Method for evaluating toxicity level of halogenated disinfection by-product based on protease

Abstract

The invention relates to the technical field of water quality detection, in particular to a method for evaluating the toxicity level of halogenated disinfection byproducts based on protease. According to the invention, fluorescence spectrum, synchronous fluorescence spectrum and ultraviolet absorption spectrum technologies are adopted to research the interaction of seven cyclic and heterocyclic halogenated disinfection byproducts with pepsin and trypsin. A fluorescence quenching mechanism is analyzed through a Stern-Volmer equation, a double logarithmic equation and the like, and the influence of the halogenated disinfection by-product on the conformation of the protease is disclosed in combination with constants and thermodynamic parameters. The binding force of the halogenated disinfection by-product and protease is in positive correlation with cytotoxicity, a new index is provided for toxicity evaluation of the halogenated disinfection by-product, and optimization of a drinking water disinfection process and formulation of a water quality standard are facilitated.

Description

Technical Field

[0001] The invention relates to the technical field of water quality detection, and in particular to a method for evaluating the toxicity level of halogenated disinfection by-products based on protease. Background Art

[0002] In the current environment, environmental pollutants can enter the human body through skin contact, eating, drinking water and other routes, and their potential hazards have been widely confirmed. Disinfection by-products (DBPs), as a typical type of environmental pollutants, pose a threat to human health that cannot be ignored. At present, the assessment of the degree of threat of DBPs to human health mainly focuses on studying the reaction relationship between DBPs concentration and the test substance and the toxic effect. The commonly used research method is to expose cell lines such as Chinese hamster ovary cells (CHO-K1), colon cells (CCD841CoN), human liver cancer cells (Hep G2), and bladder cancer cells (T24) to DBPs, and evaluate the hazards of DBPs by detecting the size of cytotoxicity. Cytotoxicity refers to the ability of exogenous substances to cause cell death or inhibit cell growth.

[0003] In the field of research on the interaction between DBPs and proteins, human serum albumin (HSA) is the main research object. Studies have found that two DBPs, 4-bromophenol and 2,4-dibromophenol, can bind to HSA through hydrogen bonds and van der Waals forces to form complexes and cause conformational changes in HSA. In addition, there is a certain correlation between the affinity between DBPs and HSA and their toxicity. In contrast, there are relatively few studies on the interaction mechanism between small molecule DBPs and digestive proteases. Understanding this mechanism is of great significance for evaluating the potential effects of DBPs after entering the gastrointestinal tract through food or drinking water.

[0004] Newly discovered cyclic DBPs in recent years have a higher toxicity index and have received widespread attention. When drinking water and food enter the stomach, digestive proteases not only participate in the digestion of proteins, but also interact with the components of the ingested substances. Since proteases may be direct binding targets of these components, they may cause changes in the microenvironment and conformation of proteases, thereby affecting protease activity and the digestion and absorption of nutrients. However, there is currently very limited research on the interaction between DBPs that enter the human gastrointestinal tract through drinking water and pepsin and trypsin. Summary of the invention

[0005] In view of this, the present invention proposes a method for evaluating the toxicity level of halogenated disinfection by-products based on protease.

[0006] The technical solution of the present invention is achieved in this way:

[0007] In a first aspect, the present invention provides a method for evaluating the toxicity level of halogenated disinfection by-products based on protease, comprising the following steps:

[0008] S1. Solution preparation: prepare pepsin solution, trypsin solution and halogenated disinfection by-product solution; the halogenated disinfection by-products include cyclic and heterocyclic compounds, and at least include two of 2,6-dichloro-1,4-benzoquinone, 2,6-dibromo-1,4-benzoquinone, 2-chlorophenylacetonitrile, 3,4-dichlorophenylacetonitrile, 5-chloro-3-pyridinol, 2-bromo-3-pyridinol and 2,6-dibromo-3-pyridinol;

[0009] S2. Spectral measurement: the protease solution is mixed with halogenated disinfection by-product solutions of different concentrations respectively; the mixed solution is measured by two-dimensional fluorescence spectrum, synchronous fluorescence spectrum and ultraviolet absorption spectrum; through these spectral data, the information about the microenvironment of amino acid residues and the structural changes of the protease is obtained;

[0010] S3. Fluorescence quenching analysis: Use the Stern-Volmer equation to fit the fluorescence data to determine whether the quenching type of the disinfection by-products on the protease is static or dynamic, and calculate the quenching constant to determine whether the disinfection by-products bind to the protease;

[0011] S4. Binding parameter analysis: The logarithmic relationship curve of fluorescence quenching was constructed by double logarithmic equation to calculate the number of binding sites and binding constant of the disinfection byproduct-protease complex; the affinity of different proteases with disinfection byproducts was compared to evaluate the interaction strength and toxicity level of disinfection byproducts;

[0012] S5. Thermodynamic parameter analysis: Enthalpy change, entropy change and Gibbs free energy were calculated using the Van't Hoff equation and the Gibbs-Helmholtz equation, and the main forces in the binding process were analyzed, including hydrogen bonding, hydrophobic interaction and electrostatic force;

[0013] S6. Structural influence analysis: Disinfection byproducts interact with proteases to form disinfection byproduct-protease complexes. During the formation process, the hydrophobic environment around tryptophan and tyrosine residues is changed, thereby affecting the compactness of the protease structure. The interaction between disinfection byproducts and proteases is characterized accordingly.

[0014] S7. Comprehensive analysis: Based on the analysis of steps S4-S7, determine whether there is a positive correlation between the interaction between the disinfection by-products and proteases and the cytotoxicity of the disinfection by-products.

[0015] In a second aspect, the present invention provides an application of the method for evaluating the impact of disinfection by-products on proteases in the digestive system of an organism, predicting the toxic effects of disinfection by-products, optimizing drinking water disinfection processes, or formulating drinking water quality standards.

[0016] The beneficial effects of the present invention include at least the following:

[0017] (1) The present invention uses fluorescence spectroscopy and ultraviolet absorption spectroscopy to clarify the interaction mechanism between three types (seven types) of halogenated DBPs, namely halogenated benzoquinones (HBQs), halogenated phenylacetonitrile (HPANs), and halogenated pyridinols (HPOLs), and pepsin and trypsin. It is determined that the quenching of endogenous fluorescence belongs to the static quenching mechanism, and it is clarified that the interaction generates a complex, changes the microenvironment and conformation near the main fluorophore of the protease, and the specific effects of each DBP on the microenvironment of the amino acid residues in different proteases. It also clarifies the thermodynamic properties and force types of the interaction, providing a comprehensive basis for a deep understanding of the interaction between DBPs and proteases.

[0018] (2) The present invention uses double logarithmic equations and fluorescence quenching experimental data to calculate that seven DBPs interact with pepsin / trypsin to form a complex with a single binding site, and obtains the order of the binding constants between DBPs and the two proteases. At the same time, the reasons for the differences in the binding tendencies of different DBPs with different proteases are analyzed, which is helpful for further studying the behavior of DBPs in vivo.

[0019] (3) The present invention found that the cytotoxicity of the same type of DBPs was positively correlated with the binding force of digestive proteases, indicating that the binding force index is expected to be used as a potential toxicity evaluation index to indicate the toxic effects of halogenated disinfection by-products, providing a new idea and method for the toxicity evaluation of halogenated disinfection by-products.

[0020] In some specific embodiments of the present invention, Explanation of terms These include:

[0021]

[0022]

[0023] BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0025] Figure 1The fluorescence emission spectra of pepsin (20 μM) at different DCBQ / DBBQ concentrations in the present application example; (ac) DCBQ, 0-6: 0-67.8 μM, concentration interval is 11.3 μM; (df) DBBQ, 0-6: 0-45 μM, concentration interval is 7.5 μM, pH = 2.2;

[0026] Figure 2 In the examples of the present application, (a) Stern-Volmer plot, (b) fluorescence quenching logarithmic plot, (c) Van't Hoff plot of the interaction between DCBQ and pepsin at different temperatures; (d) Stern-Volmer plot, (e) fluorescence quenching logarithmic plot, (f) Van't Hoff plot of the interaction between DBBQ and pepsin at different temperatures;

[0027] Figure 3 : Synchronous fluorescence spectra of pepsin (20 μM) at different DCBQ / DBBQ concentrations in the embodiment of the present application; (ab) DCBQ, 0-7: 0-79.1 μM, concentration interval is 11.3 μM; (cd) DBBQ, 0-7: 0-52.5 μM, concentration interval is 7.5 μM; (a) DCBQ, Δλ=15 nm; (b) DCBQ, Δλ=60 nm; (c) DBBQ, Δλ=15 nm; (d) DBBQ, Δλ=60 nm; temperature is 298 K, pH=2.2;

[0028] Figure 4 The UV absorption spectra of pepsin (20 μM) at different DCBQ / DBBQ concentrations in the examples of the present application; (a) DCBQ, 0-7: 0-7.91 μM, the concentration interval is 1.13 μM; (b) DBBQ, 0-7: 0-15.75 μM, the concentration interval is 2.25 μM); (c) UV absorption spectra of substances in different systems; (1) pepsin; (2) pepsin + DCBQ; (3) DCBQ; (4) [pepsin + DCBQ] -DCBQ, DCBQ (1.69 μM); the temperature is 298K, pH = 2.2;

[0029] Figure 5 The fluorescence emission spectra of pepsin (20 μM) at different CPAN / DCPAN concentrations in the examples of the present application; (ac) CPAN, 0-8: 0-2.08 mM, concentration interval 0.26 mM; (df) DCPAN, 0-8: 0-0.88 mM, concentration interval 0.11 mM, pH = 2.2;

[0030] Figure 6In the examples of the present application, (a) Stern-Volmer plot, (b) fluorescence quenching logarithmic plot, (c) Van't Hoff plot of the interaction between CPAN and pepsin at different temperatures; (d) Stern-Volmer plot, (e) fluorescence quenching logarithmic plot, (f) Van't Hoff plot of the DCPAN-pepsin system of the interaction between DCPAN and pepsin at different temperatures;

[0031] Figure 7 The synchronous fluorescence spectra of pepsin (20 μM) at different CPAN / DCPAN concentrations in the embodiments of the present application; (ab) CPAN, 0-7: 0-2.08 mM, concentration interval is 0.26 mM; (cd) DCPAN, 0-7: 0-0.88 mM, concentration interval is 0.11 mM): (a) CPAN, Δλ=15 nm; (b) DCPAN, Δλ=60 nm; (c) CPAN, Δλ=15 nm; (d) DCPAN, Δλ=60 nm; temperature is 298 K, pH=2.2;

[0032] Figure 8 The UV absorption spectra of various substances in different systems in the examples of this application are shown below; (a) pepsin (20 μM), CPAN (98.9 μM); (b) pepsin (20 μM), DCPAN (48.4 μM); the temperature is 298 K, and the pH is 2.2;

[0033] Fig. 9 : The fluorescence emission spectra of pepsin (20 μM) at different CPOL / BPOL / DBPOL concentrations in the examples of the present application; (ac) CPOL, 0-7: 0-162.12 μM, concentration interval is 23.16 μM; (df) BPOL, 0-5: 0-120.68 μM, concentration interval is 17.24 μM; (gi) DBPOL, 0-7: 0-83.02 μM, concentration interval is 11.86 μM, pH = 2.2;

