A method for assessing the toxicity level of halogenated disinfection byproducts based on protease

Abstract

This invention relates to the field of water quality testing technology, and more particularly to a method for assessing the toxicity levels of halogenated disinfection byproducts based on proteases. This invention employs fluorescence spectroscopy, synchronous fluorescence spectroscopy, and ultraviolet absorption spectroscopy to study the interactions between seven cyclic and heterocyclic halogenated disinfection byproducts and pepsin and trypsin. The fluorescence quenching mechanism, binding constant, and thermodynamic parameters are analyzed using the Stern-Volmer equation and double logarithmic equation, revealing the influence of halogenated disinfection byproducts on protease conformation. This application discloses that the binding affinity of halogenated disinfection byproducts to proteases is positively correlated with cytotoxicity, providing a new indicator for evaluating the toxicity of halogenated disinfection byproducts and contributing to the optimization of drinking water disinfection processes and the formulation of water quality standards.

Description

Technical Field

[0001] This invention relates to the field of water quality testing technology, and in particular to a method for assessing the toxicity level of halogenated disinfection byproducts based on protease. Background Technology

[0002] In the current environment, environmental pollutants can enter the human body through skin contact, ingestion, and drinking water, and their potential hazards have been widely confirmed. Disinfection byproducts (DBPs), as a typical type of environmental pollutant, pose a significant threat to human health. Currently, assessing the threat posed by DBPs to human health mainly involves studying the reaction relationship and toxic effects between DBP concentrations and test substances. A commonly used research method involves exposing 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 assessing the hazards of DBPs by detecting 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 digestive phenols (DBPs) and proteins, human serum albumin (HSA) is a primary research subject. Studies have found that 4-bromophenol and 2,4-dibromophenol, two DBPs, can bind to HSA through hydrogen bonds and van der Waals forces, forming complexes and causing conformational changes in HSA. Furthermore, the affinity between DBPs and HSA is correlated with their toxicity. In contrast, research on the interaction mechanisms between small-molecule DBPs and digestive proteases is relatively limited. Understanding this mechanism is crucial for assessing the potential impact of DBPs after entering the gastrointestinal tract via food or drinking water.

[0004] Newly discovered cyclic digestive enzymes (DBPs) with higher toxicity indices have attracted widespread attention in recent years. When drinking water and food enter the stomach, digestive proteases not only participate in the digestion of proteins but also interact with components of the ingested substance. Since proteases may be direct binding targets of these components, they can lead to changes in the protease microenvironment and conformation, thereby affecting protease activity and the digestion and absorption of nutrients. However, current research on the interactions between DBPs ingested through drinking water and pepsin and trypsin in the human gastrointestinal tract is very limited. Summary of the Invention

[0005] In view of this, the present invention proposes a method for assessing the toxicity level of halogenated disinfection byproducts based on protease.

[0006] The technical solution of this invention is implemented as follows:

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

[0008] S1. Solution preparation: Prepare pepsin solution, trypsin solution, and halogenated disinfection byproduct solution; the halogenated disinfection byproduct comprises cyclic and heterocyclic compounds, and includes at least two of the following: 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. Spectroscopic determination: The protease solution was mixed with different concentrations of halogenated sterilization byproduct solutions; the mixed solutions were subjected to two-dimensional fluorescence spectroscopy, synchronous fluorescence spectroscopy, and ultraviolet absorption spectroscopy; information about changes in the amino acid residue microenvironment and protease structure was obtained through these spectral data.

[0010] S3. Fluorescence quenching analysis: The fluorescence data were fitted with the Stern-Volmer equation to determine whether the quenching type of the disinfection byproduct on the protease was static or dynamic, and the quenching constant was calculated to determine whether the disinfection byproduct and the protease were bound.

[0011] S4. Combined parameter analysis: The logarithmic relationship curve of fluorescence quenching was constructed by constructing a double logarithmic equation, and the number of binding sites and binding constant of the disinfection by-product-protease complex were calculated; the affinity of different proteases with disinfection by-products was compared, and the interaction strength and toxicity level of disinfection by-products were evaluated.

[0012] S5. Thermodynamic parameter analysis: Enthalpy change, entropy change and Gibbs free energy are calculated using the Van't Hoff equation and the Gibbs-Helmholtz equation. The main types of forces in the bonding process are analyzed, including hydrogen bonds, hydrophobic interactions and electrostatic forces.

[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 altered, thereby affecting the compactness of the protease structure. This is used to characterize the interaction force between disinfection byproducts and proteases.

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

[0015] Secondly, the present invention provides applications of the method for evaluating the effects of disinfection byproducts on proteases in the digestive system of organisms, predicting the toxic effects of disinfection byproducts, 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) This invention, through fluorescence spectroscopy and ultraviolet absorption spectroscopy, clarified the interaction mechanism between three classes (seven types) of halogenated DBPs—halogenated benzoquinones (HBQs), halogenated phenylacetonitrs (HPANs), and halogenated pyridinols (HPOLs)—and pepsin and trypsin. It determined that the quenching of intrinsic fluorescence is a static quenching mechanism, clarified that the interaction generates a complex, alters the microenvironment and conformation near the main fluorophore of the protease, and elucidates the specific effects of each DBP on the microenvironment of amino acid residues in different proteases. Furthermore, it clarified the thermodynamic properties and force types of the interaction, providing a comprehensive basis for a deeper understanding of the interaction between DBPs and proteases.

[0018] (2) This invention uses double logarithmic equations and fluorescence quenching experimental data to calculate that seven DBPs interact with pepsin / trypsin to form complexes with a single binding site, and obtains the order of the binding constants of DBPs with the two proteases. At the same time, it analyzes the reasons for the differences in the binding tendency of different DBPs with different proteases, which helps to further study the behavior of DBPs in organisms.

[0019] (3) This invention found that the cytotoxicity of the same type of DBPs is positively correlated with the binding affinity to digestive proteases, indicating that this binding affinity index is expected to serve as a potential toxicity assessment indicator for the toxic effects of halogenated disinfection byproducts, providing a new approach and method for the toxicity assessment of halogenated disinfection byproducts.

[0020] In some specific embodiments of the present invention Terminology Explanation Including the following:

[0021]

[0022]

[0023] Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1The fluorescence emission spectra of pepsin (20 μM) at different DCBQ / DBBQ concentrations are shown in the embodiments of this application; (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 The following diagrams illustrate the interaction between DCBQ and pepsin at different temperatures in this application: (a) Stern-Volmer plot, (b) logarithmic fluorescence quenching plot, and (c) Van't Hoff plot; and (d) Stern-Volmer plot, (e) logarithmic fluorescence quenching plot, and (f) Van't Hoff plot.

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

[0028] Figure 4 The following are the UV absorption spectra of pepsin (20 μM) at different DCBQ / DBBQ concentrations in the embodiments of this application: (a) DCBQ, 0-7: 0-7.91 μM, concentration interval 1.13 μM; (b) DBBQ, 0-7: 0-15.75 μM, concentration interval 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); temperature 298 K, pH = 2.2;

[0029] Figure 5 The fluorescence emission spectra of pepsin (20 μM) at different CPAN / DCPAN concentrations are shown in the embodiments of this 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 6The following are examples of the interactions between CPAN and pepsin at different temperatures in this application: (a) Stern-Volmer plot, (b) logarithmic fluorescence quenching plot, and (c) Van't Hoff plot; and (d) Stern-Volmer plot, (e) logarithmic fluorescence quenching plot, and (f) Van't Hoff plot of the DCPAN-pepsin system at different temperatures.

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

[0032] Figure 8 The following are the ultraviolet absorption spectra of various substances in different systems in the embodiments of this application: (a) pepsin (20 μM), CPAN (98.9 μM); (b) pepsin (20 μM), DCPAN (48.4 μM); temperature 298 K, pH = 2.2;

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

[0034] Figure 10 In the embodiments of this application, the interaction analysis of pepsin (20 μM) and CPOL / BPOL / DBPOL is shown in (a) Stern-Volmer plot of the interaction between CPOL and pepsin at different temperatures; (b) logarithmic fluorescence quenching plot; (c) Van'tHoff plot; (d) Stern-Volmer plot of the interaction between BPOL and pepsin at different temperatures; (e) logarithmic fluorescence quenching plot; (f) Van'tHoff plot; (g) Stern-Volmer plot of the interaction between DBPOL and pepsin at different temperatures; (h) logarithmic fluorescence quenching plot; and (i) Van'tHoff plot.

