Study Of Interaction Between Optical Probe And Copper, Zinc Containing Superoxide Dismutase, Amine And Its Application

Aniline is an important raw material for many industrial uses such as dyes, oil paints, rubbers, pharmaceuticals and plastics but has poisonous effect on human. Natural water may be polluted by the wastewater containing aniline if not treated well.Dopamine is implicated in number of physiological and behavioral functions such as control and regulation of affective behavior, sleep, vigilance, reward system, motor and cognitive functions, as well as neuroendocrine functions regulating food intake and body temperature.Tyrosine plays an important role in the central nervous system of numerous organisms. Because of such important roles tyrosine plays in biological systems, many analytical techniques have been developed to determine its concentration in vivo。Superoxide dismutase (SOD, EC 1 .15.1.1), which catalyzes the dismutation of the superoxide anion (O2-) into hydrogen peroxide and molecular oxygen, is one of the most important metalloenzymes in the first line of defense against oxidative stress. SOD scavenges O2- and may bind one zinc (â…¡) and one copper (â…¡) .Heparin (Hep) is a natural anticoagulant. So the Hep level in the patients'blood needs to be carefully and accurately monitored during surgery and recovery.In this paper, according to above four objects, using molecule spectroscopy (fluorescence and UV-Visible spectroscopy), we study the interaction between the new fluorescent probe containing two active chloride and aniline, dopamine; the interaction between drug, drug-metal ion complex and biomolecule and its application. There are five chapters.In the first section, summarized this thesis and introduced the investigation actuality both here and abroad.In the second section, a completely new fluorescent probe 1, 5-Bis (4, 6-dicholro-1, 3, 5-triazinylamino) naphthalene (DTAN) containing two active chlorines and can react with two molecules to be determined was synthesized by one-step reaction. So, the capability of hydrogen substitution of the new fluorescent probe was enhanced and the sensitivity of the fluorescent reaction was improved. The new fluorescent probe DTAN is an electron acceptor and its electron accepting character could be adjusted after substitution of aniline, dopamine or tyrosine which is an electron donator. This should also lead to changes in the fluorescence intensity. Experimental results showed that the fluorescence intensity could be greatly enhanced by addition aniline, dopamine or tyrosine into DTAN solution, and the enhanced fluorescence intensity is proportional to the concentration of aniline, dopamine or tyrosine. Therefore a novel spectrofluorimetry method for the determination of aniline, dopamine or tyrosine was established and successfully proposed in real samples.In the third section, a new spectrofluorimetric determination of CuZnSOD was developed. Under the optimum conditions, Eu³⁺-TC formed a ternary complex in close proximity with CuZnSOD and then intramolecular energy transfer from TC- SOD complex to Eu³⁺, which resulted in the enhancement of characteristic peak of Eu³⁺ at 612nm. The enhanced fluorescence intensity is in proportion to the concentration of CuZnSOD, and the linear range was 0.0553～38.7μg mL^-1 with the limit of detection of 5.53 ng mL^-1. The developed method was practical, simple, sensitive and relatively free from interference coexisting substances and had been successfully applied to the determination of CuZnSOD in the plant and blood samples.In the fourth section, tetracycline analogues (oxytetracycline, doxycycline and tetracycline) and quinolones (norfloxacin, ciprofloxacin, enoxacine) can inactivate copper, zinc superoxide dismutase (CuZnSOD). The interactions between tetracycline analogues, quinolones and CuZnSOD were studied by using UV-visible absorption and spectrofluorimetric method. Tetracycline analogues, quinolones can make CuZnSOD's fluorescence quenching. The quenching mechanism is static quenching. Absorption spectrum and quenching constant all support this conclusion. The binding constants of tetracycline analogues, quinolones with CuZnSOD were obtained at various temperatures. According to the F?rster non-radioactive energy transfer theory; we can get the distance of donators and acceptors. We also determined the main acting force between them is electrostatic gravitation according to the thermodynamic parameter. Tetracycline analogues, quinolones can decrease the activity of CuZnSOD that purified from garlic and the whole blood from rabbits that were injected with the tetracycline. Such result provides a reference on clinical diagnosis for the level of the activity of CuZnSOD varies in different patients with different disease.In the fifth section, a new spectrofluorimetric method was developed for determination of trace amounts of heparin (Hep). Using lomefloxacin (LOM)– terbium ion (Tb3+) as a fluorescent probe, in the buffer solution of pH=8.70, Hep can remarkably enhance the fluorescence intensity of the LOM-Tb3+ complex atλ=545nm and the enhanced fluorescence intensity of Tb ³⁺ ion is in proportion to the concentration of Hep. Optimum conditions for the determination of Hep were also investigated. The linear range for the determination of Hep was 0.6².0μg/mL and the detection limit was 45.22ng/mL. This method is simple, practical and relatively free of interference from coexisting substances and can be successfully applied to assess Hep in biological samples. By the Rosenthanl graphic method, the association constant of Hep with the probe is 4.56×10⁴ L/mol and binding numbers is 18.2. Moreover, the enhancement mechanisms of the fluorescence intensity in the LOM-Tb³⁺ system and the LOM-Tb³⁺-Hep system have also been discussed.