[0034] Fig.10 For the interaction analysis between pepsin (20 μM) and CPOL / BPOL / DBPOL in the examples of the present application, (a) Stern-Volmer plot of the interaction between CPOL and pepsin at different temperatures; (b) logarithmic fluorescence quenching plot, (c) Van't Hoff plot; (d) Stern-Volmer plot of the interaction between BPOL and pepsin at different temperatures, (e) logarithmic fluorescence quenching plot, (f) Van't Hoff plot; (g) Stern-Volmer plot of the interaction between DBPOL and pepsin at different temperatures, (h) logarithmic fluorescence quenching plot, (i) Van't Hoff plot;

[0035] Fig.11 Synchronous fluorescence spectra of pepsin (20 μM) at different CPOL / BPOL / DBPOL concentrations in the present application embodiment (ab) CPOL, 0-7: 0-54.04 μM, concentration interval is 7.72 μM; (cd) BPOL, 0-7: 0-80.43 μM, concentration interval is 11.49 μM; (ef) DBPOL, 0-7: 0-55.37 μM, concentration interval is 7.91 μM); (a) BPOL, Δλ=15 nm; (b) BPOL, Δλ=60 nm; (c) CPOL, Δλ=15 nm; (d) CPOL, Δλ=60 nm; (e) DBPOL, Δλ=15 nm; (f) DBPOL, Δλ=60 nm; temperature is 298 K, pH=2.2;

[0036] Fig.12 The UV absorption spectra of pepsin (20 μM) at different concentrations of CPOL / BPOL / DBPOL in the examples of the present application; (a) CPOL, 0-7: 0-32.41 μM, the concentration interval is 4.63 μM; (b) BPOL, 0-7: 0-24.15 μM, the concentration interval is 3.45 μM; (c) DBPOL, 0-7: 0-16.59 μM, the concentration interval is 2.37 μM; the temperature is 298K, and the pH is 2.2;

[0037] Fig.13 The fluorescence emission spectra of trypsin (20 μM) at different DCBQ / DBBQ concentrations in the examples of the present application; (ac) DCBQ, 0-7: 0-19.74 μM, concentration interval of 2.82 μM; (df) DBBQ, 0-7: 0-13.16 μM, concentration interval of 1.88 μM, pH = 7.4;

[0038] Fig.14 The following is an analysis of the interaction between DCBQ and trypsin at different temperatures in the examples of the present application; (a) Stern-Volmer plot; (b) logarithmic fluorescence quenching plot; (c) Van't Hoff plot; (d) Stern-Volmer plot of the interaction between DBBQ and trypsin at different temperatures, (e) logarithmic fluorescence quenching plot, (f) Van't Hoff plot;

[0039] Fig.15Synchronous fluorescence spectra of trypsin (20 μM) at different DCBQ / DBBQ concentrations in the embodiments of the present application; (ab) DCBQ, 0-7: 0-19.74 μM, concentration interval is 2.82 μM; (cd) DBBQ, 0-7: 0-13.16 μM, concentration interval is 1.88 μM); (a) DCBQ, Δλ=15 nm; (b) DCBQ, Δλ=60 nm; (c) DBBQ, Δλ=15 nm; (d) DBBQ, Δλ=60 nm; temperature is 298 K, pH=7.4;

[0040] Fig.16 The UV absorption spectra of trypsin (40 μM) at different DCBQ / DBBQ concentrations in the examples of the present application; (a) DCBQ, 0-7: 0-7.91 μM, the concentration interval is 1.13 μM; (b) DBBQ, 0-7: 0-10.5 μM, the concentration interval is 1.5 μM; the temperature is 298K, pH = 7.4;

[0041] Fig.17 The fluorescence emission spectra of trypsin (20 μM) at different CPAN / DCPAN concentrations in the examples of the present application; (ac) CPAN, 0-7: 0-461.72 μM, concentration interval is 65.96 μM; (df) DCPAN, 0-7: 0-75.25 μM, concentration interval is 10.75 μM, pH = 7.4;

[0042] Fig.18 The interaction analysis between HPANs (CPAN / DCPAN) and trypsin at different temperatures in the present application examples; (a) Stern-Volmer plot of the interaction between CPAN and trypsin at different temperatures; (b) Log((F 0 -F) / F) and Log(1 / ([CPAN]-[trypsin](F 0 -F) / F 0 )) diagram; (c) Van't Hoff diagram; (d) Stern-Volmer diagram of the interaction between DCPAN and trypsin at different temperatures; (e) Log((F 0 -F) / F) and Log(1 / ([DCPAN]-[trypsin](F 0 -F) / F 0 )) graph; (f) Van't Hoff graph;

[0043] Fig.19For the analysis of the interaction between trypsin and CPAN / DCPAN in the examples of this application, (a) Stern-Volmer plot of the interaction between CPAN and trypsin at different temperatures; (b) logarithmic fluorescence quenching plot; (c) Van't Hoff plot; (d) Stern-Volmer plot of the interaction between DCPAN and trypsin at different temperatures; (e) logarithmic fluorescence quenching plot; (f) Van't Hoff plot;

[0044] Fig. 20 The UV absorption spectra of substances in different systems in the examples of this application; (a) trypsin (40 μM), CPAN (98.9 μM); (b) trypsin (40 μM), DCPAN (48.4 μM); temperature is 298K, pH=7.4;

[0045] Fig.21 The fluorescence emission spectra of trypsin (20 μM) at different CPOL / BPOL / DBPOL concentrations in the examples of the present application; (ac) CPOL, 0-7: 0-162.12 μM, concentration interval is 23.16 μM; (df) BPOL, 0-7: 0-120.68 μM, concentration interval is 17.24 μM; (gi) DBPOL, 0-7: 0-83.02 μM, concentration interval is 11.86 μM, pH = 7.4;

[0046] Fig. 22 Analysis of the interaction between BPOL / CPOL / DBPOL and trypsin in the examples of the present application, (a) Stern-Volmer plot of the interaction between CPOL and trypsin at different temperatures, (b) logarithmic fluorescence quenching plot, (c) Van't Hoff plot of the CPOL-trypsin system; (d) Stern-Volmer plot of the interaction between BPOL and trypsin at different temperatures; (e) logarithmic fluorescence quenching plot, (f) Van't Hoff plot; (g) Stern-Volmer plot of the interaction between DBPOL and trypsin at different temperatures; (h) logarithmic fluorescence quenching plot; (i) Van't Hoff plot;

[0047] Fig.23: This is the synchronous optical spectrum of trypsin (20 μM) at different CPOL / BPOL / DBPOL concentrations in the embodiment of the present application; (ab) CPLO, 0-7: 0-166.67 μM, concentration interval is 23.81 μM; (cd) BPOL, 0-7: 0-80.43 μM, concentration interval is 11.49 μM; (ef) DBPOL, 0-7: 0-55.37 μM, concentration interval is 7.91 μM); (a) CPOL, Δλ=15 nm; (b) CPOL, Δλ=60 nm; (c) BPOL, Δλ=15 nm; (d) BPOL, Δλ=60 nm; (e) DBPOL, Δλ=15 nm; (f) DBPOL, Δλ=60 nm; temperature is 298 K, pH=7.4;

[0048] Fig.24 This is the UV absorption spectrum of trypsin (20 μM) at different CPLO / BPOL / DBPOL concentrations in the examples of the present application; (a) CPOL, 0-7: 0-32.41 μM, concentration interval is 4.63 μM; (b) BPOL, 0-7: 0-24.15 μM, concentration interval is 3.45 μM; (c) DBPOL, 0-7: 0-16.59 μM, concentration interval is 2.37 μM; (d) UV absorption spectra of each substance in different systems; (1) trypsin; (2) trypsin + DBPOL; (3) DBPOL; (4) (trypsin + DBPOL) -DBPOL; DBPOL (16.6 μM), temperature is 298K, pH = 7.4. DETAILED DESCRIPTION

[0049] To make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme in the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to the normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0050] In some specific embodiments, the present application provides a method for evaluating the toxicity level of halogenated disinfection by-products based on protease, which comprises the following steps:

[0051] 1.1 Materials, reagents and instruments

[0052]

[0053]

[0054] 1.2 Solution configuration

[0055] The pepsin used in the experiment was stored at 4°C; the trypsin was stored at -20°C. DCBQ, DBBQ, BPOL, CPOL, and DBPOL were dissolved in ethanol to prepare five DBPs stock solutions of 1 g / L; CPAN and DCPAN were dissolved in methanol to prepare stock solutions of 20 g / L and 10 g / L respectively; they were stored at 4°C in the dark; in a specific embodiment, when diluting the DBPs stock solution, the corresponding solvent should be used.

[0056] Glycine-HCl buffer: Glycine (3.7535 g / L): HCl (4.18 mL) = 3:5 (V:V), concentration 0.2 mol / L (HCl) + Ion concentration is 0.2 mol / L); store at 4°C and dilute to 0.025 mol / L when used to make its pH 2.2.

[0057] Phosphate buffered saline (PBS): According to the "NaH 2 PO 4 (24g / L):Na 2 HPO 4 (28.39g / L)=1:3(V:V)”, concentration 0.2mol / L(PO 4 3- Ion concentration is 0.2 mol / L); store at 4°C and dilute to 0.01 mol / L when used, so that the pH is 7.4.

[0058] 1.3 Fluorescence spectrum measurement and UV absorption spectrum measurement

[0059] (1) Two-dimensional fluorescence spectroscopy: Data acquisition was performed using a Lumina fluorescence spectrometer equipped with a 150W xenon lamp. The instrument parameters were set as follows: scanning speed of 1200nm / min, excitation wavelength of 280nm, emission wavelength range of 280-450nm, slit widths of 5nm for both excitation and emission, photomultiplier tube (PMT) voltage of 700V, and scanning wavelength interval of 1nm. A 1cm quartz cuvette was used to perform fluorescence measurements within the wavelength range. A circulating water bath was used to control the temperature of the instrument.

[0060] (2) Synchronous fluorescence spectroscopy: The procedure and experimental conditions are basically the same as those for fluorescence spectroscopy. The scanning speed is 600 nm / min, and the emission and excitation spectra are scanned simultaneously, and the difference between the emission and excitation spectra is maintained. The data are recorded at a constant wavelength interval of Δλ = 15 nm (tyrosine) and 60 nm (tryptophan) and a scanning range of 250-350 nm to obtain information about the structural changes of the two residues. The data were characterized using Origin 8.5 software (OriginLab, USA).

[0061] Ideally, the fluorescence intensity should increase linearly with the increase of the concentration of the fluorescent substance, but due to the inner-filter effect of fluorescence (IFE), the growth of fluorescence intensity will gradually deviate from the linearity. IFE refers to the phenomenon that when the concentration of the fluorescent substance in the measured solution is high or it coexists with other absorbing substances, the fluorescence is weakened due to the absorption of the excitation light or emission light by the fluorescent substance or other absorbing substances. Therefore, in order to eliminate the influence of the internal filter effect (IFE) on the fluorescence intensity, the data obtained from the fluorescence emission can be corrected by the following formula:

[0062] F corr =F obs ×e ((Aex+Aem) / 2) (1-1)

[0063] In formula (1-1), F obs For the excitation wavelength λ ex , the emission wavelength is λ em The fluorescence intensity, F corr is the corrected fluorescence intensity, A ex is the absorbance at the excitation wavelength, A em is the absorbance at the emission wavelength.

