence intensity under 395nm excitation light was scanned kinetically, and the change rate of fluorescence intensity within 5-10 minutes was obtained. The results are listed in Table 2.

Example 3

The activities of elastase, chymotrypsin, and carboxypeptidase were measured by the same method as in Example 2, except that the non-peptide coumarin derivative fluorescent probe used was represented by formula (4) structure, and the results are listed in Table 2.

Formula (4)

Comparative example 2

Adopt the method identical with embodiment 2 to measure elastase, chymotrypsin, carboxypeptidase activity, difference is that the substrate probe used is Suc-Ala-Ala-Ala-pNA (purchased from Sigma-Aldrich, product number S7388).

Table 2

test case 1

This test example is used to illustrate the specificity of the elastase activity detection method provided by the present invention.

Dissolve 1mg of the sample to be tested (acetylcholinesterase, butyrylcholinesterase, bovine serum albumin, elastase, chymotrypsin, trypsin and carboxypeptidase) in 100mM Tris-HCl (containing 10mM CaCl ₂ , pH= 7.0) Prepare a solution of the sample to be tested at a concentration of 0.01 mg/L in the buffer solution and place it in a 30°C constant temperature water tank to preheat; meanwhile, place a black flat-bottom 96-well plate in a 30°C incubator to preheat. Subsequently, 40 μL of the preheated solution of the sample to be tested was placed in a preheated black flat-bottomed 96-well plate, and then 120 μL of 100 mM Tris-HCl (containing 10 mM CaCl ₂ , pH=7.0) buffer solution was added. Contact 10 μL of non-peptide coumarin derivatives with the structure shown in formula (2) with each sample to be tested, and set up a control group without the sample to be tested, and use a microplate reader to detect within 10 minutes under the excitation light of 405nm The relative rates of the hydrolysis of 10 μL of the compound represented by formula (2) catalyzed by various samples to be tested are shown in Figure 1.

Example 4

This example is used to illustrate the screening method for trypsin inhibitors according to the present invention.

Dissolve 1 mg of trypsin sample in 100 mM Tris-HCl (containing 10 mM CaCl ₂ , pH=7.0) buffer to prepare a trypsin standard solution with a trypsin concentration of 0.05 mg/mL, and soybean trypsin inhibitor (soybean trypsin inhibitor) was dissolved in 1 mL of 100 mM Tris-HCl (containing 10 mM CaCl ₂ , pH=7.0) buffer solution to prepare inhibitor solutions with concentration gradients of 0.1, 1, 4, 7, 10 and 20 μM, and 0.01 mmol of formula (2) The non-peptide coumarin derivatives of the shown structure were dissolved in 10 mL of 100 mM Tris-HCl (containing 10 mM CaCl ₂ , pH=7.0) buffer solution to prepare a fluorescent probe solution with a concentration of 1 mM, and the prepared trypsin standard The solution, soybean trypsin inhibitor solution and fluorescent probe solution were preheated in a 30°C constant temperature water tank; meanwhile, a black flat-bottomed 96-well plate was placed in a 30°C incubator to preheat. Subsequently, 40 μL soybean trypsin inhibitor solution of each concentration gradient, 100 μL 100 mM Tris-HCl (containing 10 mM CaCl ₂ , pH=7.0) buffer solution and 40 μl fluorescent probe (final concentration 5 μM) solution were added to black flat bottom 96 wells Mix in the plate as the experimental group, and set up a control group mixed with 140 μL 100 mM Tris-HCl (containing 10 mM CaCl ₂ , pH=7.0) buffer solution and 40 μL fluorescent probe solution. Quickly add 20 μL of trypsin standard solution with a row gun to start the reaction. Finally, a microplate reader was used to detect the changes in the fluorescence intensity and the UV-visible absorption values of the experimental group and the control group, respectively, and the results are listed in Table 3.

Comparative example 3

This comparative example is used to illustrate the screening method of trypsin inhibitors using the prior art.

The same method as in Example 4 was used to screen for trypsin inhibitors, except that the fluorescent probe solution was replaced by the polypeptide substrate N-α-Benzoyl-DL-Arg-pNA (CAS :911-77-3), the absorption wavelength of visible light is 405nm. The results are listed in Table 3.

table 3

As can be seen from the results of Example 1, the method provided by this method can be used for simple and sensitive detection of the activity of elastase in the sample, and as can be seen from the results of Example 1 and Comparative Example 1, by this method In the method provided by the invention, the fluorescent probe of the compound represented by the formula (2) used in the method provided by the invention has higher sensitivity and affinity than the existing fluorescent probes for polypeptide substrates.