[0035] Figure 11 In the embodiments of this application, the synchronous fluorescence spectra of pepsin (20 μM) at different CPOL / BPOL / DBPOL concentrations are as follows: (ab) CPOL, 0-7: 0-54.04 μM, concentration interval 7.72 μM; (cd) BPOL, 0-7: 0-80.43 μM, concentration interval 11.49 μM; (ef) DBPOL, 0-7: 0-55.37 μM, concentration interval 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 298 K, pH = 2.2;

[0036] Figure 12 The following are the UV absorption spectra of pepsin (20 μM) at different concentrations of CPOL / BPOL / DBPOL in the embodiments of this application: (a) CPOL, 0-7: 0-32.41 μM, concentration interval 4.63 μM; (b) BPOL, 0-7: 0-24.15 μM, concentration interval 3.45 μM; (c) DBPOL, 0-7: 0-16.59 μM, concentration interval 2.37 μM; temperature 298 K, pH = 2.2;

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

[0038] Figure 14 The following are examples of the interaction analysis between DCBQ and trypsin at different temperatures in this application: (a) Stern-Volmer plot; (b) Logarithmic fluorescence quenching plot; (c) Van'tHoff plot; (d) Stern-Volmer plot of the interaction between DCBQ and trypsin at different temperatures; (e) Logarithmic fluorescence quenching plot; (f) Van'tHoff plot.

[0039] Figure 15The synchronous fluorescence spectra of trypsin (20 μM) at different DCBQ / DBBQ concentrations are shown in the embodiments of this application; (ab) DCBQ, 0-7: 0-19.74 μM, concentration interval of 2.82 μM; (cd) DBBQ, 0-7: 0-13.16 μM, concentration interval of 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] Figure 16 The following are the UV absorption spectra of trypsin (40 μM) at different DCBQ / DBBQ concentrations in the embodiments of this application: (a) DCBQ, 0-7: 0-7.91 μM, concentration interval 1.13 μM; (b) DBBQ, 0-7: 0-10.5 μM, concentration interval 1.5 μM; temperature 298 K, pH = 7.4;

[0041] Figure 17 The fluorescence emission spectra of trypsin (20 μM) at different CPAN / DCPAN concentrations are shown in the embodiments of this 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] Figure 18 The following are analyses of the interaction between HPANs (CPAN / DCPAN) and trypsin at different temperatures in this application embodiment: (a) Stern-Volmer plot of CPAN-trypsin interaction at different temperatures; (b) Log((F0-F) / F) and Log(1 / ([CPAN]-[trypsin](F0-F) / F0)) plots; (c) Van'tHoff plot; (d) Stern-Volmer plot of DCPAN-trypsin interaction at different temperatures; (e) Log((F0-F) / F) and Log(1 / ([DCPAN]-[trypsin](F0-F) / F0)) plots; (f) Van'tHoff plot;

[0043] Figure 19 In the embodiments of this application, the interaction analysis of trypsin with CPAN / DCPAN includes: (a) Stern-Volmer plots of the interaction between CPAN and trypsin at different temperatures; (b) logarithmic fluorescence quenching plots; (c) Van'tHoff plots; (d) Stern-Volmer plots of the interaction between DCPAN and trypsin at different temperatures; (e) logarithmic fluorescence quenching plots; and (f) Van'tHoff plots.

[0044] Figure 20 The following are the ultraviolet absorption spectra of substances in different systems in the embodiments of this application: (a) trypsin (40 μM), CPAN (98.9 μM); (b) trypsin (40 μM), DCPAN (48.4 μM); temperature 298 K, pH = 7.4;

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

[0046] Figure 22 For the embodiments of this application, the interaction analysis between BPOL / CPOL / DBPOL and trypsin is shown in the following figures: (a) Stern-Volmer plot of CPOL-trypsin interaction at different temperatures; (b) logarithmic fluorescence quenching plot; (c) Van't Hoff plot of the CPOL-trypsin system; (d) Stern-Volmer plot of BPOL-trypsin interaction at different temperatures; (e) logarithmic fluorescence quenching plot; (f) Van't Hoff plot; (g) Stern-Volmer plot of DBPOL-trypsin interaction at different temperatures; (h) logarithmic fluorescence quenching plot; (i) Van't Hoff plot.

[0047] Figure 23 The following are synchronous light spectra of trypsin (20 μM) at different CPOL / BPOL / DBPOL concentrations in the embodiments of this application: (ab) CPOL, 0-7: 0-166.67 μM, concentration interval 23.81 μM; (cd) BPOL, 0-7: 0-80.43 μM, concentration interval 11.49 μM; (ef) DBPOL, 0-7: 0-55.37 μM, concentration interval 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 298 K, pH = 7.4;

[0048] Figure 24The following are the UV absorption spectra of trypsin (20 μM) at different CPLO / BPOL / DBPOL concentrations in the embodiments of this application: (a) CPOL, 0-7: 0-32.41 μM, concentration interval 4.63 μM; (b) BPOL, 0-7: 0-24.15 μM, concentration interval 3.45 μM; (c) DBPOL, 0-7: 0-16.59 μM, concentration interval 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 298 K, pH = 7.4. Detailed Implementation

[0049] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this invention, not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0050] In some specific embodiments, this application provides a method for assessing the toxicity level of halogenated disinfection byproducts based on protease, which includes the following steps:

[0051] 1.1 Materials, Reagents and Instruments

[0052]

[0053]

[0054] 1.2 Solution Preparation

[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 1 g / L stock solutions of the five DBPs; CPAN and DCPAN were dissolved in methanol to prepare 20 g / L and 10 g / L stock solutions, respectively. All solutions were stored at 4°C protected from light. In specific embodiments, the corresponding solvents should be used when diluting the DBPs stock solutions.

[0056] Glycine-hydrochloric acid buffer: Prepared according to the ratio of glycine (3.7535 g / L): hydrochloric acid (4.18 mL) = 3:5 (V:V), with a concentration of 0.2 mol / L (H₂O). +(Ion concentration is 0.2 mol / L); store at 4℃, and dilute to 0.025 mol / L before use to make its pH 2.2.

[0057] Phosphate-buffered saline (PBS): Prepared according to the ratio of NaH₂PO₄ (24 g / L): Na₂HPO₄ (28.39 g / L) = 1:3 (V:V), with a concentration of 0.2 mol / L (PO₄). 3- (Ion concentration is 0.2 mol / L); store at 4℃, and dilute to 0.01 mol / L before use to make its pH 7.4.

[0058] 1.3 Fluorescence Spectroscopy Measurement and Ultraviolet Absorption Spectroscopy Measurement

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

[0060] (2) Synchronous fluorescence spectroscopy: The procedure and experimental conditions were basically the same as those for fluorescence spectroscopy. The scan rate was 600 nm / min, simultaneously scanning the emission and excitation spectra while maintaining the difference between them. Recordings were performed at constant wavelength intervals of Δλ = 15 nm (tyrosine) and 60 nm (tryptophan) within a scan 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, fluorescence intensity should increase linearly with increasing fluorescent substance concentration. However, due to the inner-filter effect of fluorescence (IFE), the increase in fluorescence intensity gradually deviates from this linearity. IFE refers to the phenomenon where fluorescence weakens due to the absorption of excitation or emission light by the fluorophore or other absorbing substances when the concentration of the fluorophore in the test solution is high or when it coexists with other light-absorbing substances. Therefore, to eliminate the influence of the inner-filter effect (IFE) on fluorescence intensity, the data obtained from fluorescence emission can be corrected using the following formula:

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

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

[0064] (3) Ultraviolet absorption spectroscopy: Data acquisition was performed using an Evolution 220 UV-Vis spectrophotometer (ThermoFisherScientific) at a temperature of 298K. The instrument parameters were set as follows: scan speed 1200 nm / min, scan range 190-400 nm, and scan wavelength interval 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 equations )

[0066] This application is based on Fluorescence quenching principle Combination Stern-Volmer equations The interaction between proteins and exogenous small molecules was analyzed. Details are as follows:

[0067] When exogenous small molecules interact with proteins, causing a decrease in the protein's fluorescence intensity, this phenomenon is called fluorescence quenching. Small molecules that can induce endogenous fluorescence quenching in proteins are called quenchers. Fluorescence quenching or a decrease in systemic fluorescence intensity can usually be explained by excited-state reactions, molecular rearrangements, energy transfer, ground-state complex formation, and collisional quenching.