[0064] (3) UV absorption spectrum measurement: Evolution 220 UV-visible spectrophotometer (ThermoFisherScientific) was used for data acquisition, and the temperature during measurement was 298 K. The instrument parameters were set as follows: scanning speed of 1200 nm / min, scanning range of 190-400 nm, and scanning wavelength interval of 1 nm. A 1 cm quartz cuvette was used to measure the light absorption of the sample within the wavelength range.

[0065] 1.4 Fluorescence quenching ( Stern-Volmer equation )

[0066] This application is based on Fluorescence quenching principle Combination Stern-Volmer equation Analyze the interaction between proteins and exogenous small molecules. The details are as follows:

[0067] When exogenous small molecules interact with proteins, the fluorescence intensity of proteins decreases. This phenomenon is called fluorescence quenching. Small molecules that can induce endogenous fluorescence quenching of proteins are called quenchers. Fluorescence quenching or the decrease in system fluorescence intensity can usually be explained by excited state reactions, molecular rearrangements, energy transfer, ground state complex formation, and collision quenching.

[0068] The main mechanisms of fluorescence quenching are static quenching (contact quenching) and dynamic quenching (collision quenching), and mixed static / dynamic quenching may also occur. Dynamic quenching originates from the energy transfer between protein and ligand. There is no chemical reaction between the two, and no force is involved, so it will not affect the conformation and physiological activity of the protein. Static quenching is the binding of protein and ligand to form a non-fluorescent complex, which will affect the secondary and tertiary structure of the protein as well as the biological function and activity. It can be distinguished based on the fluorescence lifetime measurement value or temperature dependence.

[0069] Specifically, for dynamic quenching, the increase in temperature will increase the diffusion coefficient, particle motion and energy transfer, thereby increasing the fluorescence quenching rate constant; for static quenching, the increase in temperature will reduce the stability of non-fluorescent complexes, resulting in a decrease in the quenching rate constant. In order to further characterize the steady-state quenching mechanism of proteins and small molecules at different temperatures, the Stern-Volmer equation can be used to study and analyze fluorescence data.

[0070] F 0 / F=1+K q τ 0 [Q]=1+K SV [Q] (2-1)

[0071] In formula (2-1), F 0 and F represent the fluorescence intensity of the protein with and without the quencher molecule, respectively; K q is the quenching rate constant of the biomacromolecule; K SV is the Stern-Volmer quenching constant; τ 0 The average fluorescence lifetime of a biomolecule in the absence of a quencher is 1×10 -8 s; [Q] is the concentration of the quencher.

[0072] 1.5 Combination parameters (double logarithmic equation)

[0073] Binding parameters are important parameters for studying the interaction between small molecules and proteins. The binding constant can provide the binding strength between small molecules and proteins, which helps to understand the mechanism of action of small molecules on proteins and the functional changes of proteins. Double logarithmic equationCalculate some binding parameters between small molecules and proteins. For the static quenching process, when the small molecules independently bind to a set of equivalent sites on the protein and the free molecules and bound molecules reach equilibrium, the equation (2-2) can be used to calculate the small molecule-protein system. Binding constant (K a ) and the number of binding sites (n) :

[0074]

[0075] Among them, F 0 , F and [Q] are the same as the variables in formula (2-1), K a is the binding constant of the small molecule and protein; n is the number of binding sites of the small molecule on the protein; [P] is the total concentration of the protein. 0 -F) / F] and Log(1 / ([Q]-[P](F 0 -F) / F 0 )) curve (abbreviated as fluorescence quenching logarithmic relationship curve / fluorescence quenching logarithmic graph) will show an obvious linear relationship, and the n and K of the small molecule-protein complex a The value can be obtained from the slope and intercept of this curve. (Note: In this application, "Log[(F 0 -F) / F] and Log(1 / ([DCBQ / DBBQ]-[pepsin](F 0 -F) / F 0 )) Figure 3 is based on the principle of fluorescence quenching and Double logarithmic equation, Study on the interaction between DBBQ and pepsin; similarly, the expressions of related terms in the study of the interaction between other DBPs and pepsin / trypsin are similar)

[0076] 1.6 Thermodynamic parameters (Van't Hoff and Gibbs-Helmholtz equations)

[0077] The function of proteins is related to the specific structure, conformation and non-covalent interaction between amino acid residues. Hydrogen bonds, van der Waals forces, electrostatic forces and hydrophobic forces are usually used to explain the interaction between small molecules and proteins. Therefore, in order to further confirm the type of interaction between ligands and proteins and the stability of the complex, it is necessary to use thermodynamic parameters, namely enthalpy change (ΔH 0 ), entropy change (ΔS 0 ) and Gibbs free energy (ΔG 0 ) to characterize the interaction between the ligand and the protein. 0 , ΔS 0 and ΔG 0 The constant value of can be calculated using the Van't Hoff and Gibbs-Helmholtz equations:

[0078]

[0079] ΔG 0 =ΔH 0 -TΔS 0 =-RTlnK a (2-4)

[0080] K a is the binding constant at the corresponding temperature; R is the universal gas constant (8.3145 J mol -1 K -1 );ΔH 0 and ΔS 0 By plotting lnK a The slope and intercept of the 1 / T curve are obtained.

[0081] In some specific embodiments, the present application selects three newly discovered, representative and highly toxic cyclic disinfection byproducts (DBPs) as research objects, namely, halogenated benzoquinones (HBQs), halogenated phenylacetonitrile (HPANs), and halogenated pyridinols (HPOLs), and uses these three substances as ligands. Two-dimensional fluorescence spectroscopy, synchronous fluorescence spectroscopy and ultraviolet Absorption spectroscopy , detect the effects of three types (seven in total) of halogenated DBPs (for details, see Tables 1 and 2) on the endogenous fluorescence intensity of proteases (pepsin / trypsin); the details are as follows:

[0082] Example 1 Determination of the interaction between pepsin and HBQs

[0083] (1) Fluorescence spectroscopy

[0084] Sample preparation: Prepare 20 μM pepsin stock solution; prepare 5.65 mM and 3.76 mM DCBQ and DBBQ stock solutions respectively

[0085] Experimental operation: Pipette 3 mL of pepsin solution into a 1 cm quartz cuvette with four sides transparent to light, and then add an appropriate amount of DCBQ (DBBQ is a comparative sample) stock solution into the cuvette to reach the corresponding DBPs reaction concentration (DCBQ: 0, 11.3, 22.6, 33.9, 45.2, 56.5, 67.8 μM; DBBQ: 0, 7.5, 15, 22.5, 30, 37.5, 45 μM). Set up three experimental groups, react them at 298K, 308K, and 318K for 15 min, and then measure their fluorescence intensity. The measuring instrument and other parameter settings are as described in 1.3.

[0086] (2) Synchronous fluorescence spectroscopy: Sample preparation and experimental operation are the same as those for fluorescence spectroscopy, but only performed at a temperature of 298K.

[0087] (3) Ultraviolet absorption spectrum measurement

[0088] Sample preparation: prepare 20 μM pepsin stock solution; prepare 0.565 mM and 1.125 mM DCBQ and DBBQ stock solutions respectively.

[0089] Experimental operation: Pipette 3 mL of pepsin solution into a 1 cm quartz cuvette with two transparent surfaces, and then add an appropriate amount of DCBQ (DBBQ is a control sample) stock solution into the cuvette to reach the corresponding DBPs reaction concentration (DCBQ: 0, 1.13, 2.26, 3.39, 4.52, 5.65, 6.78, 7.91 μM; DBBQ: 0, 2.25, 4.5, 6.75, 9, 11.25, 13.5, 15.75 μM). React at 298 K for 15 min, and then measure its absorbance. The measuring instrument and other parameter settings are as described in 1.3.

[0090] (4) Fluorescence quenching analysis

[0091] Pepsin is an enzyme containing 327 amino acid residues in a single polypeptide chain, of which the five tryptophan residues are the main contributors to intrinsic fluorescence. Since pepsin is sensitive to changes in its microenvironment, the intrinsic fluorescence of the protein can be easily quenched by interacting with quencher molecules, resulting in a decrease in the fluorescence quantum yield of the fluorophore.

[0092] The present application records the fluorescence spectra of pepsin in the presence of different concentrations of DCBQ / DBBQ at an excitation wavelength of 280 nm. Figure 1 As shown in the figure, the maximum emission wavelength of pepsin is located at 340nm, and the fluorescence intensity shows a significant downward trend as the concentration of DCBQ / DBBQ gradually increases. This phenomenon indicates that the two HBQs can act as a quencher to reduce the endogenous fluorescence of pepsin, and also proves that DCBQ / DBBQ and pepsin have a combined effect.

[0093] Table 3

[0094]

[0095] The fluorescence data were fitted using the Stern-Volmer equation, as Figure 2 As shown in (a) and (d), F 0 There is a linear relationship between / F and [Q], and its slope is K SV (K SV The larger the value, the greater the degree of quenching of the protein by the quencher). SV The values ​​at different temperatures are used to understand the quenching mechanism. Figure 2As shown in the figure, the Stern-Volmer plot of DCBQ / DBBQ quenching pepsin fluorescence showed a linear trend, and the slope (K SV ) decreases significantly with increasing temperature; according to formula (2-1), K at three temperatures is calculated SV The values ​​are shown in Table 3. SV The K value of HBQs for pepsin decreases with increasing temperature, indicating that the quenching mechanism of HBQs for pepsin is static quenching, which also proves the formation of HBQs-pepsin complex. q Value greater than 2.0×10 10 M -1 S -1 (K in case of dynamic quenching q This once again proves that the quenching mechanism of HBQs on pepsin is static quenching.

[0096] (5) Combination parameter and thermodynamic parameter analysis

[0097] Based on the above fluorescence quenching analysis, it is known that HBQs binds to pepsin to form an HBQs-pepsin complex, but the binding strength between the two is still unclear.

[0098] Therefore, the present application uses the double logarithmic formula (2-2) to fit the result as follows Figure 1 As shown in (b) and (e), the logarithmic relationship curves of fluorescence quenching at three different temperatures (298K, 308K, and 318K) show an obvious linear relationship, and the n and K of the DBPs-pepsin complex a The value can be obtained from the slope and intercept of this curve, and the results are shown in Table 4. The n values ​​at different experimental temperatures are close to 1, indicating that during the interaction process, HBQs has only one independent binding site on pepsin. a The K value can characterize the binding ability of small molecules to biomacromolecules. a The larger the size, the stronger the binding ability), the binding between the two depends on the affinity of the ligand for the receptor, which can be explained by the specific properties between the biological molecule and the ligand.

[0099] As shown in Table 4, the binding constants of the two HBQs with pepsin decreased with increasing temperature, indicating that the HBQs-pepsin system was unstable at high temperatures, which again proved that HBQs reduced the endogenous fluorescence of pepsin through the static quenching effect. 4 M -1 ) has a higher binding constant than DCBQ (1.448×10 4 M -1) is slightly larger, indicating that DBBQ has a greater affinity (binding energy) for pepsin than DCBQ. Studies have found that the mammalian cytotoxicity of the two brominated phenol DBPs is correlated with their binding energy with HSA (the greater the binding energy between the two, the stronger the corresponding brominated phenol cytotoxicity).