As can be seen from the results of Examples 2, 3 and Comparative Example 2, compared with the prior art, the method provided by the present invention can effectively, quickly and easily test the activity of multiple proteolytic enzymes, and compared with the existing Compared with the method used by the technology, the fluorescence intensity of the method provided by the present invention changes more obviously, and the sensitivity and affinity of detection are higher, as can be seen from the results of Examples 2 and 3 when used in the method provided by the present invention When the compound represented by the formula (3) is used as a fluorescent probe, the change of the fluorescence intensity is more obvious, and the detection sensitivity is higher.

It can be seen from the results of Test Example 1 that the hydrolysis ability of elastase to the fluorescent probe of coumarin derivatives shown in formula (2) used in the method provided by the present invention is significantly higher than that of other types of enzymes, Therefore, the coumarin derivatives used in the method of the present invention have high specificity for enzymes.

From the results of Example 4 and Comparative Example 3, it can be seen that the method provided by the present invention can be used for the screening of trypsin inhibitors. And, it is worth mentioning that, compared with the screening method for trypsin inhibitors provided by the present invention using polypeptide substrate fluorescent probes, the variation range of the fluorescent signal is relatively large and easy to detect, while the polypeptide substrate fluorescent probes The change rate of the ultraviolet-visible absorption value of the needle is small (accurate to two digits after the decimal point), which is not easy to detect, and the coumarin derivatives provided by the present invention have higher atom economy.

In addition, considering the ease of obtaining raw materials, the fluorescent probes of coumarin derivatives provided by the present invention are easy to synthesize, while the polypeptide substrate Suc provided by Sigma-Aldrich Reagent Company used in Comparative Examples 1-3 -Ala-Ala-Ala-pNA (CAS: 70967-97-4), N-Methoxysuc-Ala-Ala-Pro-Val-pNA (CAS: 70967-90-7) and N-α-Benzoyl-DL-Arg -pNA (CAS: 911-77-3) is difficult to synthesize. Therefore, compared with the fluorescent probes provided by the prior art, the coumarin derivatives provided by the present invention have the advantages of easier implementation and lower cost.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention. These simple modifications All belong to the protection scope of the present invention.

In addition, it should be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable way if there is no contradiction. The combination method will not be described separately.

In addition, various combinations of different embodiments of the present invention can also be combined arbitrarily, as long as they do not violate the idea of the present invention, they should also be regarded as the disclosed content of the present invention.

Claims (10)

1. A method for detecting proteolytic enzyme activity, the method comprises contacting the sample to be tested containing proteolytic enzyme with a coumarin derivative fluorescent probe, obtaining the material after contact, and detecting the material after contact under excitation light The intensity of the emitted fluorescence is characterized in that the fluorescent probe of the coumarin derivative is a non-peptide coumarin derivative of the structure shown in formula (1) or formula (2); Wherein, in formula (1), R ₁ is methyl or ethyl; The proteolytic enzyme is one or more of elastase, trypsin, chymotrypsin and carboxypeptidase. 2. The method according to claim 1, wherein, in formula (1), R ₁ is ethyl. 3. The method according to claim 1, wherein the coumarin derivative is a structure shown in formula (1), and the proteolytic enzyme is elastase, trypsin, chymotrypsin and carboxypeptidase one or more of . 4. The method according to claim 1, wherein the coumarin derivative is a structure shown in formula (2), and the proteolytic enzyme is elastase. 5. The method according to any one of claims 1-4, wherein the amount of the coumarin derivative used is 1-100 μM relative to 1 μM proteolytic enzyme. 6. The method according to any one of claims 1-4, wherein the contacting conditions include: a temperature of 25-37° C., a pH value of 6-8, and a time of 5-30 min. 7. The method according to any one of claims 1-4, wherein the wavelength of the excitation light is 360-405 nm, and the emission wavelength of the fluorescence is 500-520 nm. 8. A test kit for detecting proteolytic enzyme activity in a sample, the kit comprising: a coumarin derivative fluorescent probe and a standard product of the proteolytic enzyme to be tested; the coumarin derivative fluorescent probe is of the formula (1) or non-peptide coumarin derivatives of the structure shown in formula (2); Wherein, in formula (1), R ₁ is methyl or ethyl; The proteolytic enzyme is one or more of elastase, trypsin, chymotrypsin and carboxypeptidase. 9. The kit according to claim 8, wherein, in formula (1), R ₁ is an ethyl group. 10. The use of coumarin derivative fluorescent probes in the preparation of test kits for detecting proteolytic enzyme activity in samples, the coumarin derivative fluorescent probes are of the structure shown in formula (1) or formula (2) Non-peptidic coumarin derivatives; Wherein, in formula (1), R ₁ is methyl or ethyl; The proteolytic enzyme is one or more of elastase, trypsin, chymotrypsin and carboxypeptidase.