[0068] Fluorescence quenching mechanisms mainly include static quenching (contact quenching) and dynamic quenching (collision quenching), and mixed static / dynamic quenching may also occur. Dynamic quenching originates from energy transfer between the protein and ligand; no chemical reaction occurs, and no forces are involved, so it does not affect protein conformation or physiological activity. Static quenching, on the other hand, involves the binding of the protein and ligand to form a non-fluorescent complex, which affects the protein's secondary and tertiary structure, as well as its biological function and activity. It can be distinguished based on fluorescence lifetime measurements or temperature dependence.

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

[0070] F0 / F=1+Kq τ0[Q]=1+K SV [Q] (2-1)

[0071] In formula (2-1), F0 and F represent the fluorescence intensity of the protein with and without quencher molecules, respectively; K q It is the quenching rate constant of biological macromolecules; K SV It is the Stern-Volmer quenching constant; τ0 is the average fluorescence lifetime of biomolecules without quenching agents, which is 1 × 10⁻⁶. -8 s; [Q] is the concentration of the quencher.

[0072] 1.5 Combined with parameters (double logarithmic equation)

[0073] Binding parameters are important for studying the interactions between small molecules and proteins. The binding constant provides the binding strength between small molecules and proteins, helping to understand the mechanisms of action of small molecules on proteins and changes in protein function. This application uses fluorescence intensity values ​​fitted to the concentration of the quencher to form... Double logarithmic equations The results, such as some binding parameters between small molecules and proteins, were calculated. For the static quenching process, when the small molecule independently binds to a set of equivalent sites on the protein, and the free and bound molecules reach equilibrium, the small molecule-protein system can be calculated using formula (2-2). Combining constant (K) a ) and the number of binding sites (n) :

[0074]

[0075] Where F0, F, and [Q] are the same variables as in formula (2-1), and K a is the binding constant when a small molecule binds to a protein; n is the number of binding sites of the small molecule on the protein; [P] is the total concentration of the protein. The curves of Log[(F0-F) / F] and Log(1 / ([Q]-[P](F0-F) / F0)) (referred to as the fluorescence quenching logarithmic relationship curve / fluorescence quenching logarithmic plot) will show a clear linear relationship, and 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, the "Log[(F0-F) / F] vs. Log(1 / ([DCBQ / DBBQ]-[pepsin](F0-F) / F0)) plot" is based on the principle of fluorescence quenching and Double logarithmic equations This study investigates the interaction between DBBQ and pepsin; similarly, the terminology used in studies of the interaction between other DBPs and pepsin / trypsin follows the same principle.

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

[0077] Protein function is related to specific structural, conformational, and non-covalent interactions between amino acid residues. Interactions between small molecules and proteins are typically explained using hydrogen bonds, van der Waals forces, electrostatic forces, and hydrophobic interactions. Therefore, to further confirm the type of interaction between ligands and proteins and the stability of the complex, thermodynamic parameters, namely enthalpy change (ΔH), are needed. 0 ), entropy change (ΔS) 0 ) and Gibbs free energy (ΔG 0 The sign and magnitude of ΔH are used to characterize the interaction force between the ligand and the protein. 0 ΔS 0 and ΔG 0 The constant value 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 R 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 drawing lnK a It is obtained by comparing the slope and intercept of the 1 / T curve.

[0081] In some specific embodiments, this application selected three newly discovered, representative, and highly toxic cyclic disinfection byproducts (DBPs) as research objects: halobenzoquinones (HBQs), halophenylacetonitrs (HPANs), and halopyridinols (HPOLs), using these three substances as ligands. Simultaneously, it utilized... Two-dimensional fluorescence spectroscopy, synchronous fluorescence spectroscopy and ultraviolet spectroscopy Absorption spectroscopy The effects of three classes (seven types in total) of halogenated DBPs (details in Tables 1 and 2) on the endogenous fluorescence intensity of proteases (pepsin / trypsin) were detected; details are as follows:

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

[0083] (1) Fluorescence spectroscopy determination

[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 Procedure: Transfer 3 mL of pepsin solution into a 1 cm quartz cuvette that is transparent on all four sides. Then, add an appropriate amount of DCBQ (DBBQ is the control sample) stock solution to the cuvette to reach the corresponding DBPs reaction concentrations (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 and react them at 298 K, 308 K, and 318 K for 15 min, then measure the fluorescence intensity. The measuring instruments and other parameter settings are as described in 1.3.

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

[0087] (3) Ultraviolet absorption spectroscopy 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 Procedure: Transfer 3 mL of pepsin solution into a 1 cm quartz cuvette that is translucent on both sides. Then, add an appropriate amount of DCBQ (DBBQ is the control sample) stock solution to the cuvette to achieve the corresponding DBPs reaction concentrations (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 the absorbance. The measuring instruments and other parameter settings are as described in Section 1.3.

[0090] (4) Fluorescence quenching analysis

[0091] Pepsin is an enzyme containing 327 amino acid residues in a single polypeptide chain, with the five tryptophan residues being the main contributors to intrinsic fluorescence. Because pepsin's microenvironment is highly sensitive to changes, the protein's intrinsic fluorescence is easily quenched by interaction with quenching molecules, leading to a decrease in the fluorescence quantum yield of the fluorophore.

[0092] This application records the fluorescence spectra of pepsin in the presence of different concentrations of DCBQ / DBBQ at an excitation wavelength of 280 nm. For example... Figure 1As shown, the maximum emission wavelength of pepsin is located at 340 nm. With the gradual increase of DCBQ / DBBQ concentration, the fluorescence intensity shows a significant decreasing trend. This phenomenon indicates that the two HBQs can act as quenchers, reducing the intrinsic fluorescence of pepsin, and also proves that DCBQ / DBBQ binds to pepsin.

[0093] Table 3

[0094]

[0095] The fluorescence data were fitted using the Stern-Volmer equation, such as... Figure 2 As shown in (a) and (d), there is a linear relationship between F0 / F and [Q], with a slope of K. SV (K SV The larger the value, the greater the quenching effect of the quencher on the protein. This can be achieved by recording K. SV To understand its quenching mechanism, we need to examine the values ​​at different temperatures. For example... Figure 2 As shown, the Stern-Volmer plot of pepsin fluorescence quenched by DCBQ / DBBQ exhibits a linear trend, with a slope (K) SV The K value decreases significantly with increasing temperature; K at the three temperatures is calculated according to formula (2-1). SV The values ​​are shown in Table 3, K SV The values ​​decrease with increasing temperature, indicating that the quenching mechanism of HBQs on pepsin is static quenching, which also proves the formation of the HBQs-pepsin complex. The Kc value of HBQs on pepsin was calculated. q Value greater than 2.0 × 10 10 M -1 S -1 (K under dynamic quenching conditions) q The maximum value of HBQs further proves that the quenching mechanism of pepsin is static quenching.

[0096] (5) Analysis combining parameters and thermodynamic parameters

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

[0098] Therefore, the fitting result obtained by this application using the double logarithmic formula (2-2) is 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) exhibit a clear linear relationship, and the n and K values ​​of the DBPs-pepsin complex are also significantly linear. aThe values ​​can be obtained from the slope and intercept of this curve, as shown in Table 4. The n values ​​at different experimental temperatures are close to 1, indicating that HBQs have only one independent binding site on pepsin during the interaction process. K a The K-value can characterize the binding ability of small molecules to biological macromolecules (K). a The larger the ligand, the stronger its binding ability. The binding between the two depends on the affinity of the ligand for the receptor, which can be explained by specific properties between the biomolecule and the ligand.

[0099] As shown in Table 4, the binding constants of the two HBQs with pepsin decrease with increasing temperature, indicating that the HBQs-pepsin system is unstable at high temperatures. This further proves that HBQs reduce the intrinsic fluorescence of pepsin through a static quenching effect; and it can be seen that DBBQ (1.578 × 10⁻⁶) 4 M -1 The binding constant ratio of DCBQ to pepsin is 1.448 × 10⁻⁶. 4 M -1 The slightly larger value indicates that DBBQ has a greater affinity (binding energy) for pepsin than DCBQ. Studies have found a correlation between the mammalian cytotoxicity of the two bromophenol DBPs and their binding energies with HSA (the greater the binding energy, the stronger the corresponding bromophenol cytotoxicity).