[0100] Studies have shown that the cytotoxic IC values ​​of DCBQ and DBBQ are 50 The (μM) values ​​are: 27.3 and 19.8 respectively (refer to Wang W, Qian Y, Li J, et al. Analytical and toxicity characterization of halo-hydroxyl-benzoquinones as stable halobenzoquinone disinfection byproducts in treated water [J]. Analytical Chemistry, 2014, 86 (10): 4982-4988.). This is consistent with the binding energy law of HBQs and pepsin in this application, from which it can be inferred that the binding energy of HBQs and pepsin may be positively correlated with the cytotoxicity of HBQs. The K of pepsin and HBQs a Value is about 10 4 Lmol -1 , indicating that the binding strength between HBQs and pepsin is medium ( Note: This application focuses on in vitro toxicity rather than in vivo toxicity; therefore, the mechanism of developmental toxicity cannot be used to explain explain ).

[0101] In terms of thermodynamic analysis, the results calculated by formulas (2-3) and (2-4) are shown in Table 4. 0 <0, indicating that the HBQs-pepsin complex is formed spontaneously. According to the theoretical analysis of Ross and Subramanian, ΔH 0 <0,ΔS 0 When >0, it indicates that hydrophobic force, electrostatic force and hydrogen bond may exist simultaneously in this process.

[0102] The hydrophobic force is formed by many hydrophobic amino acid residues near the active site of pepsin and the benzene ring of HBQs. In addition, the study of Ross and Subramanian also showed that the protein and the ligand first contacted each other by hydrophobic force, and then combined by other forces (hydrogen bond, van der Waals force, electrostatic force) to form a complex; hydrogen bond may be formed by oxygen atoms and halogen atoms in HBQs and certain amino acid residues in pepsin; at pH 2.2, pepsin (pI = 2.9, pI refers to the pH of the solution when the amino acid is at zero net charge) is an acidic protein with almost no positive charge. Therefore, hydrophobic force and hydrogen bond are the main forces in the binding process of HBQs and pepsin.

[0103] Table 4

[0104]

[0105] (6) Synchronous fluorescence spectroscopy analysis

[0106] Effects of two HBQs on synchronous fluorescence spectra of pepsin Figure 3 As shown in Figure 2, with the increase of HBQs concentration, the trend of synchronous fluorescence intensity is generally consistent with the above fluorescence spectrum ( Figure 1 ), which also shows the quenching effect of DCBQ / DBBQ on pepsin. Figure 3 As shown in (ab), with the increase of DCBQ concentration, the synchronous fluorescence peaks corresponding to tyrosine and tryptophan residues undergo obvious red shifts (tyrosine: 299nm-304nm; tryptophan: 342nm-348nm). The results show that the binding of DCBQ to pepsin leads to an increase in the polarity of the microenvironment of tyrosine and tryptophan residues and a decrease in hydrophobicity, which causes a change in the conformation of pepsin. The fluorescence quenching degree of DCBQ on tryptophan residues is greater than that of tyrosine, and the decrease ratios are 71.6% and 43.8%, respectively. This indicates that tryptophan plays an important role in the fluorescence quenching process of DCBQ on pepsin. Figure 3 As shown in (cd), with the increase of DBBQ concentration, the synchronous fluorescence peak corresponding to tyrosine has no obvious displacement, while the synchronous fluorescence peak corresponding to tryptophan has a 3nm blue shift. The results show that the binding of DBBQ to pepsin has little effect on the microenvironment around the tyrosine residue, but changes the polarity of the tryptophan microenvironment and induces an increase in the hydrophobicity of the chromophore microenvironment, which further indicates that the two bind due to a strong hydrophobic interaction.

[0107] (7) Ultraviolet absorption spectrum analysis

[0108] This application uses ultraviolet absorption spectroscopy to study the interaction model between HBQs and pepsin and the structural modification of pepsin by HBQs. Figure 4As shown, the UV absorption spectra of pepsin in DCBQ / DBBQ systems with different concentrations, the strong absorption peak appearing at 275nm is the characteristic absorption peak of aromatic amino acid residues in pepsin. Figure 4 In (a), 275 nm is a characteristic absorption peak of DCBQ, which coincides with the characteristic absorption peak of pepsin. Therefore, it is impossible to predict whether the increase in absorbance at 275 nm is caused by the formation of a DCBQ-pepsin complex or by an increase in DCBQ concentration. Therefore, the difference between the absorbances of [pepsin+DCBQ] and [DCBQ] and the difference in absorbance of [pepsin] were analyzed. The results are shown in Figure 2. Figure 4 As shown in the green curve in (c), it was found that the spectra of the two were significantly different, so the increase in absorbance at 275 nm was caused by the formation of a complex between pepsin and DCBQ. Figure 4 In (b), 290 nm is a characteristic absorption peak of DBBQ. With the addition of DBBQ, the absorbance at 275 nm gradually increases and undergoes an obvious red shift (275 nm-283 nm).

[0109] The above phenomena indicate that DBBQ can bind to pepsin to form a DBBQ-pepsin complex, leading to a change in the conformation of pepsin, which is consistent with the experimental results of fluorescence quenching.

[0110] Example 2 Determination of the interaction between pepsin and HPANs

[0111] (1) Fluorescence spectroscopy

[0112] The differences between the sample preparation and the experimental operation and those in Example 1 are as follows: 130 mM (CPAN), 55 mM (DCPAN), the reaction concentrations were adjusted to CPAN: 0, 0.26, 0.52, 0.78, 1.04, 1.3, 1.56, 1.82, 2.08 mM; DCPAN: 0, 0.11, 0.22, 0.33, 0.44, 0.55, 0.66, 0.77, 0.88 mM. Other parameter settings refer to Example 1.

[0113] (2) Synchronous fluorescence spectroscopy: Sample preparation and experimental operation are the same as those for fluorescence spectroscopy, but only performed at a temperature of 298K.

[0114] (3) Ultraviolet absorption spectrum measurement

[0115] Sample preparation: The sample preparation was the same as that in the UV absorption spectrum determination section of Example 1, and 32.97 mM and 16.13 mM CPAN and DCPAN stock solutions were prepared respectively.

[0116] Experimental operation: Pipette 3mL pepsin solution into a 1cm quartz cuvette with light transmission on both sides, then add an appropriate amount of CPAN (DCPAN is a comparative sample) stock solution to the cuvette to reach the corresponding DBPs reaction concentration (CPAN: 98.9μM; DCPAN: 48.4μM). React for 15min at a temperature of 298K, and then measure its absorbance. Since HPANs has a more obvious effect on the peptide bond of pepsin, the difference between the [pepsin] curve and the [(HPANs+pepsin)-HPANs] curve is taken for analysis. Other parameter settings refer to Example 1.

[0117] (4) Fluorescence quenching analysis

[0118] Figure 5 The results showed that the fluorescence intensity of pepsin at three temperatures (298K, 308K, and 318K) decreased regularly with the gradual increase of the concentration of the two HPANs. This phenomenon indicates that the two HPANs can be used as a quencher to quench the intrinsic fluorescence of pepsin. The fluorescence data were calculated using the Stern-Volmer equation, and the KSV of the two HPANs for pepsin decreased with the increase of temperature, indicating that HPANs reduce the intrinsic fluorescence of pepsin through a static quenching effect, and also prove the formation of the HPANs-pepsin complex.

[0119] (5) Combination parameter and thermodynamic parameter analysis

[0120] Based on the quenching analysis, it was found that pepsin and HPANs combined to produce a ground state complex, and then the double logarithmic equation calculation and thermodynamic analysis were used to further obtain the force type and binding energy (K a ), the number of binding sites (n). The results are shown in Table 5. The n values ​​at different experimental temperatures are close to 1, indicating that during the interaction process, HPANs have only one independent binding site on pepsin. a Value is about 10 2 L / mol, the binding force is weak, and the binding constant gradually decreases with the increase of temperature, indicating that the CPAN / DCPAN-pepsin system is unstable at high temperature, which once again proves that the quenching type of CPAN / DCPAN on pepsin is static quenching. It is also known that the binding constant of DCPAN and pepsin (8.352×10 2 M -1 ) is greater than the binding constant of CPAN (3.560×10 2 M -1 ), indicating that DCPAN has a stronger affinity for pepsin.

[0121] Based on the above analysis, the cytotoxicity data (LC50) of HPANs were consulted and found: DCPAN (83μM), CPAN (148μM). It can be found that the higher the affinity of DCPAN for pepsin, the stronger its toxicity. Therefore, according to the size of the binding constant obtained in the experiment (DBBQ>DCBQ>DCPAN>CPAN) and the change law of the existing toxicity data, it can be inferred that the binding energy of DBPs with pepsin is positively correlated with the cytotoxicity of DBPs.

[0122] Table 5

[0123]

[0124]

[0125] Table 6

[0126]

[0127] According to Ross and Subramanian, when ΔH 0 <0 and ΔS 0 >0, there are usually hydrophobic forces and electrostatic forces. The hydrophobic force is formed by the presence of many hydrophobic amino acid residues in pepsin and HPANs containing aromatic rings; the hydrogen bond is formed by the nitrogen atom in HPANs and the amino group of pepsin, as well as ΔH 0 The negative value of ΔG can also explain the involvement of hydrogen bonds in this interaction; pepsin does not ionize in a system with a pH of 2.2. Therefore, hydrophobic forces and hydrogen bonds are the main forces in the binding process between HPANs and pepsin. 0 <0 indicates that the formation of HPANs-pepsin complex occurs spontaneously.

[0128] (6) Synchronous fluorescence spectroscopy analysis

[0129] By detecting the compactness of fluorophores such as tyrosine and tryptophan residues, the structural changes of the CPAN / DCPAN-pepsin system can be simultaneously analyzed. Figure 7 As shown in Figure 2, with the increase of the concentration of the two HPANs, the fluorescence intensity of the maximum emission peaks of the two amino acids decreased regularly. Figure 7 As shown in (ab), as the CPAN content in the pepsin solution increases, the synchronous fluorescence peak corresponding to tyrosine undergoes a 2nm blue shift, while the synchronous fluorescence peak corresponding to tryptophan does not shift. The results show that the binding of CPAN to pepsin leads to a decrease in the polarity of the microenvironment where the tyrosine residues of pepsin are located, and an increase in hydrophobicity, but has no effect on the microenvironment where the tryptophan residues are located. Figure 7As shown in (cd), with the increase of DCPAN content in the pepsin solution, the synchronous fluorescence peak corresponding to tyrosine blue-shifted by 1 nm, and the synchronous fluorescence peak corresponding to tryptophan red-shifted by 2 nm. The results showed that the binding of DCPAN to pepsin caused the polarity of the microenvironment of the tyrosine residue to decrease and the hydrophobicity to increase, and the polarity of the microenvironment of the tryptophan residue to increase and the hydrophobicity to decrease, that is, the amino acids in the non-polar hydrophobic cavity were moved to a more hydrophobic / hydrophilic environment, indicating that the hydrophobic cavity inside and outside the pepsin was unbalanced, resulting in a change in the conformation of the pepsin.

