Investigation On The Binding Mechanism Of Pesticides And Plasticizer With Protein

In recent years, because of the great consumption of pesticides and plasticizers in agricultural and industrial industry, and their uncontrolled or improper uses, the adverse impact of these two kinds of important chemical pollutants on food safety and human health have draw much attention. Protein,as the necessary components of living organisms, its structure changes may cause structural or functional damage to organism, and lead to some diseases. When contaminants enter the human body through direct or indirect way, they may produce toxic effects to the protein by interacting with the protein, and induce structure changes of protein, which will finally affect the biological function of protein.In this study, the interaction mechanism between several pesticides and phthalic acid esters(PAEs) with protein were investigated with the application of multiple spectroscopy methods and molecular simulation by using HSA and trypsin as protein model. Also, the conformational changes of protein induced by these chemical pollutants were determined. The present work is expected to provide some insights into the distribution, transporting, metabolism and toxicity effect of the pollutants in body from the molecular level.The main contents were summarized as follows:1. A brief introduction concerning the structure, function and biological properties of protein was presented in the first chapter. Also, the investigating methods about the interaction between small molecules and protein were summarized in this chapter.2. The binding properties of prometryn with HSA and the protein structural changes were determined under simulative physiological conditions(pH 7.4) by multispectroscopic methods including fluorescence, UV–vis absorption, circular dichroism(CD) and Fourier transform infrared(FT–IR) spectroscopy, coupled with molecular modeling technique. The result of fluorescence titration suggested that the fluorescence quenching of HSA by prometryn was considered as a static quenching procedure, the binding constant between them was 103 orders of magnitude, revealing that prometryn can bind to HSA with moderate affinity. The negative enthalpy change(ΔH°) and positive entropy change(ΔS°) values indicated that the binding process was governed mainly by hydrophobic interactions and hydrogen bonds. The site marker displacement experiments suggested the location of prometryn binding to HSA was Sudlow’s site I in subdomain IIA. Furthermore, molecular docking studies revealed prometryn can bind in the large hydrophobic activity of subdomain IIA. Analysis of UV–vis absorption, synchronous fluorescence, CD and FT–IR spectra demonstrated that the addition of prometryn resulted in partial unfolding of the polypeptides and conformational alteration of HSA with reduction of α-helix content and increases in β-sheet, β-turn and random coil structures.3. The interaction mode of Di-n-octyl phthalate(DnOP) and Di-(2-ethylhexyl) phthalate(DEHP) with HSA in aqueous solution at pH 7.4 were determined using multispectroscopic methods along with molecular simulation technique. Analysis of the fluorescence titration data at different temperatures suggested that the fluorescence quenching mechanisms of HSA by DnOP and DEHP were static, which were driven mainly by hydrophobic interactions and hydrogen bonds, and the binding affinity of DEHP to HSA was slightly stronger than DnOP. The binding of DnOP and DEHP to HSA primarily took place in subdomain IIA of HSA, indicating that the binding sites of DnOP and DEHP to HSA were both located in site I, and the binding interaction changed the secondary structure of HSA with a loss of α-helix content. Furthermore, the unfolding degree of the polypeptides of HSA induced by DEHP was more remarkable than DnOP. Protein surface hydrophobicity(PSH) tests indicated that DnOP and DEHP binding to HSA caused an increase in the PSH of HSA, demonstrating that the exposed hydrophobic patches on protein surface increased in the presence of DnOP and DEHP. At the same time, the fluorescence spectrometry was carried out to quantitatively analyze DnOP and DEHP with acridine orange(AO) as a fluorescence probe after optimizing the experimental conditions. The fluorescence intensities of AO were proportional to the amounts of DnOP and DEHP in the range of 1.20-11.76 × 10-5 mol L–1,with detection limits of 1.15 × 10-5 mol L–1 and 1.02 × 10-5 mol L–1, respectively.4. The effects of plasticizer dimethyl phthalate(DMP) and dibutyl phosphate(DBP) on the spectral properties, structure and catalytic activity of trypsin were investigated using fluorescence, UV–vis absorption and circular dichroism(CD) spectroscopy along with atomic force microscopy(AFM) and molecular simulation under simulative physiological conditions(pH 7.4). The result of fluorescence quenching indicated that ground state complex were formed between DMP and DBP with trypsin, resulting in the intrinsic fluorescence quenching of trypsin, and there was a single class of binding sites on trypsin for both DMP and DBP. The binding constants between DMP and DBP with trypsin at 298 K were 3.92 × 103 L mol-1 and 4.94 × 103 L mol-1, respectively, which indicated that DBP led to relatively higher degree of fluorescence quenching of trypsin, namely DBP showed slightly stronger affinity with trypsin than DMP. Analysis of synchronous fluorescence, UV–vis absorption, CD and FT–IR spectra demonstrated that the addition of DMP and DBP to trypsin both led to the conformational alteration of trypsin and resulted in the rearrangement of the polypeptides of protein. The molecular docking and trypsin activity assay showed that DMP and DBP primarily interacted with the catalytic triad of trypsin(His57,Asp102 and Ser195) and led to the inhibition of trypsin activity. The AFM topography image showed that the binding of DMP and DBP with trypsin caused the surface morphology change of trypsin and the protein aggregation. Moreover, according to the fact that the fluorescence intensitis of DMP and DBP in the buffer solution of BR at 7.0 were proportional to the amount of DMP and DBP, the fluorescent photometric method was used for the determination of DMP and DBP. It was found that in the linear range of 3.32-47.62 × 10–7 mol L-1,the detection limits for DMP and DBP were 1.58 × 10–7 mol L-1 and 2.43 × 10–7 mol L–1, respectively.