[0100] Studies have shown that DCBQ and DBBQ have existing cytotoxic IC50 values. 50 The μM values ​​were 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 by products in treated water[J]. Analytical Chemistry, 2014, 86(10): 4982-4988.). This is consistent with the binding energy pattern of HBQs and pepsin in this application, suggesting that the binding energy of HBQs and pepsin may be positively correlated with the cytotoxicity of HBQs. The Kc of pepsin and HBQs... a The value is approximately 10 4 Lmol -1 This indicates that HBQs and pepsin have a moderate binding affinity. Note: This application focuses on in vitro toxicity, not in vivo toxicity; therefore, the mechanism of developmental toxicity cannot be used to... explain ).

[0101] In terms of thermodynamic analysis, the results calculated by formulas (2-3) and (2-4) are shown in Table 4, ΔG0 A value <0 indicates that the HBQs-pepsin complex is spontaneously formed. Based on the theoretical analysis of Ross and Subramanian, ΔH... 0 <0, ΔS 0 When the value is greater than 0, it indicates that hydrophobic forces, electrostatic forces, and hydrogen bonds may coexist in this process.

[0102] The hydrophobic interaction is formed by numerous hydrophobic amino acid residues near the active site of pepsin and the benzene ring of HBQs. Furthermore, the study by Ross and Subramanian indicates that proteins and ligands initially contact each other via hydrophobic forces, then combine through other forces (hydrogen bonds, van der Waals forces, and electrostatic forces) to form a complex. Hydrogen bonds may be formed by oxygen and halogen atoms in HBQs with certain amino acid residues in pepsin. At pH 2.2, pepsin (pI = 2.9, where pI refers to the pH of the solution when the amino acid has a net charge of zero) is an acidic protein with almost no positive charge. Therefore, hydrophobic interactions and hydrogen bonds are the main forces in the binding process between HBQs and pepsin.

[0103] Table 4

[0104]

[0105] (6) Synchronous fluorescence spectroscopy analysis

[0106] The effects of two HBQs on the synchronous fluorescence spectrum of pepsin are as follows: Figure 3 As shown, the trend of synchronous fluorescence intensity with increasing HBQs concentration is generally consistent with the fluorescence spectrum described above. Figure 1 This also demonstrates the quenching effect of DCBQ / DBBQ on pepsin. For example... Figure 3 As shown in (ab), with the increase of DCBQ concentration, the synchronous fluorescence peaks corresponding to tyrosine and tryptophan residues showed a significant red shift (tyrosine: 299nm-304nm; tryptophan: 342nm-348nm). The results indicate that the binding of DCBQ to pepsin leads to an increase in the polarity and a decrease in the hydrophobicity of the microenvironment of tyrosine and tryptophan residues, resulting in a conformational change in pepsin. The fluorescence quenching degree of tryptophan residues by DCBQ is greater than that of tyrosine, with a decrease ratio of 71.6% and 43.8%, respectively. This indicates that tryptophan plays an important role in the fluorescence quenching process of pepsin by DCBQ. Figure 3As shown in (cd), with the increase of DBBQ concentration, the synchronous fluorescence peak corresponding to tyrosine did not shift significantly, while the synchronous fluorescence peak corresponding to tryptophan underwent a 3 nm blue shift. The results indicate that the binding of DBBQ to pepsin has little effect on the microenvironment around tyrosine residues, but it changes the polarity of the tryptophan microenvironment and induces an increase in the hydrophobicity of the chromophore microenvironment, further indicating that the two bind due to strong hydrophobic interactions.

[0107] (7) Ultraviolet absorption spectroscopy analysis

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

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

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

[0111] (1) Fluorescence spectroscopy determination

[0112] The sample preparation and experimental procedures differ from those in Example 1 as follows: 130 mM (CPAN) and 55 mM (DCPAN) were used, and the reaction concentrations were adjusted as follows: 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 are the same as in Example 1.

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

[0114] (3) Ultraviolet absorption spectroscopy measurement

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

[0116] Experimental procedure: Transfer 3 mL of pepsin solution into a 1 cm quartz cuvette that is translucent on both sides. Then, add an appropriate amount of CPAN (DCPAN is the control sample) stock solution to the cuvette to reach the corresponding DBPs reaction concentration (CPAN: 98.9 μM; DCPAN: 48.4 μM). React at 298 K for 15 min, and then measure the absorbance. Since HPANs have a significant effect on the peptide bonds of pepsin, the difference between the [pepsin] curve and the [(HPANs + pepsin) - HPANs] curve is used for analysis. Other parameter settings are the same as in Example 1.

[0117] (4) Fluorescence quenching analysis

[0118] Figure 5 The fluorescence intensity of pepsin at three temperatures (298K, 308K, and 318K) showed a regular decrease with increasing concentrations of both HPANs. This phenomenon indicates that the two HPANs can act as quenchers, quenching the intrinsic fluorescence of pepsin. Calculations using the Stern-Volmer equation revealed that the KSV of both HPANs for pepsin decreased with increasing temperature, indicating that HPANs reduce the intrinsic fluorescence of pepsin through a static quenching effect, and also demonstrating the formation of the HPANs-pepsin complex.

[0119] (5) Analysis combining parameters and thermodynamic parameters

[0120] Quenching analysis revealed that pepsin and HPANs bound to form a ground-state complex. Further calculations using a double logarithmic equation and thermodynamic analysis were then employed to determine the type of force involved in the binding process and the binding energy (K). a The number of binding sites (n) was calculated. The results are shown in Table 5. The n values ​​at different experimental temperatures were close to 1, indicating that HPANs had only one independent binding site on pepsin during the interaction process. The K-values ​​of the two HPANs with pepsin were also discussed. a The value is approximately 10 2The binding strength is weak, and the binding constant gradually decreases with increasing temperature, indicating that the CPAN / DCPAN-pepsin system is unstable at high temperatures. This further proves that the quenching type of pepsin by CPAN / DCPAN is static quenching. The binding constant of DCPAN with pepsin is also known to be 8.352 × 10⁻⁶ L / mol. 2 M -1 The binding constant of CPAN is greater than that of CPAN (3.560 × 10⁻⁶). 2 M -1 This indicates that DCPAN has a greater affinity for pepsin.

[0121] Based on the above analysis, reviewing the cytotoxicity data (LC50) of HPANs revealed that DCPAN (83 μM) and CPAN (148 μM) exhibited high affinity for pepsin, but also strong cytotoxicity. Therefore, based on the experimentally obtained binding constants (DBBQ > DCBQ > DCPAN > CPAN) and the existing cytotoxicity data, it can be inferred that the binding energy of DBPs to 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, typically exhibiting both hydrophobic and electrostatic interactions. The hydrophobic interactions arise from the presence of numerous hydrophobic amino acid residues in pepsin and their interaction with aromatic ring-containing HPANs; the hydrogen bonds are formed by the nitrogen atom in HPANs interacting with the amino group of pepsin, and ΔH... 0 A negative value can also explain the involvement of hydrogen bonds in this interaction; pepsin does not ionize in a system at pH 2.2. Therefore, hydrophobic forces and hydrogen bonds are the main forces in the binding process of HPANs to pepsin. ΔG 0 A value less than 0 indicates that the formation of the HPANs-pepsin complex is spontaneous.

[0128] (6) Synchronous fluorescence spectroscopy analysis

[0129] By detecting the density of fluorophores such as tyrosine and tryptophan residues, structural changes in the CPAN / DCPAN-pepsin system can be analyzed simultaneously. Figure 7 As shown, with increasing concentrations of the two HPANs, the fluorescence intensity of the maximum emission peaks of the two amino acids decreases systematically. Figure 7 As shown in (ab), with the increase of CPAN content in the pepsin solution, the synchronous fluorescence peak corresponding to tyrosine showed a 2 nm blue shift, while the synchronous fluorescence peak corresponding to tryptophan did not shift. The results indicate that the binding of CPAN to pepsin reduces the polarity and increases the hydrophobicity of the microenvironment containing tyrosine residues in pepsin, but has no effect on the microenvironment containing tryptophan residues. Figure 7 As shown in (cd), with the increase of DCPAN content in the pepsin solution, the synchronous fluorescence peak corresponding to tyrosine undergoes a 1 nm blue shift, and the synchronous fluorescence peak corresponding to tryptophan undergoes a 2 nm red shift. The results indicate that the binding of DCPAN to pepsin leads to a decrease in the polarity and an increase in the hydrophobicity of the microenvironment in which the tyrosine residues are located, and an increase in the polarity and a decrease in the hydrophobicity of the microenvironment in which the tryptophan residues are located. In other words, amino acids in the nonpolar hydrophobic cavity are moved to a more hydrophobic / more hydrophilic environment, indicating an imbalance between the internal and external hydrophobic cavities of pepsin, which leads to a change in the conformation of pepsin.