[0130] (7) Ultraviolet absorption spectrum analysis

[0131] The results of UV absorption spectroscopy are as follows Figure 8 As shown in (a), 206nm is the characteristic absorption peak of the peptide bond in pepsin, and 197nm is the characteristic absorption peak of CPAN. When pepsin is mixed with CPAN, the peak value presented changes significantly, and the absorption wavelengths of [pepsin] and [pepsin + CPAN]-CPAN also show obvious differences, which indicates that pepsin interacts with CPAN to generate a CPAN-pepsin complex, which affects the peptide chain structure of pepsin, further confirming the experimental results of fluorescence quenching. When pepsin is mixed with DCPAN, the absorbance of pepsin is as follows: Figure 8 (b) shows the phenomenon and theoretical explanation of the mixture of pepsin and CPAN. Figure 8 (a) Similar, the conclusions are the same.

[0132] Example 3 Determination of the interaction between pepsin and HPOLs

[0133] (1) Fluorescence spectroscopy

[0134] The difference between the sample preparation and Example 1 is that 7.72mM, 5.74mM, and 3.95mM CPOL, BPOL, and DBPOL stock solutions were prepared respectively. Other parameter settings refer to Example 1

[0135] The difference between the experimental operation and Example 1 is that the reaction concentration range and gradient are different, as follows:

[0136] CPOL: 0, 23.16, 46.32, 69.48, 92.64, 115.8, 138.96, 162.12 μM; BPOL: 0, 17.24, 34.48, 51.72, 68.96, 86.2 μM; DBPOL: 0, 23.72, 35.58, 47.44, 59.3, 71.16, 83.02 μM. Other parameter settings refer to Example 1

[0137] (2) Synchronous fluorescence spectrometry (sample preparation is the same as fluorescence spectrometry)

[0138] Experimental operation: Pipette 3 mL of pepsin solution into a 1 cm quartz cuvette with four sides transparent to light, then add an appropriate amount of CPOLs stock solution (BPOLs or DBPOL as a control sample) into the cuvette to reach the corresponding DBPs reaction concentration (CPOL: 0, 7.72, 15.44, 23.16, 30.88, 38.6, 46.32, 54.04 μM; BPOL: 0, 11.49, 22.98, 34.47, 45.96, 57.45, 68.94, 80.43 μM; DBPOL: 0, 7.91, 15.82, 23.73, 31.64, 39.55, 47.46, 55.37 μM). Set up three experimental groups, react them at 298K, 308K, and 318K for 15 min, and then measure their fluorescence intensity. For other parameter settings, refer to Example 1.

[0139] (3) Ultraviolet absorption spectrum measurement

[0140] The difference between the sample preparation and Example 1 is that 2.32 mM, 1.73 mM, and 1.19 mM stock solutions of CPOL, BPOL, and DBPOL were prepared respectively.

[0141] Experimental operation: Pipette 3 mL of pepsin solution into a 1 cm quartz cuvette with two transparent surfaces, and then add an appropriate amount of CPOL (BPOL or DBPOL as a comparison sample) stock solution into the cuvette to reach the corresponding DBPs reaction concentration (CPOL: 0, 4.63, 9.26, 13.89, 18.52, 23.15, 27.28, 32.41 μM; BPOL: 0, 3.45, 6.9, 10.35, 13.8, 17.25, 20.7, 24.15 μM; DBPOL: 0, 2.37, 4.74, 7.11, 9.48, 11.85, 14.22, 16.59 μM). React at a temperature of 298 K for 15 min, and then measure its absorbance. The measuring instrument and other parameter settings refer to Example 1.

[0142] (4) Fluorescence quenching analysis

[0143] like Fig. 9 As shown in Table 7, with the increase of the concentration of three HPOLs, the overall fluorescence intensity of pepsin showed a downward trend, indicating that HPOLs can be used as a quencher to reduce the intrinsic fluorescence of pepsin. The results of Stern-Volmer equation processing are shown in Table 7. It can be found that KSV, K qThe values all decreased with the increase of temperature, indicating that HPOLs reduced the endogenous fluorescence of pepsin through static quenching effect, and also indicating the formation of HPOLs-pepsin complex.

[0144] Table 7

[0145]

[0146]

[0147] (5) Analysis of binding parameters and thermodynamic parameters

[0148] As shown in Table 8, the n values at different experimental temperatures were close to 1, indicating that during the interaction process, HPOLs had only one independent binding site on pepsin. The K a values of the three HPOLs and pepsin were negatively correlated with temperature, indicating that the HPOLs-pepsin system was unstable at high temperature and not conducive to the formation of the complex, which was consistent with the changing trend of the quenching constant and could be interpreted as one of the demonstrations of static quenching. In addition, the results showed that the binding constant of DBPOL (6.121×10 3 M -1 ) was the largest, followed by BPOL (4.009×10 3 M -1 ), and the smallest was CPOL (3.763×10 3 M -1 ), indicating that pepsin had a higher affinity for DBPOL.

[0149] Based on the above conclusions on the relationship between the binding constants and toxicity of HBQs and HPANs, the developmental toxicity levels (EC 50 , μM) (the median effect concentration refers to the concentration at which a certain toxicant can cause a certain effect (developmental abnormalities, deformities, etc.) in 50% of the tested cell lines) of HPOLs were respectively: BPOL (294.0 μM) < CPOL (292.5 μM) < DBPOL (13.8 μM). It can be seen that the toxicity levels of CPOL and BPOL were similar, but in this application, the binding constant of BPOL (4.009×10 3 M -1 ) was slightly larger than that of CPOL (3.763×10 3 M -1 ), indicating that the conclusions in this experiment could not be explained by the mechanism of developmental toxicity. The reason might be: the tested substances were different. The toxicity rules measured in this application (an in vitro experiment) were consistent with those measured in CHO (Chinese hamster ovary cells) (DBBQ (19.8 μM) > DCBQ (27.3 μM) > DCPAN (83 μM) > CPAN (148 μM)), while the research on zebrafish embryos was an in vivo toxicity experiment.

[0150] In thermodynamic analysis, due to ΔG 0 <0, indicating that the formation of HPOLs-pepsin complex is spontaneous. According to Ross and Subramanian, when ΔS 0 >0,ΔH 0 <0, it indicates that electrostatic force and hydrophobic force may exist in this process. The hydrophobic force is due to the generation of hydrophobic amino acids in pepsin, and since pepsin has almost no positive charge in the system of pH 2.2, the electrostatic force can be ignored.

[0151] Table 8

[0152]

[0153]

[0154] (6) Synchronous fluorescence spectroscopy analysis

[0155] like Fig.11 As shown in the figure, the intervals of Δλ=15nm and Δλ=60nm are the fluorescence spectra of the specific amino acid residues of pepsin: tyrosine and tryptophan. As the concentration of HPOLs increases, the fluorescence intensity of the maximum emission peak shows a downward trend. Fig.11 As shown in (ab), with the increase of CPOL content, the synchronous fluorescence peak corresponding to tyrosine undergoes a 1nm red shift, and when the CPOL content reaches more than 10μM, its maximum emission peak position becomes another peak (307nm) and undergoes a red shift; the synchronous fluorescence peak corresponding to tryptophan undergoes an obvious blue shift (342nm-336nm), and the results show that the binding of CPOL to pepsin leads to an increase in the polarity and a decrease in the hydrophobicity of the microenvironment where the tyrosine residue is located, while the polarity and the hydrophobicity of the microenvironment where the tryptophan residue is located are decreased; the peak position value of tyrosine changes suddenly, and its quenching degree is relatively strong, and it can be considered that the binding site of the two is closer to the tyrosine residue. Fig.11 As shown in (cd), with the increase of BPOL content, the synchronous fluorescence peak corresponding to tyrosine red-shifted by 1 nm, while the synchronous fluorescence peak corresponding to tryptophan did not shift significantly. The results showed that the binding of BPOL to pepsin increased the polarity of the microenvironment of the tyrosine residue and decreased its hydrophobicity. Fig.11 As shown in (ef), with the increase of DBPOL content, the synchronous fluorescence peak corresponding to tyrosine blue-shifted by 1nm, and the synchronous fluorescence peak corresponding to tryptophan blue-shifted by 2nm. The results showed that the binding of DBPOL to pepsin caused the polarity of the microenvironment of tyrosine and tryptophan residues to decrease and the hydrophobicity to increase. The amino acids buried in the non-polar hydrophobic cavity were moved to a more hydrophobic environment, indicating that the hydrophobic cavity inside and outside the pepsin may be unbalanced, leading to changes in the conformation and function of the pepsin.

[0156] (7) Ultraviolet absorption spectrum analysis

[0157] like Fig.12 As shown in (ac), 275nm is the characteristic absorption peak of amino acids in pepsin. Fig.12 In (a), 291 nm is the characteristic absorption peak of CPOL. As the CPOL content increases, the absorbance at 275 nm gradually increases and a red shift occurs (275 nm-289 nm). Fig.12 In (b), 283 nm is the characteristic absorption peak of BPOL. As the BPOL content increases, the absorbance at 275 nm gradually increases and a red shift occurs (275 nm-281 nm). Fig.12 In (c), 293nm is the characteristic absorption peak of DBPOL. As the content of DBPOL increases, the absorbance at 275nm gradually increases and red-shifts (275nm-288nm). The above phenomena all indicate that pepsin interacts with HPOLs to form an HPOLs-pepsin complex, which may cause modification of the pepsin structure, further confirming the experimental conclusion of fluorescence quenching.

[0158] It can be seen from Examples 1-3 that :

[0159] (1) Through fluorescence quenching experiments and the Stern-Volmer equation, it was found that three types (seven kinds) of halogenated DBPs can quench the endogenous fluorescence of pepsin by forming a DBPs-pepsin complex, which belongs to the static quenching mechanism.

[0160] (2) The double logarithmic equation showed that the seven halogenated DBPs had only one binding site with pepsin and were not affected by temperature. The binding constants of DBPs with pepsin were: HBQs (DBBQ>DCBQ, 10 4 )>HPOLs(DBPOL>BPOL>CPOL, 10 3 )>HPANs(DCPAN>CPAN,10 2 ). Based on the existing studies on the relationship between the binding constant of DBPs and proteins and the toxicity of DBPs, the cytotoxicity of DBPs obtained by using CHO as the test substance (DBBQ>DCBQ>DCPAN>CPAN) is consistent with the binding constant of pepsin-HBQs / HPANs obtained in this application.

[0161] (3) The main interaction forces between pepsin and HBQs are hydrophobic force and hydrogen bond through thermodynamic analysis; the interaction forces between pepsin and HPANs are mainly hydrophobic force and hydrogen bond; the interaction forces between pepsin and HPOLs are mainly hydrophobic force. This indicates that hydrophobic force is the main interaction force between pepsin and DBPs. The binding process ΔG of DBPs and pepsin selected in this application is 0 <0, indicating that the formation of these seven DBPs-pepsin complexes is a spontaneous process.

[0162] (4) After the seven DBPs interacted with pepsin, the maximum emission peaks (λ max ) position has moved, indicating that the polarity and hydrophobicity of the microenvironment near the amino acid residues have changed, resulting in a change in the conformation of pepsin, which may further cause changes in the function of pepsin.

