Study On The Interaction Mechanism Between Nitrogenous Disinfection By-product Haloacetonitrile And Typical Biological Macromolecules

Since the 21st century,water pollution has not only aggravated the water crisis,but also caused a variety of diseases,which has become a major security problem for the development of our country.Disinfection is an important part of drinking water treatment and reducing drinking water pollution,it can kill harmful microorganisms in water and reduce the harm of water pollution to human body.Meanwhile,new pollution problems such as disinfection by-products（DBPs）also occur.Haloacetonitriles（HANs）is an emerging class of nitrogenous disinfection by-products（N-DBPs）with low content in drinking water.However,HANs has attracted much attention due to its genotoxicity and genotoxicity.It is necessary to comprehensively evaluate the environmental risks of HANs.At present,the studies of HANs mainly focus on their toxicity,little is known about the interaction mechanism for HANs in human body.Therefore,the study of HANs and biological macromolecules in vivo is helpful to comprehensively evaluate the interaction processes of HANs in human body.In this study,fluorescence spectroscopy,high-resolution mass spectrometry and MOE（Molecular Operating Environment）molecular simulation technology were used to analyze6 HANs（IAN,DBAN,BAN,TCAN,DCAN,CAN）and biological macromolecules（hemoglobin-Hb,Serum albumin-HSA,deoxyribonucleic acid-DNA）.By combining basic experiments and theoretical simulations,key mechanism for HANs and Hb,HSA and DNA were clarified.（1）In the first part,the interaction mechanism between IAN,DBAN,BAN,TCAN,DCAN,CAN and Hb was investigated.By analyzing fluorescence spectrum,it was found that the fluorescence intensity of Hb decreased with the addation of HANs.According to the fluorescence spectrum of Hb,the fluorescence quenching mechanism between Hb and HANs was static quenching,and the quenching order was TCAN>DBAN>DCAN>IAN>BAN>CAN（I substitution>Br substitution>Cl substitution;Three substitution>two substitution>single substitution）.According to thermodynamic analysis,the binding of HANs to Hb was a spontaneous process,which is influenced by both“van der Waals force”and“hydrogen bond”during the binding process.By synchronous fluorescence spectrum analysis,it was found that the binding sites for HANs and Hb were closed to Tyr/Trp residue region on the surface of Hb,which was speculated that n≥2.The binding ability of HANs to Hb was sequenced as follows:Hb-TCAN system>Hb-DBAN system>Hb-DCAN system>Hb-IAN system>Hb-BAN system>Hb-CAN system.The binding number of the Hb-HANs system was fixed at 1-4.The correlation analysis between the conversion rate of mass spectra and fluorescence quenching constant K_SV and HANs showed that there was a significant positive correlation between them（p≤0.05）.On this basis,the interaction model between HANs and Hb was constructed by MOE（Molecular Operating Environment）software.N←Lys₁₃₉（α₁）、X→Asp₁₂₆（α₁）、N←Lys₁₂₇（α₁）、N←His₄₅（α₁）、X→Tyr₄₂（β₁）、X→Asp₁₂₆（α₂）、N←Lys₁₂₇（α₂）、X→Tyr₄₂（α₂）、N←Tyr₃₅（β₂）,and other key combination were selected.The binding frequency,binding energy and binding area of HANs to Hb were calculated.In order to further clarify the binding mechanism of HANs and Hb,the correlations between the key parameters and the fluorescence quenching constant K_SV/mass spectrometry conversion rate were analyzed.The"hydrogen bond"X_HANs→O(Asp₁₂₆,α₁),N_HANs←N(Lys₁₂₇,α₁),X_HANs→O(Asp₁₂₆,α₂)and N_HANs←N(Lys₁₂₇,α₂)were identified as the key interactions between HANs and Hb.His₁₀₃,His₁₂₂,Leu₁₀₆,Phe₁₂₈,Ser₁₃₁ and Ser₁₂₄ were the key amino acids for the interactions between HANs and Hb to form the van der Waals force.In addition,the binding energy of Heme-HANs complex and the oxygen carrying capacity of Hb before and after adding HANs were measured.It was found that the binding energy of Heme complex-Hans was positive and the oxygen carrying capacity of Hb was not affected after adding HANs.Therefore,HANs has no effect on the ability of Hb to transport oxygen in humans.