[0130] (7) Ultraviolet absorption spectroscopy analysis

[0131] The results of ultraviolet absorption spectroscopy are as follows: Figure 8 As shown in (a), 206 nm is the characteristic absorption peak of the peptide bond in pepsin, and 197 nm is the characteristic absorption peak of CPAN. When pepsin is mixed with CPAN, the peak values ​​change significantly, and the absorption wavelengths of [pepsin] and [pepsin + CPAN]-CPAN also show significant differences. This indicates that pepsin interacts with CPAN to form a CPAN-pepsin complex, affecting the peptide chain structure of pepsin, further confirming the results of the fluorescence quenching experiment. When pepsin is mixed with DCPAN, the absorbance of pepsin is as follows... Figure 8 As shown in (b), the phenomenon and theoretical explanation are related to the mixture of pepsin and CPAN. Figure 8 (a) Similar, the conclusions are consistent.

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

[0133] (1) Fluorescence spectroscopy determination

[0134] The sample preparation differed from that in Example 1 in that 7.72 mM, 5.74 mM, and 3.95 mM stock solutions of CPOL, BPOL, and DBPOL were prepared respectively. Other parameter settings were the same as in Example 1.

[0135] The experimental procedure differs from that in Example 1 in that the reaction concentration range and gradient are different, as detailed below:

[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 are the same as in Example 1.

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

[0138] Experimental Procedure: Transfer 3 mL of pepsin solution into a 1 cm quartz cuvette that is transparent on all four sides. Then, add an appropriate amount of CPOL stock solution (BPOLs or DBPOL as control samples) to the cuvette to achieve the corresponding DBPs reaction concentrations (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 and react them at 298 K, 308 K, and 318 K for 15 min, then measure the fluorescence intensity. Other parameter settings are the same as in Example 1.

[0139] (3) Ultraviolet absorption spectroscopy measurement

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

[0141] Experimental Procedure: Transfer 3 mL of pepsin solution into a 1 cm quartz cuvette that is translucent on both sides. Then, add an appropriate amount of CPOL (BPOL or DBPOL as a control sample) stock solution to the cuvette to achieve the corresponding DBPs reaction concentrations (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 298 K for 15 min, and then measure the absorbance. The measuring instruments and other parameter settings are the same as in Example 1.

[0142] (4) Fluorescence quenching analysis

[0143] As Figure 9 shown, with the increase of the concentrations of the three HPOLs, the overall fluorescence intensity of pepsin showed a downward trend, indicating that HPOLs can act as a quencher to reduce the intrinsic fluorescence of pepsin. The results of the Stern-Volmer equation treatment are shown in Table 7. It can be found that both the KSV and K q values decreased with the increase of temperature, indicating that HPOLs reduced the endogenous fluorescence of pepsin through a static quenching effect, and also indicating the formation of HPOLs-pepsin complexes.

[0144] Table 7

[0145]

[0146]

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

[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 temperatures and was not conducive to the formation of complexes, which was consistent with the change trend of the quenching constant and could be interpreted as one of the arguments for 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 found to be: 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 higher than that of CPOL (3.763×10 3 M -1The slightly larger value indicates that the conclusions of this experiment cannot be explained by the mechanism of developmental toxicity. The reason may be that the test substances are different. The toxicity pattern measured in this application (an in vitro experiment) is consistent with that of CHO (Chinese hamster ovary cells) (DBBQ (19.8 μM) > DCBQ (27.3 μM) > DCPAN (83 μM) > CPAN (148 μM)), while the study on zebrafish embryos is an in vivo toxicity experiment.

[0150] In thermodynamic analysis, due to ΔG 0 A value <0 indicates that the formation of the HPOLs-pepsin complex is spontaneous. According to Ross and Subramanian, when ΔS... 0 >0, ΔH 0 If the value is less than 0, it indicates that electrostatic and hydrophobic forces may exist during this process. The hydrophobic force is due to the production of hydrophobic amino acids in pepsin, while the electrostatic force is negligible because pepsin carries almost no positive charge in a system with a pH of 2.2.

[0151] Table 8

[0152]

[0153]

[0154] (6) Synchronous fluorescence spectroscopy analysis

[0155] like Figure 11 As shown, the fluorescence spectra of specific amino acid residues in pepsin, tyrosine and tryptophan, are at intervals of Δλ = 15 nm and Δλ = 60 nm, respectively. The fluorescence intensity of the maximum emission peak decreases with increasing HPOL concentration. Figure 11 As shown in (ab), with the increase of CPOL content, the synchronous fluorescence peak corresponding to tyrosine showed a red shift of 1 nm. When the CPOL content reached more than 10 μM, its maximum emission peak position changed to another peak (307 nm) and showed a red shift. The synchronous fluorescence peak corresponding to tryptophan showed a significant blue shift (342 nm-336 nm). The results indicate that the binding of CPOL to pepsin leads to an increase in the polarity and a decrease in the hydrophobicity of the microenvironment of tyrosine residues, and a decrease in the polarity and an increase in the hydrophobicity of the microenvironment of tryptophan residues. The peak value of tyrosine changed abruptly and its quenching degree was strong. It can be considered that the binding sites of the two are closer to the tyrosine residues. Figure 11 As shown in (cd), with the increase of BPOL content, the synchronous fluorescence peak corresponding to tyrosine was red-shifted by 1 nm, while the synchronous fluorescence peak corresponding to tryptophan did not shift significantly. The results indicate that the binding of BPOL to pepsin leads to an increase in the polarity of the microenvironment of tyrosine residues and a decrease in hydrophobicity. Figure 11As shown in (ef), with the increase of DBPOL content, the synchronous fluorescence peak corresponding to tyrosine undergoes a 1 nm blue shift, and the synchronous fluorescence peak corresponding to tryptophan undergoes a 2 nm blue shift. The results indicate that the binding of DBPOL to pepsin leads to a decrease in the polarity and an increase in the hydrophobicity of the microenvironment in which tyrosine and tryptophan residues are located. Amino acids buried in nonpolar hydrophobic cavities are moved to a more hydrophobic environment, indicating that the hydrophobic cavities inside and outside pepsin may be unbalanced, leading to changes in the conformation and function of pepsin.

[0156] (7) Ultraviolet absorption spectroscopy analysis

[0157] like Figure 12 As shown in (ac), 275nm is the characteristic absorption peak of amino acids in pepsin. Figure 12 (a) 291nm is the characteristic absorption peak of CPOL. As the content of CPOL increases, the absorbance at 275nm gradually increases and a red shift occurs (275nm-289nm). Figure 12 (b) 283nm is the characteristic absorption peak of BPOL. As the BPOL content increases, the absorbance at 275nm gradually increases and a red shift occurs (275nm-281nm). Figure 12 (c) shows that 293 nm is the characteristic absorption peak of DBPOL. As the DBPOL content increases, the absorbance at 275 nm gradually increases and a red shift occurs (275 nm - 288 nm). These phenomena 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] As can be seen from Examples 1-3 :

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

[0160] (2) Through double logarithmic equations, it was found that the seven halogenated DBPs have only one binding site with pepsin, and this binding site is not affected by temperature; the binding constants of DBPs and pepsin are: HBQs(DBBQ>DCBQ, 10 4 HPOLs(DBPOL>BPOL>CPOL, 10) 3 >HPANs(DCPAN>CPAN, 10) 2Based on existing research on the relationship between the binding constants of DBPs and proteins and the toxicity of DBPs, the cytotoxicity pattern of DBPs obtained using CHO as the test substance (DBBQ>DCBQ>DCPAN>CPAN) is consistent with the binding constants of pepsin-HBQs / HPANs obtained in this application.