[0163] Example 4 Determination of the interaction between trypsin and HBQs

[0164] (1) Fluorescence spectroscopy

[0165] Sample preparation: prepare 20 μM trypsin stock solution; prepare 2.82 mM and 1.88 mM DCBQ and DBBQ stock solutions respectively.

[0166] The experimental operation differs from that of Example 1 in that: reaction concentration (concentration range of DCBQ: 0, 2.82, 5.64, 8.46, 11.28, 14.1, 16.92, 19.74 μM; DBBQ: 0, 1.88, 3.76, 5.64, 7.52, 9.4, 11.28, 13.16 μM); other parameters and conditions are the same as in Example 1.

[0167] (2) Synchronous fluorescence spectroscopy: Sample preparation and experimental operation are the same as those for fluorescence spectroscopy, but only performed at a temperature of 298K.

[0168] (3) Ultraviolet absorption spectrum measurement

[0169] Sample preparation: prepare 40 μM trypsin stock solution; prepare 0.565 mM and 0.75 mM DCBQ and DBBQ stock solutions respectively.

[0170] The experimental operation differs from that of Example 1 in that: reaction concentration (DCBQ: 0, 1.13, 2.26, 3.39, 4.52, 5.65, 6.78, 7.91 μM; concentration range of DBBQ: 0, 1.5, 3, 4.5, 6, 7.5, 9, 10.5 μM); other parameters and conditions are the same as those of Example 1.

[0171] (4) Fluorescence quenching analysis

[0172] Based on the above fluorescence spectrum analysis method and quenching mechanism, the effect of HBQs on trypsin fluorescence quenching at three temperatures (298K, 303K, 308K) was studied. The maximum emission wavelength of trypsin is located at 336nm. Fig.13 As shown in Table 9, with the increase of the concentration of the two HBQs, the fluorescence intensity of trypsin showed a decreasing trend. The results showed that HBQs, as a quencher, caused the decrease of the endogenous fluorescence of trypsin. The quenching constant (KSV) at three temperatures was calculated using the Stern-Volmer equation to determine its quenching mechanism. The calculation results are shown in Table 9. KSV, K q It showed a decreasing trend with the increase of temperature, which showed that HBQs reduced the intrinsic fluorescence of trypsin through static quenching effect, and also indicated the formation of HBQs-trypsin complex.

[0173] Table 9

[0174] system T / K <![CDATA[K SV (×10 4 M)]]> <![CDATA[K q (×10 12 M -1 S -1 )]]> DCBQ-Trypsin 298 3.640 3.640 303 3.233 3.233 308 2.963 2.963 DBBQ-Trypsin 298 4.484 4.484 303 4.294 4.294 308 3.991 3.991

[0175] (5) Combination parameter and thermodynamic parameter analysis

[0176] The results are shown in Table 10. The n values ​​at different experimental temperatures are close to 1, indicating that during the interaction process, HBQs has only one independent binding site on trypsin. The binding constants of both HBQs decrease with increasing temperature, indicating that the HBQs-trypsin system is unstable at high temperatures and is not conducive to the formation of the complex, which is consistent with the trend of the quenching constant, once again proving that the quenching mechanism of HBQs on trypsin is static quenching; K a For 10 4 L / mol level, indicating that the two have a medium binding affinity. In addition, the binding constant of DBBQ and trypsin (9.115×10 4 M -1 ) is greater than the binding constant of DCBQ and trypsin (6.420×10 4 M -1 ), indicating that trypsin is more likely to bind to DBBQ, which is consistent with the size law of the binding constant of pepsin-HBQs.

[0177] The binding constants of the two HBQs with trypsin were greater than those of HBQs with pepsin, indicating that trypsin has a higher affinity for DBPs than pepsin, which is consistent with the results of Wu et al. on trypsin and pepsin with small molecules.

[0178] Table 10

[0179]

[0180] (6) Synchronous fluorescence spectroscopy analysis

[0181] Synchronous fluorescence technology is used to analyze the two main fluorophores in protease molecules: tyrosine and tryptophan residues. Fig.15 As shown in the figure, the binding of HBQs to trypsin causes the tryptophan residue buried in the non-polar hydrophobic cavity to be moved to a more hydrophilic / hydrophobic environment, resulting in a conformational change in trypsin. The change in hydrophobicity further demonstrates that the binding of HBQs to trypsin is dominated by hydrophobic forces.

[0182] (7) Ultraviolet absorption spectrum analysis

[0183] like Fig.16 The strong absorption peak at 280 nm is the characteristic absorption peak of aromatic amino acid residues in trypsin. Fig.16 In (a), 274 nm is the characteristic absorption peak of DCBQ. As the DCBQ content increases, the absorbance at 280 nm gradually increases and a blue shift occurs (280 nm-276 nm). Fig.16 In (b), 292nm is the characteristic absorption peak of DBBQ. As the DBBQ content increases, the absorbance at 280nm gradually increases and an obvious red shift occurs (280nm-282nm). These phenomena indicate that HBQs can bind to trypsin to form an HBQs-trypsin complex, causing conformational changes in pepsin, further confirming the results of the fluorescence quenching experiment.

[0184] Example 5 Determination of the interaction between trypsin and HPANs

[0185] (1) Fluorescence spectroscopy

[0186] Sample preparation: Prepare 20 μM trypsin stock solution; prepare 32.98 mM and 5.38 mM CPAN and DCPAN stock solutions respectively.

[0187] The experimental operation is different from that of Example 1 in that: the corresponding HPANs reaction concentrations (CPAN: 0, 65.96, 131.92, 197.88, 263.84, 329.8, 395.76, 461.72 μM; DCPAN: 0, 10.75, 21.5, 32.25, 43, 53.75, 64.5, 75.25 μM); other parameters and conditions are the same as in Example 1.

[0188] (2) Synchronous fluorescence spectroscopy

[0189] Sample preparation: Prepare 20 μM trypsin stock solution; prepare 130 mM and 55 mM CPAN and DCPAN stock solutions respectively.

[0190] Experimental operation: The difference from Example 1 is: the corresponding HPANs reaction concentrations (CPAN: 0, 0.26, 0.52, 0.78, 1.04, 1.3, 1.56, 1.82 mM; DCPAN: 0, 0.11, 0.22, 0.33, 0.44, 0.55, 0.66, 0.77 mM); other parameters and conditions are the same as in Example 1.

[0191] (3) Ultraviolet absorption spectrum measurement

[0192] Sample preparation: Prepare a 40 μM trypsin stock solution; prepare 32.97 mM and 16.13 mM CPAN and DCPAN stock solutions respectively.

[0193] Experimental operation: The difference from Example 1 is: the corresponding HPANs reaction concentration (CPAN: 98.9 μM; DCPAN: 48.4 μM); since HPANs has a significant effect on the peptide bond of trypsin, the difference between the [trypsin] curve and the [(HPANs+trypsin)-HPANs] curve is used for analysis. Other parameters and conditions are the same as in Example 1.

[0194] (4) Fluorescence quenching analysis

[0195] like Fig.17 As shown in the figure, with the increase of HPANs concentration, the fluorescence intensity of trypsin showed a decreasing trend, and the maximum emission peak position showed an obvious red shift (336nm-340nm), and this change also occurred at the other two temperatures. This phenomenon shows that HPANs can be used as a quencher to reduce the intrinsic fluorescence of trypsin and change the microenvironment of the amino acid residues. The fluorescence data were calculated using the Stern-Volmer equation to obtain the quenching rate constants K of the two HPANs. q All of them decrease with the increase of temperature and are greater than the maximum collision rate constant (2.0×10 10 M -1 S -1 ), indicating that HPANs reduced the fluorescence intensity of trypsin through a static quenching effect, and also indicating the formation of an HPANs-trypsin complex.

[0196] Table 11

[0197]

[0198] (5) Combination parameter and thermodynamic parameter analysis

[0199] The results are shown in Table 12. The n values ​​at different experimental temperatures are close to 1, indicating that HPANs have only one independent binding site on trypsin during the interaction process. The binding constants of both HPANs decrease with increasing temperature, indicating that the HPANs-trypsin system is unstable at high temperatures and is not conducive to the formation of the complex. This is consistent with the trend of the quenching constant, which once again proves that the quenching mechanism of HPANs on trypsin is static quenching. It was also found that the binding constant of DCPAN to trypsin is (4.774×10 3 M -1 ) is much larger than the binding constant of CPAN and trypsin (0.751×10 3 M -1 ), this result shows that trypsin has a stronger affinity for DCPAN, while from the perspective of binding strength, the binding force is weaker, which is consistent with the binding constant size law of pepsin and HPANs. And from Table 15, it can be seen that the binding energy of trypsin and HPANs is greater than the binding energy of pepsin and HPANs, indicating that the affinity of HPANs for trypsin is greater than that for pepsin.

[0200] Table 12

[0201]

[0202] According to the theory of Ross and Subramanian, ΔH 0 <0, ΔS 0 >0, indicating that hydrophobic forces and hydrogen bonds are involved in the formation of the HPAN-trypsin complex. The hydrophobic forces are formed by the presence of many hydrophobic amino acid residues in trypsin and the HPANs containing aromatic rings; the hydrogen bonds are formed by the nitrogen atoms in HPANs and the amino groups of trypsin. The system containing DCPAN has a lower ΔG than the system containing CPAN. 0 The more negative the value is, the more likely it is that the HPANs-trypsin complex is formed spontaneously, and this also proves once again that the binding energy between DCPAN and trypsin is greater than that between CPAN and trypsin, and the more stable the DCPAN-trypsin complex system is.

[0203] (6) Synchronous fluorescence spectroscopy analysis

[0204] Fig.19 As shown in Figure 2, as the concentration of HPANs increases, the fluorescence intensity of tyrosine and tryptophan residues decreases regularly. Fig.19As shown in (ab), with the increase of CPAN concentration, the synchronous fluorescence peak (309nm) corresponding to tyrosine did not shift, but when the CPAN content reached above 160μM, a peak appeared at 290nm, and as CPAN continued to increase, the fluorescence intensity of this peak gradually increased and blue-shifted, while the synchronous fluorescence peak corresponding to tryptophan did not shift. This phenomenon indicates that the binding of CPAN to trypsin leads to a decrease in the polarity and an increase in the hydrophobicity of the microenvironment where the tyrosine residues are located, while the microenvironment where the tryptophan residues are located is not affected. Fig.19 As shown in (cd), with the increase of DCPAN concentration, the synchronous fluorescence peak (309nm) corresponding to tyrosine did not shift, and the synchronous fluorescence peak corresponding to tryptophan showed a 3nm red shift, which indicated that the binding of DCPAN to trypsin led to the polarity enhancement and hydrophobicity reduction of the microenvironment where the tryptophan residue was located, making it easier to be exposed to the solvent. The above phenomena all indicate that the microenvironment of trypsin has changed, affecting the functional group skeleton that maintains trypsin, leading to changes in the conformation of trypsin.

[0205] (7) Ultraviolet absorption spectrum analysis

[0206] like Fig. 20 As shown in Figure 2, 203 nm is the characteristic absorption peak of the peptide bond in trypsin. Fig. 20 As shown in (a), 197nm is the characteristic absorption peak of CPAN. When trypsin is mixed with CPAN, the peak value changes significantly, and the absorption wavelengths of [trypsin] and [trypsin + CPAN]-CPAN are also significantly different. Fig. 20 (b), 198nm is the characteristic absorption peak of DCPAN. When trypsin is mixed with DCPAN, the absorbance of trypsin decreases and red shifts (203nm-210nm). The above phenomena all indicate that the formation of CPAN / DCPAN-trypsin complex affects the peptide chain structure of trypsin, further confirming the experimental results of fluorescence quenching.

