Studies On The Interaction Between Pesticids (Veterinaries) And Protein By Spectrometry And Its Analytical Determine

In the dissertation, sdudying the quantitative analysis of pesticides (veterinary medicine) small molecules, fluorescence spectrometry, chemometrics with fluorescence spectrometry and resonance scattering technology and other methods were used in several pesticide (veterinary medicine) residue analysis of actual sample; meanwhile under the condition of physiological pH, fluorescence spectroscopy combined with UV-visible absorption spectroscopy and circular dichroism were used to investigate the interaction of several small pestieide (veterinary) molecules with protein. Detection of these harmful compounds to provide a new method, while the level of understanding of the molecular mass in the body of harmful compounds in the storage, transport to provide a theoretical basis.The main investigations in this paper were listed as follows, respectively:1. The study found that metolcarb and bovine serum albumin (BSA) had happened the fluorescence enhancement effect by fluorescence spectroscopy. At the same time determine the binding constants were obtained at different temperatures. In addition, the high value of fluorescence anisotropy suggested that the probe molecule is located in motional restricted environment of the protein; The thermodynamic function enthalpy and entropy for the reaction were calculated according to vant's Hoff equation, and determin the main force of between the metolcarb and BSA were hydrogen bonding and van der Waals force. The sychronous fluorescence spectra, three-dmensional fluorescenee contour map and fluorescence polarization experiments showed that metolcarb and BSA combination makes the protein structure changes;Under a Britton-Robinson buffer medium of pH 4.0, The fluorescence intensity can be enhanced separately in the presence of (3-cyclodextrin (β-CD) with△λ=30 nm, and the fluorescence intensity is directly proprotional to the concentration of the pesticides used. The linear ranges for metolcarb, propoxur and carbofuran are 0.2-2.0, 0.02-0.38 and 0.04-0.56μg-mL-1, respectively; the limits of detection was set at 0.0828,0.0145 and 0.02μg·mL-1. The different chemometric methods as the classical least squares (CLS) method, the principal component regression (PCR) method and the partial least squares (PLS) method were applied to the these compounds. Build a new method to simultaneously detection three carbamate pesticides of propoxur, metolcarb and carbofuran were found to possess intrinsic fluorescence, but the spectrum measured were severely overlapped. The analytic ability of these compounds were also compared, the result shows that the partial least squares (PLS) method is a powerful chemometric tool to the determination of mixtures of three carbamate pesticides, which was then applied in the determination of a set of synthetic validation samples and some practice food samples with satisfactory results.2. Study the interaction of pirimicarb and the Lysozyme (LYS) by the fluorescence method, the experimental results revealed that the pirimicarb quench the intrinsic fluorescence of LYS through a dynamic quenching procedure; the non-radiative energy transfer is attributed to LYS intrinsic fluorescence being quenched also; The thermodynamic parameters results showed that hydrophobic forces can be the main type of driving forces in pirimicarb-LYS interaction; the sychronous fluorescence spectra and three-dmensional fluorescenee contour map showed that pirimicarb and LYS combination makes the Lysozyme structure changes;Using the fluorescence spectrometry quantitative determination the pesticide pirimicarb. Primicarb has a strong fluorescence emission peaked at 397nm after being excited with a wavelength of 252 nm in the B-R buffer at pH 7.0. The fluorescence intensity is sensitized by the cetyltrimethyl ammonium bromide (CTMAB) and proportional to the concentration of primicarb. The linear range is 0.006-0.14μg-mL-1, the limit of detection is 5.7 ng-mL-1. The method is simple, rapid and sensitive. It has been applied to the determination of primicarb in the real food samples with satisfactory results.3. Study the interaction between furazolidone and BSA by fluorescence Spectroscopy, UV-visible absorption spectroscopy and circular dichroism at the physiological pH. The results of the fluorescence Spectroscopy and UV-visible absorption spectroscopy showed that the furazolidone strongly quenched the fluorescence of BSA and the quench was the static quenching. The average number of binding sites and the binding constant for binding furazolidone to BSA at different temperatures were determined. The thermodynamic parameters were also calculated. According to the results, the binding patterm is considered to be mainly the reflection of hydrogen bond and van der Waals forces. The binding locality was an area 3.51 nm away from tryptophan residue in BSA based on Forster nonradiation energy transfer mechanism. In addition, the effect of furazolidone on the conformation of BSA was also studied by synchronous fluorescence spectrometry, three-dmensional fluorescenee contour map and circular dichroism. The competitive probes, such as warfarin, ibuprofen and digitoxin (siteⅠ,siteⅡand siteⅢprobes, respectively), revealed that the binding location of furazolidone to BSA in the site I of the hydrophobic pocket;It was found that in a B-R buffer of pH 7.0, acridine orange has a strong fluorescence emission peaked, but the furazolidone quench the intrinsic fluorescence of acridine orange, The fluorescence quench intensity is proportional to the concentration of furazolidone. The linear range is 0.06-0.60μg-mL-1, the limit of detection is 0.0521μg-mL-1. The method is simple, rapid and sensitive. It has been applied to the determination of furazolidone in the eggs, pig kidneys and shrimps with satisfactory results.4. Study the binding characteristic between fenpropathrin and human serum albumin (HSA) by the fluorescence method. The experimental results revealed that the fenpropathrin quench the intrinsic fluorescence of HSA through a dynamic quenching procedure, The thermodynamic parameters results showed that the process of FPR-HSA interaction is a spontaneous molecular process in which entropy increases and Gibbs free energy decreases, and hydrophobic forces can be the main type of driving forces in fenpropathrin-HSA interaction; the sychronous fluorescence spectra, three-dmensional fluorescenee contour map and circular dichroism showed that fenpropathrin and HSA combination makes the HSA structure changes;It was found that in a HCl-NaAC buffer solution of pH 1.7, intensity of the resonance scattering at 421nm due to the reaction between fenpropathrin and Chromotrope 2R (CT2R) was enhanced significantly, and that linear relationship between magnitude of intensity of the resonance scattering signal and concentration of fenpropathrin was obtained in the range of 0.03-0.66μg-mL-1detection limit of 0.0249μg-mL-1. Established a new methods, use the resonance scattering technological measurement pesticide fenpropathrin residues. The proposed method F been applied to the determination of fenpropathrin in some samples of fruits and vegetable, giving values of recovery in the range of 80.0-116.7%, and of RSD (n=5) in the range 1.0-2.2%.