[0161] (3) Thermodynamic analysis revealed that the main interaction forces between pepsin and HBQs are hydrophobic interactions and hydrogen bonds; the main interaction forces between pepsin and HPANs are hydrophobic interactions and hydrogen bonds; and the main interaction force between pepsin and HPOLs is hydrophobic interactions. This indicates that hydrophobic interactions are the primary force driving the binding of pepsin to DBPs. The binding process ΔG between DBPs and pepsin selected in this application... 0 <0 indicates that the formation of these seven DBPs-pepsin complexes is a spontaneous process.

[0162] (4) Synchronous fluorescence spectroscopy and ultraviolet absorption spectroscopy revealed the maximum emission peak (λ) of the main fluorophore residues (tryptophan and tyrosine) after the interaction of seven DBPs with pepsin. max The shift in position indicates a change in the polarity and hydrophobicity of the microenvironment near the amino acid residues, leading to a conformational change in pepsin, which may further cause changes in pepsin function.

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

[0164] (1) Fluorescence spectroscopy determination

[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 procedures differed from those in Example 1 in the following ways: reaction concentration (DCBQ concentration range: 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 were the same as in Example 1.

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

[0168] (3) Ultraviolet absorption spectroscopy 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 procedures differed from those in Example 1 in the following ways: reaction concentrations (DCBQ: 0, 1.13, 2.26, 3.39, 4.52, 5.65, 6.78, 7.91 μM; DBBQ concentration range: 0, 1.5, 3, 4.5, 6, 7.5, 9, 10.5 μM); other parameters and conditions were the same as in Example 1.

[0171] (4) Fluorescence quenching analysis

[0172] Based on the aforementioned fluorescence spectroscopy analysis methods and quenching mechanisms, the effects of HBQs on trypsin fluorescence quenching at three temperatures (298K, 303K, and 308K) were investigated. The maximum emission wavelength of trypsin was located at 336nm. Figure 13 As shown, the fluorescence intensity of trypsin decreased with increasing concentrations of both HBQs, indicating that HBQs act as a quencher, causing a decrease in the intrinsic fluorescence of trypsin. The quenching constant (KSV) at three temperatures was calculated using the Stern-Volmer equation to determine the quenching mechanism. The calculation results are shown in Table 9. q The fluorescence of HBQs decreased with increasing temperature, indicating that HBQs reduced the intrinsic fluorescence of trypsin through static quenching, and also indicating the formation of the 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) Analysis combining parameters and thermodynamic parameters

[0176] The results are shown in Table 10. The n values ​​at different experimental temperatures are close to 1, indicating that HBQs have only one independent binding site on trypsin during the interaction process. The binding constants of both HBQs decrease with increasing temperature, indicating that the HBQs-trypsin system is unstable at high temperatures, which is unfavorable for complex formation. This is consistent with the trend of the quenching constant, further proving that the quenching mechanism of HBQs on trypsin is static quenching; K a 10 4 The L / mol level indicates a moderate binding affinity between the two. Additionally, the binding constant of DBBQ with trypsin (9.115 × 10⁻⁶)... 4 M -1 The binding constant of DCBQ to trypsin is greater than that of DCBQ (6.420 × 10⁻⁶). 4 M -1This indicates that trypsin binds more readily to DBBQ, which is consistent with the general trend of the binding constant of pepsin-HBQs.

[0177] The binding constants of the two HBQs with trypsin were greater than those with pepsin, indicating that trypsin has a higher affinity for DBPs than pepsin. This is consistent with the findings 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 was used to analyze the two main fluorophores in protease molecules: tyrosine and tryptophan residues. For example... Figure 15 As shown, the binding of HBQs to trypsin causes tryptophan residues embedded in a nonpolar hydrophobic cavity to move to a more hydrophilic / hydrophobic environment, resulting in a conformational change in trypsin. This change in hydrophobicity further demonstrates that the binding of HBQs to trypsin is dominated by hydrophobic interactions.

[0182] (7) Ultraviolet absorption spectroscopy analysis

[0183] like Figure 16 The strong absorption peak at 280 nm is a characteristic absorption peak of aromatic amino acid residues in trypsin. Figure 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). Figure 16 (b) shows that 292 nm is the characteristic absorption peak of DBBQ. As the DBBQ content increases, the absorbance at 280 nm gradually increases and a significant red shift (280 nm - 282 nm) occurs. These phenomena indicate that HBQs can combine with trypsin to form an HBQs-trypsin complex, causing a conformational change in pepsin, which further confirms the results of the fluorescence quenching experiment.

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

[0185] (1) Fluorescence spectroscopy determination

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

[0187] The experimental procedures differed from those in Example 1 in the following ways: 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 were the same as in Example 1.

[0188] (2) Synchronous fluorescence spectroscopy determination

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

[0190] Experimental procedure: The difference from Example 1 is the corresponding HPANs reaction concentration (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 spectroscopy measurement

[0192] Sample preparation: 40 μM trypsin stock solution was prepared; 32.97 mM and 16.13 mM CPAN and DCPAN stock solutions were prepared respectively.

[0193] Experimental procedure: The difference from Example 1 is the corresponding HPANs reaction concentration (CPAN: 98.9 μM; DCPAN: 48.4 μM). Since HPANs have a significant effect on the peptide bonds 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 Figure 17 As shown, with increasing HPANs concentration, the fluorescence intensity of trypsin decreased, and the maximum emission peak position showed a significant red shift (336 nm-340 nm). This change also occurred at the other two temperatures. This phenomenon indicates that HPANs can act as a quencher, reducing the intrinsic fluorescence of trypsin and altering the microenvironment of the amino acid residues. The quenching rate constant K for the two HPANs was calculated using the Stern-Volmer equation. q All decrease with increasing temperature, and all are greater than the maximum collision rate constant (2.0 × 10⁻⁶). 10 M-1 S -1 The results indicate that HPANs reduce the fluorescence intensity of trypsin through a static quenching effect, and also indicate the formation of the HPANs-trypsin complex.

[0196] Table 11

[0197]

[0198] (5) Analysis combining parameters and thermodynamic parameters

[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, which is unfavorable for complex formation. This is consistent with the trend of the quenching constant, further proving that the quenching mechanism of HPANs on trypsin is static quenching. It was also found that the binding constant of DCPAN with trypsin is (4.774 × 10⁻⁶). 3 M -1 The value is much larger than the binding constant of CPAN to trypsin (0.751 × 10⁻⁶). 3 M -1 This result indicates that trypsin has a stronger affinity for DCPAN, while the binding strength is weaker, consistent with the binding constant of pepsin and HPANs. Table 15 shows that the binding energy between trypsin and HPANs is greater than that between pepsin and HPANs, indicating that HPANs have a stronger affinity for trypsin than for pepsin.

[0200] Table 12

[0201]

[0202] According to the Ross and Subramanian theory, ΔH 0 <0、ΔS 0 A value >0 indicates that hydrophobic interactions and hydrogen bonds are involved in the formation of the HPAN-trypsin complex. The hydrophobic interactions are due to the presence of many hydrophobic amino acid residues in trypsin and their interaction with aromatic ring-containing HPANs; the hydrogen bonds are due to the interaction between the nitrogen atom in HPANs and the amino group in trypsin. The system containing DCPAN has a higher ΔG value than the system containing CPAN. 0 The more negative the value, the more spontaneous the formation of the HPANs-trypsin complex is, which also proves that the binding energy of DCPAN to trypsin is greater than that of CPAN to trypsin, and that the DCPAN-trypsin complex system is more stable.

[0203] (6) Synchronous fluorescence spectroscopy analysis

[0204] Figure 19 As shown, the fluorescence intensity of tyrosine and tryptophan residues decreases systematically with increasing HPAN concentration. Figure 19 As shown in (ab), with increasing CPAN concentration, the synchronous fluorescence peak corresponding to tyrosine (309 nm) did not shift. However, when the CPAN content reached above 160 μM, a peak appeared at 290 nm. With further increases in CPAN, the fluorescence intensity of this peak gradually increased and underwent a blue shift. 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 surrounding tyrosine residues, while the microenvironment surrounding tryptophan residues remained unaffected. Figure 19 As shown in (cd), with increasing DCPAN concentration, the synchronous fluorescence peak corresponding to tyrosine (309 nm) did not shift, while the synchronous fluorescence peak corresponding to tryptophan showed a 3 nm red shift. This phenomenon indicates that the binding of DCPAN to trypsin leads to increased polarity and decreased hydrophobicity of the microenvironment of tryptophan residues, making them more easily exposed to solvents. All of these phenomena indicate that the trypsin microenvironment changes, affecting the maintenance of the trypsin functional group backbone and leading to a change in trypsin conformation.