[0207] Example 6 Determination of the interaction between trypsin and HPOLs

[0208] (1) Fluorescence spectroscopy

[0209] Sample preparation: Prepare 20 μM trypsin stock solution; prepare 7.72 mM, 5.74 mM, and 3.95 mM CPOL, BPOL, and DBPOL stock solutions respectively.

[0210] The experimental operation is different from that of Example 1 in that: the corresponding HPOLs reaction concentrations (CPOL: 0, 23.16, 46.32, 69.48, 92.64, 115.8, 138.96, 162.12 μM; BPOL: 0, 17.24, 34.48, 51.72, 68.96, 86.2, 103.44, 120.68 μM; DBPOL: 0, 11.86, 23.72, 35.58, 47.44, 59.3, 71.16, 83.02 μM); other parameters and conditions are the same as in Example 1.

[0211] (2) Synchronous fluorescence spectroscopy:

[0212] Sample preparation: Prepare 20 μM trypsin stock solution; prepare 11.91 mM, 5.74 mM, and 3.95 mM CPOL, BPOL, and DBPOL stock solutions, respectively.

[0213] Experimental operation: The difference from Example 1 is: the corresponding HPOLs should be concentrated (CPOL: 0, 23.81, 47.62, 71.43, 95.24, 119.05, 142.86, 166.67 μM; BPOL: 0, 11.49, 22.98, 34.47, 45.96, 57.45, 68.94, 80.43 μM; DBPOL: 0, 7.91, 15.82, 23.73, 31.64, 39.55, 47.46, 55.37 μM); other parameters and conditions are the same as in Example 1.

[0214] (3) Ultraviolet absorption spectrum measurement

[0215] Sample preparation: Prepare 20 μM trypsin stock solution; prepare 2.32 mM, 1.73 mM, 1.19 mM CPOL, BPOL, and DBPOL stock solutions respectively.

[0216] Experimental operation: Pipette 3 mL of trypsin solution into a 1 cm quartz cuvette with two transparent surfaces, then add an appropriate amount of CPOL (BPOL or DBPOL as a comparison sample) stock solution into the cuvette to reach the corresponding reaction concentration (CPOL: 0, 4.63, 9.26, 13.89, 18.52, 23.15, 27.28, 32.41 μM; BPOL: 0, 3.45, 6.9, 10.35, 13.8, 17.25, 20.7, 24.15 μM; DBPOL: 0, 2.37, 4.74, 7.11, 9.48, 11.85, 14.22, 16.59 μM). React at a temperature of 298 K for 15 min, and then measure its absorbance. Other parameters and conditions are the same as in Example 1.

[0217] (4) Fluorescence quenching analysis

[0218] like Fig.21 As shown. With the increase of HPOLs concentration, the fluorescence intensity of trypsin showed a decreasing trend, and the maximum emission peak positions of BPOL and DBPOL both showed obvious red shift (BPOL: 336nm-340nm, DBPOL: 336nm-341nm), and the higher the temperature, the more obvious the shift phenomenon. This phenomenon shows that HPOLs can be used as a quencher to reduce the intrinsic fluorescence of trypsin and change the microenvironment of the amino acid residues. The fluorescence data were calculated using the Stern-Volmer equation, and the quenching constants KSV of the three HPOLs all decreased with the increase of temperature, indicating that HPOLs reduce the intrinsic fluorescence of trypsin through the static quenching effect, and also prove the formation of HPOLs-trypsin complex.

[0219] Table 13

[0220] system T / K <![CDATA[K SV (×10 3 M -1 )]]> <![CDATA[K q (×10 11 M -1 S -1 )]]> BPOL-Trypsin 298 1.657 1.657 308 1.562 1.562 318 1.362 1.362 BPOL-Trypsin 298 3.749 3.749 308 3.382 3.382 318 3.211 3.211 DBPOL-Trypsin 298 4.416 4.416 308 3.943 3.943 318 3.641 3.641

[0221] (5) Combination parameter and thermodynamic parameter analysis

[0222] As shown in Table 14, the n values ​​at different experimental temperatures are close to 1, indicating that during the interaction process, HPOLs have only one independent binding site on trypsin. As shown in Table 14, the binding constant of HPOLs and trypsin decreases with increasing temperature, indicating that the HPOLs-trypsin system is unstable at high temperatures and is not conducive to the formation of the complex, which is consistent with the change trend of the quenching constant, once again proving that the quenching mechanism of HPOLs on trypsin is static quenching. In addition, the binding constant of CPOL and trypsin is the smallest (1.507×10 3 M -1 ), the binding constant of DBPOL to trypsin is the largest (6.569×10 3 M -1 ), the results showed that trypsin was more likely to bind to DBPOL, and the binding strength was weaker, which was consistent with the law of the binding constant between pepsin and HPOLs.

[0223] Table 14

[0224]

[0225]

[0226] Table 15 shows that the binding energy of DBPOL to trypsin is greater than that of DBPOL to pepsin, while the binding constants of BPOL and CPOL to trypsin are smaller than those to pepsin, which is consistent with the research results of Xiao et al. The reason may be that trypsin contains 11 amino acid residues that participate in the binding of small molecules to trypsin, and pepsin contains 21 amino acid residues that participate in the binding of small molecules to pepsin, resulting in CPOL / BPOL having a greater affinity for pepsin than trypsin.

[0227] Table 15

[0228]

[0229] Non-covalent forces are the main necessary condition for the formation of ground state complexes between ligands and proteins. The four main forces have been mentioned above. The results calculated using formulas (2-3) and (2-4) are shown in Table 14. ΔG 0 <0, indicating that the formation of HPOLs-trypsin complex is spontaneous. Among the thermodynamic parameters, ΔH 0 , ΔS 0 The value is mainly used to determine the type of force in the interaction between small molecules and proteins. According to the Ross and Subramanian theory, when ΔS 0 Greater than 0,ΔH 0 When pK is less than or close to 0, hydrophobic force and electrostatic force are the main forces. The hydrophobic force is generated by many hydrophobic amino acids in trypsin; in the pH = 7.4 system, trypsin will be positively charged, CPOL (pK a1 =3.39, pK a2 =7.97)BPOL(pK a1＝ 1.67, pK a2 =7.29)DBPOL(pK a1 =-1.02, pK a2 =6.84) part will be negatively charged. Therefore, hydrophobic force and electrostatic force are the main forces in the binding process of HPOLs and trypsin.

[0230] (6) Synchronous fluorescence spectroscopy analysis

[0231] Fig.23 As shown in the figure, as the concentration of HPOLs increases, the fluorescence intensity of tyrosine and tryptophan residues decreases regularly, and the degree of fluorescence quenching of tyrosine is greater than that of tryptophan, which indicates that tyrosine contributes more to the quenching of trypsin fluorescence and the binding site is closer to tyrosine. Fig.23As shown in (ab), as the concentration of CPOL gradually increases, the corresponding synchronous fluorescence peaks of tyrosine and tryptophan both shift blue (tyrosine: 309nm-307nm, tryptophan: 343nm-341nm), which indicates that the binding of CPOL to trypsin leads to a decrease in the polarity and an increase in the hydrophobicity of the microenvironment where the amino acid residues are located. Fig.23 As shown in (cd), with the increase of BPOL content, the synchronous fluorescence peak corresponding to tyrosine did not shift, while the synchronous fluorescence peak corresponding to tryptophan shifted 2 nm blue. This phenomenon indicates that the binding of BPOL to trypsin leads to a decrease in polarity and an increase in hydrophobicity of the microenvironment where the tryptophan residues are located, while the microenvironment where the tyrosine residues are located is not disturbed. Fig.23 As shown in (ef), as the concentration of DBPOL gradually increases, the synchronous fluorescence peak corresponding to tyrosine shows a 2nm blue shift, and the synchronous fluorescence peak corresponding to tryptophan shows a 2nm red shift. This phenomenon indicates that the binding of DBPOL to trypsin leads to a decrease in the polarity and increase in the hydrophobicity of the microenvironment where the tyrosine residues are located, and an increase in the polarity and decrease in the hydrophobicity of the microenvironment where the tryptophan residues are located. The above phenomena all indicate that the binding of HPOLs to trypsin changes the microenvironment where the amino acid residues are located, and the hydrophobic cavity inside and outside the trypsin may be unbalanced, which in turn leads to changes in the conformation and function of pepsin.

[0232] (7) Ultraviolet absorption spectrum analysis

[0233] As shown in Figure 4-12, 280 nm is the characteristic absorption peak of amino acids in trypsin, and 203 nm is the absorption peak of peptide bonds in trypsin. Fig.24 In (a), 291 nm is a characteristic absorption peak of CPOL. As the CPOL content increases, the absorbance at 280 nm gradually increases and a red shift occurs (280 nm-283 nm). Fig.24 In (b), 240nm and 304nm are the two characteristic absorption peaks of BPOL. As the BPOL content increases, the absorbance at 280nm gradually increases and a red shift occurs (280nm-284nm). The results show that the interaction between trypsin and CPOL and BPOL changes the microenvironment of the amino acid residues, affects the functional groups that maintain the skeletal structure of trypsin, and causes the conformation of trypsin to change. Fig.24 In (c), 248 nm and 317 nm are two characteristic absorption peaks of DBPOL. As the content of DBPOL increases, the absorbance at 280 nm changes slightly. Therefore, the difference between the absorbance of [trypsin + DBPOL] and [DBPOL] and the difference of the absorbance of [trypsin] were analyzed. The results are shown in Figure 2. Fig.24 As shown by the green curve in (d), it is found that the spectra of the two are significantly different; Fig.24As shown in the red curve in (d), when DBPOL is added, the absorbance at 203 nm, the absorption peak of the peptide bond in trypsin, increases. This phenomenon indicates that trypsin interacts with DBPOL to form a DBPOL-trypsin complex, which may affect the microenvironment around the peptide bond and lead to changes in the polypeptide backbone structure, further confirming the conclusion of the fluorescence quenching experiment.

[0234] It can be seen from Examples 4-6 that :

[0235] (1) Three types (seven kinds) of halogenated DBPs: DCBQ, DBBQ, CPAN, DCPAN, CPOL, BPOL, and DBPOL, can all quench the endogenous fluorescence of trypsin by forming a DBPs-trypsin complex, which belongs to the static quenching mechanism.

[0236] (2) The double logarithmic equation showed that the seven halogenated DBPs had only one binding site with trypsin and was not affected by temperature. The binding constants of DBPs with trypsin were: DBBQ>DCBQ, (10 4 )>DBPOL>DCPAN>BPOL>CPOL>CPAN, (10 3 ). Through the speculation of the relationship between the binding constant of DBPs and proteins and the toxicity of DBPs in existing studies, the toxicity data of DBPs selected in this application were consulted and it was found that the cytotoxicity of DBPs obtained by using CHO as the test substance (DBBQ>DCBQ>DCPAN>CPAN) was consistent with the binding force of trypsin-HBQs / HPANs obtained in this experiment.