（2）In the first part,the interaction mechanism between IAN,DBAN,BAN,TCAN,DCAN,CAN and HSA was investigated.By analyzing fluorescence spectrum,it was found that the fluorescence intensity of HSA decreased with the addation of HANs.According to the fluorescence spectrum of HSA,the fluorescence quenching mechanism between HSA and HANs was static quenching,and the quenching order was TCAN>DBAN>DCAN>IAN>BAN>CAN（I substitution>Br substitution>Cl substitution;three substitutions>two substitutions>single substitution）.According to thermodynamic analysis,the binding of HANs to HSA was a spontaneous process,which is influenced by both“van der Waals force”and“hydrogen bond”during the binding process.By synchronous fluorescence spectrum analysis,it was found that the binding sites for HANs and HSA were closed to Tyr/Trp residue region on the surface of HSA,which was speculated that n≥2.The binding ability of HANs to HSA was sequenced as follows:HSA-TCAN system>HSA-DBAN system>HSA-DCAN system>HSA-IAN system>HSA-BAN system>HSA-CAN system.The binding number of the HSA-HANs system was fixed at 1-4.The correlation analysis between the conversion rate of mass spectra and fluorescence quenching constant K_SV and HANs showed that there was a significant positive correlation between them（p≤0.05）.On this basis,the interaction model between HANs and HSA was constructed by MOE（Molecular Operating Environment）software.N←Lys199,N→Asp108,X←Arg₁₈₆,X→Ser₂₀₂,N→Leu₁₈₂ and X←Phe₁₃₄,and other key combination were selected.The binding frequency,binding energy and binding area of HANs to HSA were calculated.In order to further clarify the binding mechanism of HANs and HSA,the correlations between the key parameters and the fluorescence quenching constant K_SV/mass spectrometry conversion rate were analyzed.The“hydrogen bond”NHANs←N（Lys199）and NHANs→N（Asp108）were identified as the key interactions between HANs and HSA.Ala₁₉₄、Ser₁₉₃、His₁₄₆、Val₄₆₂、Lys₁₀₆ and His₁₀₅ were the key amino acids for the interactions between HANs and HSA to form the van der Waals force.In addition,by measuring the oxygen carrying capacity of HSA with the addition of HANs,it was found that the interactions between HANs and HSA did not affect the oxygen carrying capacity of HSA.（3）In the first part,the interaction mechanism between IAN,DBAN,BAN,TCAN,DCAN,CAN and DNA was investigated.Because the fluorescence intensity of DNA molecule is low,it is necessary to use fluorescent probe ethidium bromide（EB）to complete the fluorescence spectrum experiment.The fluorescence intensity of DNA-EB system decreased gradually with the addition of HANs,but the position of fluorescence peak remained unchanged.By analyzing K_SV,the quenching type of DNA-EB by HANs was static quenching,and the quenching order was TCAN>DBAN>DCAN>IAN>BAN>CAN（I substitution>Br substitution>Cl substitution;Three substitution>two substitution>single substitution）.According to the thermodynamic analysis,the binding of DNA to HANs was a spontaneous process,in which“van der Waals force”and“hydrogen bond”played a major role in its binding.On the basis of the above studies,MOE molecular simulation technology was used to evaluate the molecular mechanism for the interactions between HANs and DNA.DNA fragments were selected for the study with PDB code1EJ9 and base order 1-22（T-thymidine,C-cytosine,a-adenine,G-guanine）.Since a DNA helix structure contains about 10 base pairs,the structure of base 5-15 in the middle range was selected for optimization modeling,and the most stable binding site in the DNA fragment was obtained by EB screening,which was closed to the base set sequence A11-G12-A13（large groove binding region）.In this region,all HANs also stably bound to DNA.Considering the complexity of molecular diameter（6（?））and DNA base pair changes in HANs,the A11-G12-A13 bases in the above large groove region were mutated based on the homology modeling strategy in this study,and the DNA was modeled according to the order of T→C→A→G（PDB code 1EJ9,base order1-22）.HANs was docked to 64 DNA spatial configurations to obtain relevant parameters such as binding energy and binding area.It was clear that“hydrogen bonds”N_HANs-N（C/G）and X_HANs-N（G）and van der Waals forces formed with the surrounding bases are involved in the binding of HANs to DNA.By analyzing the correlations between docking data and fluorescence quenching constant K_SV,ACC,AGC,ACG,AGG,GCC,GGC,GCG,GGG were identified as the most likely binding sequences of DNA-HANs.