[0205] (7) Ultraviolet absorption spectroscopy analysis

[0206] like Figure 20 As shown, 203 nm is the characteristic absorption peak of peptide bonds in trypsin. Figure 20 As shown in (a), 197 nm 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 also show significant differences. Figure 20 (b) 198 nm is the characteristic absorption peak of DCPAN. When trypsin is mixed with DCPAN, the absorbance of trypsin decreases and undergoes a red shift (203 nm - 210 nm). The above phenomena all indicate that the formation of the 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 determination

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

[0210] The experimental procedures differed from those in Example 1 in the following ways: the corresponding HPOL 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 were the same as in Example 1.

[0211] (2) Synchronous fluorescence spectroscopy determination:

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

[0213] Experimental procedure: The difference from Example 1 is that the corresponding HPOL concentrations should be (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 spectroscopy measurement

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

[0216] Experimental procedure: Transfer 3 mL of trypsin solution into a 1 cm quartz cuvette that is translucent on both sides. Then, add an appropriate amount of CPOL (BPOL or DBPOL as a control sample) stock solution to the cuvette to reach the corresponding reaction concentrations (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 298 K for 15 min, and then measure the absorbance. Other parameters and conditions are the same as in Example 1.

[0217] (4) Fluorescence quenching analysis

[0218] like Figure 21 As shown, with increasing HPOL concentration, the fluorescence intensity of trypsin decreased, and the maximum emission peaks of BPOL and DBPOL showed significant redshifts (BPOL: 336nm-340nm, DBPOL: 336nm-341nm), with the shift becoming more pronounced at higher temperatures. This phenomenon indicates that HPOLs can act as a quencher, reducing the intrinsic fluorescence of trypsin and altering the microenvironment of amino acid residues. Calculations using the Stern-Volmer equation revealed that the quenching constants (KSV) of the three HPOLs decreased with increasing temperature, indicating that HPOLs reduce the intrinsic fluorescence of trypsin through a static quenching effect, and also demonstrating the formation of the 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) Analysis combining parameters and thermodynamic parameters

[0222] As shown in Table 14, the n values ​​at different experimental temperatures are close to 1, indicating that HPOLs have only one independent binding site on trypsin during the interaction process. Table 14 also shows that the binding constant of HPOLs to trypsin decreases with increasing temperature, indicating that the HPOLs-trypsin system is unstable at high temperatures, which is unfavorable for complex formation. This is consistent with the trend of the quenching constant, further proving that the quenching mechanism of HPOLs on trypsin is static quenching. Furthermore, CPOL has the smallest binding constant with trypsin (1.507 × 10⁻⁶). 3 M -1 DBPOL exhibits the largest binding constant to trypsin (6.569 × 10⁻⁶). 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 binding constant of pepsin to HPOLs.

[0223] Table 14

[0224]

[0225]

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

[0227] Table 15

[0228]

[0229] Non-covalent forces are a major necessary condition for the formation of ground-state complexes between ligands and proteins, and 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 A value <0 indicates that the formation of the HPOLs-trypsin complex is spontaneous. In the thermodynamic parameters, ΔH... 0 ΔS 0 The value is primarily 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 the pH is less than or close to 0, hydrophobic and electrostatic forces are the dominant forces. The hydrophobic forces are generated by many hydrophobic amino acids in trypsin; in a system with pH = 7.4, trypsin carries a positive charge, 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) The portion will carry a negative charge. Therefore, hydrophobic and electrostatic forces are the main forces in the binding process of HPOLs to trypsin.

[0230] (6) Synchronous fluorescence spectroscopy analysis

[0231] Figure 23 As shown, with increasing HPOL concentration, the fluorescence intensity of tyrosine and tryptophan residues decreased systematically, and the fluorescence quenching degree of tyrosine was greater than that of tryptophan. This indicates that tyrosine contributes more to the quenching of trypsin fluorescence and that its binding site is closer to tyrosine residues. Figure 23As shown in (ab), with the gradual increase of CPOL concentration, the corresponding synchronous fluorescence peaks of tyrosine and tryptophan both exhibited a blue shift (tyrosine: 309nm-307nm, tryptophan: 343nm-341nm). This phenomenon indicates that the binding of CPOL to trypsin leads to a decrease in the polarity and an increase in the hydrophobicity of the microenvironment of the amino acid residues. Figure 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 showed a 2 nm blue shift. This phenomenon indicates that the binding of BPOL to trypsin leads to a decrease in the polarity and an increase in the hydrophobicity of the microenvironment of tryptophan residues, while the microenvironment of tyrosine residues remains undisturbed. Figure 23 As shown in (ef), with the gradual increase of DBPOL concentration, the synchronous fluorescence peak corresponding to tyrosine showed a 2 nm blue shift, and the synchronous fluorescence peak corresponding to tryptophan showed a 2 nm red shift. This phenomenon indicates that the binding of DBPOL to trypsin leads to a decrease in the polarity and an increase in the hydrophobicity of the microenvironment surrounding tyrosine residues, and an increase in the polarity and a decrease in the hydrophobicity of the microenvironment surrounding tryptophan residues. All of these phenomena suggest that the binding of HPOLs to trypsin alters the microenvironment surrounding amino acid residues, potentially causing an imbalance in the hydrophobic cavities inside and outside trypsin, thereby leading to changes in the conformation and function of pepsin.

[0232] (7) Ultraviolet absorption spectroscopy analysis

[0233] As shown in 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. Figure 24 (a) 291nm is a characteristic absorption peak of CPOL. As the content of CPOL increases, the absorbance at 280nm gradually increases and a red shift occurs (280nm-283nm). Figure 24 (b) 240 nm and 304 nm are two characteristic absorption peaks of BPOL. As the content of BPOL increases, the absorbance at 280 nm gradually increases and a red shift occurs (280 nm - 284 nm). The results indicate that the interaction between trypsin and CPOL and BPOL changes the microenvironment of amino acid residues, affects the functional groups that maintain the trypsin skeleton structure, and causes a change in the conformation of trypsin. Figure 24 (c) 248 nm and 317 nm are two characteristic absorption peaks of DBPOL. With increasing DBPOL content, the absorbance at 280 nm changes little. Therefore, the difference between the absorbance of [trypsin + DBPOL] and [DBPOL] and the difference in absorbance of [trypsin] were analyzed. The results are as follows: Figure 24 As shown by the green curve in (d), the spectra of the two are significantly different; Figure 24As shown by the red curve in (d), the absorbance at the absorption peak of peptide bonds in trypsin increased at 203 nm after the addition of DBPOL. This phenomenon indicates that trypsin interacts with DBPOL to form a DBPOL-trypsin complex, which may affect the microenvironment around the peptide bonds, leading to changes in the peptide backbone structure, and further confirming the conclusion of the fluorescence quenching experiment.

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

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

[0236] (2) The double logarithmic equation shows that the seven halogenated DBPs have only one binding site with trypsin, and this binding is not affected by temperature; while the binding constants of DBPs and trypsin are: DBBQ > DCBQ, (10 4 )>DBPOL>DCPAN>BPOL>CPOL>CPAN, (10 3 Based on existing research on the relationship between the binding constants of DBPs and proteins and the toxicity of DBPs, and after reviewing the toxicity data of the DBPs selected in this application, it was found that the cytotoxicity pattern of DBPs obtained using CHO as the test substance (DBBQ>DCBQ>DCPAN>CPAN) is consistent with the binding affinity of trypsin-HBQs / HPANs obtained in this experiment.

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

[0238] (4) After determining the interaction between seven DBPs and trypsin through synchronous fluorescence spectroscopy and ultraviolet absorption spectroscopy, the formation of the DBPs-trypsin complex was further confirmed, and the maximum emission peak (λ) of the major fluorophores (tryptophan and tyrosine) residues of trypsin was found. max The shift in position indicates a change in the microenvironment near the amino acid residues, leading to a conformational change in trypsin and potentially altering its function.

