Analysis Of DNA By The Fluorescence Probe Of Methylene Blue And Its Application In The Study Of The Interactions Between Heavy Metals And Paraquat With DNA

Deoxyribonucleic acid (DNA) is the carrier of genetic information and the material basis of gene expression. It has very important functions in life processes, such as breeding, heredity and aberrance. Abundant amounts of poisonous chemicals in the environment, such as heavy metals, organic dyes, pesticides and herbicides, enter into the body by inhalation, ingestion or absorbtion through skin. They can interact with DNA directly or indirectly in vivo, which may change DNA structure and induce oxidative damage to DNA, and then affect its function or genetic characteristic. Considering physiological importance of DNA, we firstly developed a novel method for sensitive determination of DNA, and then investigated the interactions of heavy metals and paraquat with calf thymus DNA (ctDNA) using methylene blue (MB) as the probe molecule. The dissertation consists of the following four parts.In the first part, the fluorescence intensity of MB quenched by DNA in the pH range of 6.5⁸.2 was studied by means of synchronous fluorescence technology. Therefore, a novel method for sensitive determination of DNA was developed. The decreased fluorescence intensity at 680 nm was proportional to the concentration of DNA in the range of 0.1⁴.0 μg·mL^-1 for ctDNA, 0.05³.0 ug·mL^-1 for thermally denatured ctDNA and 0.1³.0μg·mL^-1 for herring sperm DNA. The detection limits were 41.1, 13.2 and 14.5 ng·mL^-1, respectively. This method is simple, rapid, and the reagents are low toxic. It has been applied to the determination of DNA in synthetic sample with satisfactory results.In the second part, the characteristics of the interaction between MB and ctDNA were studied by absorption spectrum, fluorescence spectrum and fluorescence polarization. In the presence of increasing amounts of ctDNA, remarkable hypochromism was observed in the absorption maximum of MB in the visible region till γ=4 (γ=[DNA_p]/[MB]), and MB fluorescence was quenched efficiently by ctDNA. Thereafter the large increase in the MB polarization upon binding to ctDNA with a red shift in absorption spectrum of MB were observed at γ>6. Results from the above spectral measurements indicated that at low γ ratios (γ<4) the cationic MB molecule binded to the negatively charged phosphates on ctDNA by electrostatic interaction, stacking on the surface of ctDNA helix, while at high γ ratios (γ>6) intercalation of MB molecules between the nucleic acid bases is the preferred mode. The effects of K₄Fe(CN)₆ and thermally denatured ctDNA on the quenching fluorescence ofMB-ctDNA system at low and high y ratios were also performed, which were consistent with the above conclusion. The intrinsic binding constants of the MB binding to ctDNA were 2.94 X105 L- mol'1 for electrostatic binding and 1.13 X106 L- mol" for intercalative binding, respectively.In the third part, the interactions of Cd(II), Pb(II) and Cr(III) ions with ctDNA have been carried out using MB as the probe molecule by fluorescence and absorption spectroscopy. MB binded to double helical ctDNA via the intercalative mode, and its fluorescence was efficiently quenched by ctDNA and remarkable hypochromism was observed in the absorption spectrum of MB. The fluorescence intensity of the probe molecule was enhanced differently when Cd(II), Pb(II) and Cr(III) ions were added to the MB-ctDNA solution system respectively. Hyperchromism was also observed in the absorption spectrum of MB-ctDNA system in presence of Cd(II), Pb(II) and Cr(III) ions. These results indicated that the heavy metal ions may be complexed with ctDNA helix and probably binded at phosphate moieties and N-7 of adenine or guanine;consequently, some intercalated MB molecules were released due to the above binding. The binding affinity of heavy metal ions to ctDNA helix were compared as follow: Cr(III)>Cd(II) > Pb(II).In the last part, the interaction of paraquat with ctDNA has been performed using MB as the probe molecule by absorption spectrum, fluorescence spectrum and fluorescence polarization. MB primarily binded to double helical ctDNA via the intercalative mode. Scatchard curves indicated the non-competitive inhibition of MB binding to ctDNA in presence of paraquat. Therefore, non-intercalation binding occurred between paraquat and ctDNA, which was conformed by fluorescence polarization. The effect of sodium chloride showed that sodium ion can markedly constrain the above interaction. The above results suggested that positively charged paraquat ion can interact with the negatively charged phosphate moieties on ctDNA through electrostatic attraction and stack on the surface of double stranded ctDNA, which may cause ctDNA shrinked and reduce the binding affinity of MB to ctDNA in the surrounding site. It indicated that sodium ion might neutralize the negatively charged phosphate backbone of ctDNA, and then weaken the electrostatic attraction between paraquat and ctDNA. The binding constant of paraquat binding to ctDNA was l.gOxK^...