Investigation On Innocuous Treatment Of Carcinogen Or Mutagen By The Catalysis Of Metal Porphyrin

Ethidium Bromide (EB) is a dangerous chemical to the environment. It has serious toxic effects on health of people and induced abnormality, cancer and chromosomal mutation. Ethidium bromide has being widly used in nucleic acid analysis, because it can intercalate with DNA, which lead to obvious increase in fluorescence intensity. Considering the harmfulness of EB, there should be studies devoted to the development of effective methods for the treatment of ethidium bromide. This thesis aim at the establishement of a novel method for the trasfromation of EB catalyzed by a biomimetic compound, hemin, into specices with less toxicity in the presence of H₂O₂.First, hemin was selected as the catalyst for the transformation reaction. Optimal conditions were investigated, including the pH of the reaction system, the concentrations of EB, H₂O₂, and Hemin. New fluorescence behavior was observed in the hemin-H₂O₂-EB system that the emission peak and excitation peak showed to be obvious blue-shift, compared with EB in free form, which implied the appearence of new fluorescence species in the reaction system.Second, hemin supported on silica gel was prepared and its catalytic activity was investigated. Being supported on a carrier, the catalyst was easily to be separated from the solution thus can be used repeatly. Besides, the supported hemin bears the merits of simplicity in manipulateion and lowering the cost.For discussing the mechanism of transformation, the experimental results came from two reaction system (Hem-H₂O₂-EB and Fenton system) were compared. It was found that the results of the two systems are consistent, so it can be deduced that the Hemin-H₂O₂-EB system was one kind of Fenton reaction.Based on the phenomena discovered above, a novel fluorimetric method for homogeneous detection of hydroxyl radical was developed. The hydroxyl radical in the solution could be detected with reliability and simplicity.Third, silica gel TLC and HPLC were employed in the separation and purification of the product produced by Hemin-H₂O₂-EB sysytem. Scale prearation of product was carried out by silica gel column chromatography for structure conmfirmation, study on molecular biology and the experiments in toxicity. Product purified by column chromatography was also identified by molecular spectroscopy including UV absorption spectraand fluorescence spectra.Finally, less toxicity of EB product transformed by Hemin-H₂O₂-EB system was affirmed by investigation on molecular biology and microbiology. Fluorescence anisotropy of EB and its product was detected in theabsence and presence of DNA. Gel filtration chromatography and gelelechtrophoresis were performed for further study on the interaction betweenthe EB product and DNA. The results of all of the above-mentionedexperiments revealed that the interaction between EB product and DNA wasfar weaker than that of EB-DNA. At last, comparison on the mutagenicity of EB and the product was carried out by Ames test, a classical test for mutagens. The results indicated that mutagenicity of the productwas greatly reduced.