[0239] In summary, this application selected two emerging cyclic and one heterocyclic DBPs—halobenzoquinones (HBQs), halophenylacetonitrs (HPANs), and halopyridinols (HPOLs)—as ligands, and investigated the interactions between DBPs and pepsin and trypsin using fluorescence spectroscopy and ultraviolet absorption spectroscopy. The main findings are as follows:

[0240] This application uses fluorescence spectroscopy and the Stern-Volmer equation to obtain three classes (seven types) of halogenated DBPs: DCBQ, DBBQ, CPAN, DCPAN, CPOL, BPOL, and DBPOL. All of them can quench the endogenous fluorescence of pepsin and trypsin by forming DBPs-pepsin and trypsin, which belongs to the static quenching mechanism.

[0241] Ultraviolet absorption spectroscopy and synchronous fluorescence spectroscopy revealed that seven DBPs interact with pepsin and trypsin to form DBPs-pepsin / trypsin complexes, altering the microenvironment near the major fluorophores (tryptophan and tyrosine residues) of pepsin and trypsin, leading to conformational changes in the proteases. In the pepsin / trypsin system, DCBQ decreased the hydrophobicity of the microenvironment near tryptophan residues, while DBBQ increased it. CPAN and DCPAN both increased the hydrophobicity of the microenvironment near tyrosine residues and decreased the hydrophobicity near tryptophan residues, with the binding site closer to the tryptophan residues, indicating that the tryptophan residues were moved to a more hydrophilic environment. CPOL and DBPOL increased the hydrophobicity of the microenvironment near tryptophan residues in pepsin, CPOL and BPOL increased the hydrophobicity of the microenvironment near tyrosine residues in trypsin, and BPOL increased the hydrophobicity of the microenvironment near tryptophan residues in trypsin, with the binding site closer to the tyrosine residues. The above results indicate that the binding of DBPs to both proteases alters the amino acid microenvironment, placing the amino acids in a more hydrophilic / hydrophobic environment, resulting in a more loose / compact structure of the proteases. This also provides important evidence for characterizing the interaction between DBPs and proteases.

[0242] Thermodynamic analysis revealed that the interactions between the seven DBPs and pepsin are spontaneous and elucidated the force types in the DBPs-pepsin system. Specifically, the HBQs-pepsin system primarily involves hydrophobic interactions and hydrogen bonds; the HPANs-pepsin system primarily involves hydrophobic interactions and hydrogen bonds; and the HPOLs-pepsin system primarily involves hydrophobic interactions and electrostatic forces. This indicates that hydrophobic interactions are the dominant force in the formation of the DBPs-pepsin / trypsin complex.

[0243] Calculations using double logarithmic equations and fluorescence quenching experimental data revealed that seven DBPs can interact with pepsin / trypsin at a single binding site to form DBPs-pepsin / trypsin complexes. The binding constants of DBPs with pepsin are in the following order: DBBQ > DCBQ > DBPOL > BPOL > CPOL >

[0244] The binding constants of DBPs to trypsin were: DCPAN > CPAN; the order was: DBBQ > DCBQ > DBPOL > DCPAN > BPOL > CPOL > CPAN. The results indicate that DCBQ, DBBQ, DBPOL, DCPAN, and CPAN bind more readily to trypsin. This is because trypsin has three catalytic residues (His-57, Asp-102, and Ser-195), while pepsin has two (Asp-32 and Asp-215). The binding sites of DBPs to proteases may be closer to the catalytic active site of trypsin, leading to a stronger affinity of trypsin for DBPs. CPOL and BPOL bind more readily to pepsin because trypsin contains 11 amino acid residues involved in the binding of DBPs to trypsin, while pepsin contains 21 amino acid residues involved in the binding of DBPs to pepsin. This results in a greater affinity of CPOL / BPOL for pepsin than for trypsin.

[0245] The investigation of toxicity data for the selected DBPs revealed the following pattern of cytotoxicity using CHO as the test substance: DBBQ (19.8 μM) > DCBQ (27.3 μM) > DCPAN (83 μM) > CPAN (148 μM). This pattern is consistent with the binding affinity of pepsin / trypsin-HBQs / HPANs obtained in this experiment. However, the developmental toxicity data of BDPs using zebrafish embryos as the test substance (DCBQ > DBBQ > DBPOL > CPOL > BPOL) is significantly inconsistent with the binding affinity of pepsin / trypsin-HBQs / HPOLs in this application, indicating that the conclusions in this application are consistent with the pattern of cytotoxicity. Overall, for the same class of DBPs, the cytotoxicity of DBPs is positively correlated with the binding affinity of digestive proteases, suggesting that this indicator may serve as a potential toxicity assessment indicator for the toxic effects of halogenated disinfection byproducts.

[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 within the protection scope of the present invention.

Claims

1. A method for assessing the toxicity level of halogenated disinfection byproducts based on protease, characterized in that, Includes the following steps: S1. Solution preparation: Prepare pepsin solution, trypsin solution, and halogenated disinfection byproduct solution; the halogenated disinfection byproduct comprises cyclic and heterocyclic compounds, and includes at least two of the following: 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. Spectroscopic determination: The protease solution was mixed with halogenated disinfection byproduct solutions of different concentrations. Two-dimensional fluorescence spectroscopy, synchronous fluorescence spectroscopy, and ultraviolet absorption spectroscopy were performed on the mixed solution; These spectral data provide information about changes in the amino acid residue microenvironment and protease structure. S3. Fluorescence quenching analysis: The fluorescence data were fitted with the Stern-Volmer equation to determine whether the quenching type of the disinfection byproduct on the protease was static or dynamic, and the quenching constant was calculated to determine whether the disinfection byproduct and the protease were bound. S4. Combined parameter analysis: The logarithmic relationship curve of fluorescence quenching was constructed by constructing a double logarithmic equation, and the number of binding sites and binding constant of the disinfection by-product-protease complex were calculated; the affinity of different proteases with disinfection by-products was compared, and the interaction strength and toxicity level of disinfection by-products were evaluated. S5. Thermodynamic parameter analysis: Enthalpy change, entropy change and Gibbs free energy are calculated using the Van't Hoff equation and the Gibbs-Helmholtz equation. The main types of forces in the bonding process are analyzed, including hydrogen bonds, hydrophobic interactions and electrostatic forces. 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 altered, thereby affecting the compactness of the protease structure. This is used to characterize the interaction force between disinfection byproducts and proteases. S7. Comprehensive Analysis: Based on the analysis in steps S4-S7, determine whether there is a positive correlation between the interaction between disinfection byproducts and proteases and the cytotoxicity of disinfection byproducts.

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 determined, respectively; the protease solutions were mixed with solutions of different concentrations of halogenated disinfection byproducts. The mixed solution was then subjected to two-dimensional fluorescence spectroscopy, synchronous fluorescence spectroscopy, and ultraviolet absorption spectroscopy.

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

4. The method according to claim 1, characterized in that, Step S2 also includes the following: In the simultaneous fluorescence spectroscopy determination, the fluorescence spectrometer scans at a speed of 600 nm / min, simultaneously scanning the emission and excitation spectra while maintaining the difference between the emission and excitation spectra. Recordings are performed at constant wavelength intervals of Δλ = 15 nm, Δλ = 60 nm, and a scanning range of 250-350 nm, 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, a UV-Vis spectrophotometer was used for data acquisition. The temperature during the measurement was 298K. The scanning speed was 1200nm / min, the scanning range was 190-400nm, and the scanning wavelength interval was 1nm. A 1cm quartz cuvette was 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 further includes: if the Stern-Volmer plot of the fluorescence of the disinfection byproduct quenching the protease shows a linear relationship, it indicates that the quenching mechanism of the disinfection byproduct on pepsin is static quenching.

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

8. The method according to claim 1, characterized in that, Step S7 also includes: reference to the mammalian cytotoxicity IC50 of disinfection byproducts. 50 Whether the values ​​and their binding energies with serum proteins are consistent can be used to determine whether the binding energy of disinfection byproducts to proteases is positively correlated with their cytotoxicity.

9. The application of the method according to any one of claims 1-8, characterized in that, Used to assess the effects of disinfection byproducts on proteases in the digestive system of organisms.

10. The application of the method according to any one of claims 1-8, characterized in that, Used to predict the toxic effects of disinfection byproducts, optimize drinking water disinfection processes, or develop drinking water quality standards.

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