[0237] (3) The main interaction forces between trypsin and HBQs are hydrophobic force, electrostatic force, and hydrogen bond through thermodynamic analysis; the interaction forces between trypsin and HPANs include hydrophobic force, electrostatic force, and hydrogen bond; the interaction forces between trypsin and HPOLs are mainly hydrophobic force and electrostatic force. This indicates that hydrophobic force is also the main interaction force between trypsin and DBPs. The binding process ΔG of DBPs and trypsin selected in this application is 0 <0, indicating that the formation of these seven DBPs-trypsin complexes is a spontaneous process.

[0238] (4) After the interaction between seven DBPs and trypsin was obtained by synchronous fluorescence spectroscopy and UV absorption spectroscopy, the formation of DBPs-trypsin complex was confirmed again, and the maximum emission peak (λ max ) position has moved, which indicates that the microenvironment near the amino acid residues has changed, resulting in a change in the conformation of trypsin, which may cause a change in the function of trypsin.

[0239] In summary, this application selected two emerging cyclic and one heterocyclic DBPs: halogenated benzoquinones (HBQs), halogenated phenylacetonitrile (HPANs), and halogenated pyridinols (HPOLs) as ligands, and studied the interaction between DBPs and pepsin and trypsin by fluorescence spectroscopy and ultraviolet absorption spectroscopy. The main research conclusions are as follows:

[0240] The present application uses fluorescence spectroscopy and the Stern-Volmer equation to obtain three types (seven kinds) of halogenated DBPs: DCBQ, DBBQ, CPAN, DCPAN, CPOL, BPOL, and DBPOL, all of which can quench the endogenous fluorescence of pepsin and trypsin in the form of DBPs-pepsin and trypsin, which belongs to the static quenching mechanism.

[0241] Using ultraviolet absorption spectroscopy and synchronous fluorescence spectroscopy, it was found that the seven DBPs interacted with pepsin and trypsin to generate DBPs-pepsin / trypsin complexes, which changed the microenvironment near the main fluorophores (tryptophan and tyrosine residues) of pepsin and trypsin, resulting in changes in the conformation of the proteases. In the pepsin / trypsin system, DCBQ reduced the hydrophobicity of the microenvironment near the tryptophan residue, and DBBQ increased the hydrophobicity of the microenvironment near the tryptophan residue; both CPAN and DCPAN increased the hydrophobicity of the microenvironment near the tyrosine residue, weakened the hydrophobicity near the tryptophan residue, and the binding site was closer to the tryptophan residue, indicating that the tryptophan residue was moved to a more hydrophilic environment. CPOL and DBPOL increased the hydrophobicity of the microenvironment near the tryptophan residue in pepsin, CPOL and BPOL increased the hydrophobicity of the microenvironment near the tyrosine residue in trypsin, and BPOL increased the hydrophobicity near the tryptophan residue in trypsin, and the binding site was closer to the tyrosine residue. The above results show that the binding of DBPs to the two proteases changes the microenvironment of amino acids, making the amino acids in a more hydrophilic / hydrophobic environment, resulting in a looser / tighter structure of the proteases. This also provides an important basis for characterizing the interaction between DBPs and proteases.

[0242] Thermodynamic analysis showed that the interaction between the seven DBPs and pepsin was spontaneous and the force type of the DBPs-pepsin system was clarified. The HBQs-pepsin system was mainly hydrophobic force and hydrogen bond; the HPANs-pepsin system was mainly hydrophobic force and hydrogen bond; the HPANs-pepsin system was mainly hydrophobic force; the interaction between the seven DBPs and trypsin was spontaneous and the force type of the DBPs-trypsin system was clarified. The hydrophobic force was the main force in the formation of the DBPs-pepsin / trypsin complex.

[0243] The double logarithmic equation and fluorescence quenching experimental data showed that the seven DBPs can interact with pepsin / trypsin at a single binding site to form a DBPs-pepsin / trypsin complex. The binding constants of DBPs and pepsin are: DBBQ>DCBQ>DBPOL>BPOL>CPOL>

[0244] DCPAN>CPAN; the binding constants of DBPs and trypsin are: DBBQ>DCBQ>DBPOL>DCPAN>BPOL>CPOL>CPAN. The results show that DCBQ, DBBQ, DBPOL, DCPAN, and CPAN are more likely to bind to trypsin because trypsin has three catalytic residues (His-57, Asp-102, and Ser-195), and pepsin has two catalytic residues (Asp-32, Asp-215). The binding site of DBPs and proteases may be closer to the catalytic active center of trypsin, resulting in a stronger affinity of trypsin for DBPs. CPOL and BPOL are more likely to bind to pepsin because trypsin contains 11 amino acid residues that participate in the binding of DBPs and trypsin, and pepsin contains 21 amino acid residues that participate in the binding of DBPs and pepsin, resulting in a greater affinity of CPOL / BPOL for pepsin than for trypsin.

[0245] Through the investigation of the toxicity data of the selected DBPs, it was found that the cytotoxicity size law of DBPs obtained using CHO as the test substance: DBBQ (19.8μM)>DCBQ (27.3μM)>DCPAN (83μM)>CPAN (148μM)) is consistent with the binding force of pepsin / trypsin-HBQs / HPANs obtained in this experiment; while the developmental toxicity data of BDPs obtained using zebrafish embryos as the test substance (DCBQ>DBBQ>DBPOL>CPOL>BPOL) is obviously inconsistent with the binding force of pepsin / trypsin-HBQs / HPOLs in this application, indicating that the conclusion in this application is consistent with the cytotoxicity size law. In general, for the same type of DBPs, the cytotoxicity of DBPs is positively correlated with the binding force of digestive proteases, indicating that this indicator is expected to be used as a potential toxicity evaluation indicator to indicate the toxic effects of halogenated disinfection by-products.

[0246] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for evaluating the toxicity level of halogenated disinfection by-products based on protease, characterized in that: The steps include: S1. Solution preparation: prepare pepsin solution, trypsin solution and halogenated disinfection by-product solution; the halogenated disinfection by-products include cyclic and heterocyclic compounds, and at least include two of 2,6-dichloro-1,4-benzoquinone, 2,6-dibromo-1,4-benzoquinone, 2-chlorophenylacetonitrile, 3,4-dichlorophenylacetonitrile, 5-chloro-3-pyridinol, 2-bromo-3-pyridinol and 2,6-dibromo-3-pyridinol; S2, spectral measurement: the protease solution is mixed with halogenated disinfection byproduct solutions of different concentrations; The mixed solution is measured by two-dimensional fluorescence spectrum, synchronous fluorescence spectrum and ultraviolet absorption spectrum; Through these spectral data, information about the microenvironment of amino acid residues and structural changes of proteases can be obtained; S3. Fluorescence quenching analysis: Use the Stern-Volmer equation to fit the fluorescence data to determine whether the quenching type of the disinfection by-products on the protease is static or dynamic, and calculate the quenching constant to determine whether the disinfection by-products bind to the protease; S4. Binding parameter analysis: The logarithmic relationship curve of fluorescence quenching was constructed by double logarithmic equation to calculate the number of binding sites and binding constant of the disinfection byproduct-protease complex; the affinity of different proteases with disinfection byproducts was compared to evaluate the interaction strength and toxicity level of disinfection byproducts; S5. Thermodynamic parameter analysis: Enthalpy change, entropy change and Gibbs free energy were calculated using the Van't Hoff equation and the Gibbs-Helmholtz equation, and the main forces in the binding process were analyzed, including hydrogen bonding, hydrophobic interaction and electrostatic force; S6. Structural influence analysis: Disinfection byproducts interact with proteases to form disinfection byproduct-protease complexes. During the formation process, the hydrophobic environment around tryptophan and tyrosine residues is changed, thereby affecting the compactness of the protease structure. The interaction between disinfection byproducts and proteases is characterized accordingly. S7. Comprehensive analysis: Based on the analysis of steps S4-S7, determine whether there is a positive correlation between the interaction between the disinfection by-products and proteases and the cytotoxicity of the disinfection by-products.

2. The method according to claim 1, characterized in that: Step S2 also includes the following: The interactions of pepsin and trypsin with 2,6-dichloro-1,4-benzoquinone, 2,6-dibromo-1,4-benzoquinone, 2-chlorophenylacetonitrile, 3,4-dichlorophenylacetonitrile, 5-chloro-3-pyridinol, 2-bromo-3-pyridinol and 2,6-dibromo-3-pyridinol were measured respectively; the protease solution was mixed with different concentrations of halogenated disinfection byproduct solutions; The mixed solution was then measured by two-dimensional fluorescence spectrum, synchronous fluorescence spectrum and ultraviolet absorption spectrum.

3. The method according to claim 1, characterized in that: Step S2 also includes the following: In the measurement of the two-dimensional fluorescence spectrum, the scanning speed of the fluorescence spectrometer is 1200nm / min, the excitation wavelength is 280nm, the emission wavelength range is 280-450nm, the slit widths of excitation and emission are both 5nm, the photomultiplier tube voltage is 700V, and the scanning wavelength interval is 1nm; a 1cm quartz cuvette is used to perform fluorescence measurement within the wavelength range.

4. The method according to claim 1, characterized in that: Step S2 also includes the following: In the measurement of the synchronous fluorescence spectrum, the scanning speed of the fluorescence spectrometer is 600nm / min, and the emission and excitation spectra are scanned simultaneously, and the difference between the emission and excitation spectra is maintained, and the recording is performed at constant wavelength intervals of Δλ=15nm, Δλ=60nm and a scanning range of 250-350nm, thereby obtaining information about the structural changes of two residues; the two residues are tyrosine and tryptophan.

5. The method according to claim 1, characterized in that: Step S2 also includes the following: In the determination of the ultraviolet absorption spectrum, an ultraviolet-visible spectrophotometer is used for data collection, the temperature during the determination is 298K; the scanning speed is 1200nm / min, the scanning range is 190-400nm, and the scanning wavelength interval is 1nm; a 1cm quartz cuvette is used to measure the light absorption of the sample within the wavelength range.

6. The method according to claim 1, characterized in that: Step S3 also includes: if the Stern-Volmer plot of the disinfection by-product quenching protease fluorescence presents a linear relationship, it indicates that the disinfection by-product quenching mechanism of pepsin is static quenching.

7. The method according to claim 1, characterized in that: Step S4 also includes: fitting the fluorescence quenching logarithmic graph at 298K, 308K and 318K temperatures by a double logarithmic formula to obtain the number of binding sites and binding constant of the disinfection by-product-protease complex; the binding constant can characterize the size of the binding energy; and further prove whether the quenching type of the disinfection by-product on the protease is static.

8. The method according to claim 1, characterized in that: Step S7 also includes: referring to the mammalian cell toxicity IC of the disinfection byproducts 50 The value and its size pattern of binding energy with serum protein are consistent, and based on this, it can be judged whether the binding energy of disinfection by-products with proteases is positively correlated with their cytotoxicity.

9. Use of the method according to any one of claims 1 to 8, characterized in that Used to evaluate the impact of disinfection by-products on proteases in the digestive system of organisms.

10. Use of the method according to any one of claims 1 to 8, characterized in that Used to predict the toxic effects of disinfection by-products, optimize drinking water disinfection processes or establish drinking water quality standards.

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