Study On The Apparent And Microscopic Effect Of The Interaction Between Chlorophenols And Bovine Serum Albumin

Chlorophenols are a class of the most important persistent organic pollutants, which have high toxicity, persistence, and possible carcinogenicity. In many countries, some of chlorophenols are included in the list of priority toxic pollutants. Because of its properties of high bioaccumulation coefficient and stability, recent investigations showed that chlorophenols were detected in many environmental systems such as surface water, sediment, organism, and even in the blood of human being. Research on the toxicity mechanism of chlorophenol is of vital important. However, most of the recent researches on the toxicity mechanism of chlorophenols are focus on the relation of toxicity-dose and toxicity effect on cell, the effectiveness and microscopic mechanism for the interaction between chlorophenols and biomacromolecules were seldom reported.Interaction of environmental chemical pollutants with protein plays an important role in the transport, distribution, toxicity and metabolism for pollutants in the organism. Research on the microcosmic and molecular mechanism of the interaction will be of important scientific interest and application value in further exploring and forecasting the mechanism of toxicity, metabolism, defense and detoxification. In our dissertation, bovine serum albumin was selected as a model protein to study the interaction between chlorophenols and protein at the molecular level. The contents and conclusions of the research are as follows:(1) The interaction of five chlorophenols (2-chlorophenol,4-chlorophenol,2,4-dichlorophenol, 2,4,6-trichlorophenol and pentachlorophenol) and bovine serum albumin was investigated by fluorescence spectroscopy. Fluorescence analysis results showed that 2,4-dichlorophenol, 2,4,6-trichlorophenol and pentachlorophenol cause distinct quenching on the intrinsic fluorescence of BSA, the quenching mechanism is static quenching mode with the formation of non-fluorescent complexes. The magnitude order of binding constants for three chlorophenols is pentachlorophenol,2,4,6-trichlorophenol and 2,4-dichlorophenol. Calculated thermodynamic parameters implied that the main interaction force is hydrophobic contact, but electrostatic interaction cannot be excluded. Site competitive experiments indicated that the main binding site located in the hydrophobic cavity of BSA (siteⅠ), combined with the structure of chlorophenols we can inferred that benzene ring of chlorophenols interact with the hydrophobic cavity of BSA by hydrophobic force. Otherwise, ionized phenolic hydroxyl group may also interact with positive charged amino acid residue by electrostatic interaction. The binding affinity is related to the number of substituted chlorine atom on the benzene ring of chlorophenols. Forster non-radiative energy transfer distance was calculated according to the spectra overlap between the fluorescence spectra of chlorophenols and the UV absorption spectrum of BSA.2-chlorophenol and 4-chlorophenol did not result in the quenching of the intrinsic fluorescence of BSA. Whether there exists the interaction between the two chlorophenols and BSA need to be confirmed by other experiments.(2) The binding of Cu2+ and Pb2+ to BSA and the combined effect of inorganic-organic mixture pollutants on the fluorescence of BSA was study by fluorescence quenching technology. Results indicated that with the presence of quencher Cu2+, both the binding constant and quenching mechanism between chlorphenols and BSA were changed, Cu2+ may compete with chlorophenols on the binding site of BSA. While for Pb2+, only the binding constant between chlorphenol and BSA was influenced with the presence of Pb2+, Pb2+-BSA-chlorophenol ternary complex may be formed in the process.(3) Thermodynamic properties of chlorophenol-BSA reaction system were investigated by means of isothermal titration calorimetry. Evident heat emission was observed during the binding process of 2,4-dichlorophenol,2,4,6-trichlorophenol and pentachlorophenol to BSA. Fitted association constant by independent-site mode based on iterative least squares algorithm is in accordance with that calculated by fluorescence quenching data, which verified that hydrophobic force is the main driving force for the three chlorophenols binding to BSA. The weak exothermal phenomenon was also observed in the process of 2-chlorophenol and 4-chlorophenol titrating to BSA, which indicated that there may exist weak interaction,2-chlorophenol and 4-chlorophenol may bind on the surface of BSA.(4) The microcosmic mechanism of chlorophenols on BSA was studied by UV-vis spectroscopy, synchronous and three-dimensional fluorescence spectroscopy, circular dichroism spectroscopy, and Fourier-transform infrared spectroscopy. Results showed that the amino acids microenvironment of BSA was sensitive to the binding of chlorophenols to BSA, while the secondary structure of BSA kept predominant a-helix.(5)1H and 1H-1H COSY nuclear magnetic resonance spectroscopy was used to explore the chemical shift of hydrogen atom on chlorophenols. Results showed that the chemical shift of hydrogen atom on chlorophenols was changed to different extent, which confirmed the interaction between chlorophenols and BSA. Analysis of the chemical shift demonstrated that with the increase of chlorophenol atom on benzene ring, the binding of chlorophenols to BSA changed from the H-bond interaction on the surface of BSA to the hydrophobic interaction in the inner hydrophobic cavity of BSA.(6) The spatial binding mode of chlorophenols to BSA was modeled by Sybyl 8.1 software. Result indicated that 2-chlorophenol and 4-chlorophenol binding on the the groove of BSA surface.2,4-dichlorophenol,2,4,6-trichlorophenol and pentachlorophenol inserted into the inner hydrophobic space of BSA, interacting with the amino acid residue by H-bond. Hydrophobic and electrostatic forces play an important role in stabilizing the formed complex. The result from quantum chemistry theory indicated that the stability sequence of formed complex between chlorophenols and BSA is pentachlorophenol,2,4,6-trichlorophenol and 2,4-dichlorophenol.