Study On The Interaction Of Organic Compounds To Biomolecules By Capillary Electrophoresis And Its Application

At present, the toxicity of pollutants in the environment is estimated by themethods of biologic experiments, but these methods have some disadvantages, such astime-consuming, high expense, lack of pertinence. Furthermore, they are not thestraightforward research on the functionary mechanism. Therefore, it is necessary tobuild a kind of instrumental analytical method that can evaluate the toxicity ofpollutants immediately, in order to fast obtain the exact data.In this thesis, affinity capillary electrophoresis (ACE) is used to study the toxicityof pollutants and the harm of them to people and the environment. The principle isaccording to the interaction of pollutants to biomacromolecule, because the toxicity ofpollutants lies on the interaction of pollutants to target, as the receptor. In general, thestronger the interaction is, the more the toxicity is. The binding constant is the primaryparameter that characterizes the interaction, so the binding constants of pollutants andreceptor can reflect the toxicity of pollutants indirectly.At first in our reseach, the interaction of three isomeric compounds,m -nitroaniline, p -nitroaniline and o -nitroaniline, to bovine serum albumin (BSA)was studied. In the phosphate buffer, the acidity was similar to that of the physiologiccircumstance of the interaction of biomacromolecule to ligand. The result shows that theorder of binding constants of three compounds to BSA is o -nitroaniline >p -nitroaniline>m -nitroaniline and the values are 9.238×10~3L/mol, 9.263×10~3L/moland 10.793×10~3L/mol respectively. The result from the change of the travioletabsorption of three nitroanilines agreed well with the experimental results by obtainingfrom ACE. Correlation between the binding constants and some data including sometoxicological data from the biological experiments and molecular structural paramentersof nitroanilines were studied. It is found that they are related to some extent.Affinity capillary electrophoresis was used to study the interaction of eight anilinecompounds to bovine serum albumin. The sequence of the binding constants wasaniline< o-toluidino< p-toluidino< m-nitroaniline< p-nitroaniline< o-nitroaniline< N,N-dimethylaniline< a-naphthylamine. The binding constants were compared with themolecule structure parameters and some acute toxicologic data from the biologicalexperiments of eight aniline compounds, and it was found that they were relative to aconsiderable degree. The result demonstrates that it is feasible to estimate thebiotoxicity of aniline compounds by affinity capillary electrophoresis. According to the experimental results, it is concluded that it is feasible to study thebiotoxicity of pollutants by affinity capillary electrophoresis. In the end, capillary electrophoresis/ improved frontal analysis (CE/IFA) was builtin order to overcome the shortcoming of the routine frontal analysis, and it couldeffectively reduce the measurement error between the height of ligand's peak used toprotrat the standard curve and that of the free ligand's peak in the equilibrium systemand improve the accuracy of the result. The binding constant of the typical organicpollutant, m-nitroaniline, to BSA was 1.629×103 L/mol, and the number of binding siteswas 3.90. After it was compared with the peak shifting method of capillaryelectrophoresis on calculating the binding constant of molecules, a satisfactory resultwas obtained. Also the interaction of the carcinogenic pollutant, benzidine to bovinehemoglobin was studied by CE/IFA, and the binding parameters were processed withboth Scatchard linear regrossion analysis and Klotz linear regrossion analysis, and thedatas agreed well with each other. So it is concluded that CE/IFA is reliable andadvanced.