Research On The Method For Determining Peroxynitrite By Peroxidase And Its Kinetic Reaction Mechanism

Peroxynitrite(ONOO?), as a kind of high activity free radical in vivo, can injury a variety of amino acids, proteins, DNA, lipids and other biological macromolecules, causing a series of major diseases, which is of great significance to determine its content in vivo and reactive mechanism. At present, scientists focused on researching the reaction mechanism of 3-nitrotyrosine produced by ONOO? and tyrosine without enzyme, while it had relatively few researches on the method to determinate ONOO?. What's more, it is very difficult to capture the dynamic process of peroxynitrite due to its active property and fast reacted rate. Up to now, the methods of fluorescence probe and catalytic reaction without enzymatic have been the main methods for the determination. Peroxidase mimic enzyme with haem as a prosthetic group is a kind of oxide-reductase, which had a unique activation function to the hydrogen acceptor and could efficiently catalyze the hydrogen acceptor material; it was extracted from plant, animal and microbial. ONOO? could be taken as a kind of hydrogen receptor due to its strong oxidation, according to the reactions of which peroxidase mimic enzyme could catalyze hydrogen acceptor(ONOO?) and ONOO? could oxidize tyrosine; we established the new method to determinate ONOO?. The method had overcome the disadvantage of the difficulties in the synthesis of fluorescent probes, poor selectivity and sensitivity.Based on the principle that peroxynitrite can oxidize tyrosine to produce dimer tyrosine catalyzed by peroxidase mimic enzyme, we selected three kinds of peroxidase mimic enzyme myoglobin, hemoglobin and hemin to establish the catalytic system of mimic enzyme—ONOO?—tyrosine and to determinate the content of peroxynitrite by using fluorescence spectrometer, the results showed that we could accurately determinate ONOO? concentration by using the catalytic action to ONOO? catalyzed by peroxidase mimic enzyme; it was a static quenching process to tyrosine catalyzed by peroxidase mimic enzyme; Under the catalytic role of peroxidase mimic enzyme, tyrosine could quickly be oxidized to dimer tyrosine by ONOO?, rather than being nitrated to three nitrotyrosine. Determination method catalyzed by myoglobin were concluded that detection limit was 4.8×10-8 mol·L-1, Linear range was 4.8×10-6~4.8×10-4 mol·L-1; Average recovery rate was 95.85 %; hemoglobin were concluded that detection limit was 2.4×10-8 mol·L-1, Linear range was 2.2×10-6~8.8×10-4 mol·L-1; Average recovery rate was 98.85 %; While hemin were concluded that detection limit was 9.2×10-8 mol·L-1,Linear range 9.6×10-6~4.2×10-4 mol·L-1; Average recovery rate was 90.42%,which all had better effects. What's more, by optimizing the reaction conditions respectively to compare the different experimental conditions, linear range and detection limit of the three kinds of peroxidase mimic enzyme to determinate the content of peroxynitrite, finding that hemoglobin had the best catalytic effect, which has important significance to study the method to determinate free radicals produced by organisms and the mechanism of the catalytic reaction.In this paper, we firstly explored the kinetic characteristics of peroxynitrite and tyrosine in the presence of myoglobin, hemoglobin and hemin by using the flow injection analyzer. The results show that: the oxidation processes of peroxynitrite and tyrosine catalyzed by myoglobin, hemoglobin and hemin were followed by Michaelis-Menten's dynamics law; Based on the Michaelis constant(Km) and the maximum initial rate(Vmax), we deduced the reaction mechanism was that peroxynitrite catalyzed by mimic enzymes directly oxidized tyrosine combined with mimic enzymes, rather than decomposed to reactive intermediates ·OH and·NO2,while without O2·-; In addition, we monitored rate constant in different temperature and p H in order to investigate the impact on the reaction catalyzed by three kinds of different mimic enzymes, resulting that the optimum conditions of myoglobin to catalyze this system was 30 ? and p H 8.5, the rate constant was 8.750×105 mol·L-1·s-1, and hemoglobin were 25 ? and p H 8.0, the rate constant was 1.035×106 mol·L-1·s-1, and while hemin was 37 ? and p H 9.5, the rate constant was 6.842×105 mol·L-1·s-1, all the results were in agreement with the optimal reaction conditions after being optimized through fluorescence spectrum; Comparting kinetic parameters, discovering that Km Hb(4.46 ?mol·L-1)<Km Mb(4.78 ?mol·L-1) <Km Hemin(4.90 ?mol·L-1),Vmax Hb(0.072 ?IF/s)>Vmax Mb(0.036 ?IF/s)>Vmax Hemin(0.026 ?IF/s), the rate constant of hemoglobin in optimum condition was greater than hemin; We concluded that the rate constants under optimum condition were hemoglobin> myoglobin>hemin, and found hemoglobin's catalytic activity was higher than myoglobin and hemin.All these results provided kinetic parameters and theoretical basis for the study of determining peroxynitrite's content in enzyme catalysis and its reaction mechanism, what's more, for developing new diagnosis technology to prevent and cure related diseases caused by high activity free radicals in vivo.