Study On The Effects Of Cobalt Salts And Cobalt Porphyrins On Tyrosine Nitration By Peroxynitrite

Peroxynitrite (ONOO-) is an active molecule, which can be formed through multiple pathways in vivo. ONOO- can modify amino acids, proteins, DNA, lipids and other biological molecules in vivo and result in serious biological consequences. The nitration of tyrosine is one of the main manners to exhibit the toxicity of peroxynitrite. Metalloporphyrins and other transition metal complexes catalyze ONOO- decomposition to scavenge it in vitro and in vivo, and they can catalyze tyrosine nitration induced by peroxynitrite. Cobalt is an important transition metal element with significant biological functions, which exists in multiple forms in vitro and in vivo. To study the influence of cobalt complexes on peroxynitrite decomposition and peroxynitrite-mediated nitration of tyrosine is quite essential. The research concerning the scavenging effect of cobalt porphyrins on ONOO- provides the theory basis and experimental data to explore peroxynitrite scavengers.Firstly, the catalysis of cobalt salts on peroxynitrite decomposition and tyrosine nitration were studied by UV-Vis spectrum and stopped-flow apparatus. Through the analysis of the products after cobalt salts reacting with peroxynitrite, it was concluded that cobalt salts influenced the reactions of peroxynitrite through Co(III)OONO intermediates. Using stopped-flow apparatus, the catalytic activities (kcat) of cobalt salts for peroxynitrite decomposition at different temperatures were obtained. The activation energy for spontaneous peroxynitrite decomposition was 80.8 kJ·mol-1. The activation energies of catalyzed peroxynitrite decomposition were 62.3 kJ·mol-1 and 61.5 kJ·mol-1, repectivly for CoCl2 and Co(Ac)2. The catalytic effects of cobalt salts on peroxynitrite-mediated nitration were investigated qualitatively and quantitatively.Cobalt salts catalyzed the nitration of tyrosine, tryptophan and BSA. The change of three-dimensional fluorescence spectrum (3D fluorescence spectrum) indicated that the damage to BSA was extended by cobalt salts. Cobalt salts accelerated the nitration rate of tyrosine induced by peroxynitrite. The activation energy for spontaneous peroxynitrite-mediated nitration was 75.3 kJ·mol-1. The activation energies of catalyzed nitration were 56.5 kJ·mol-1 and 56.1 kJ·mol-1, repectivly for CoCl2 and Co(Ac)2. Cobalt salts decreased the energies for peroxynitrite decomposition and tyrosine nitration by peroxynitrite.Secondly, the catalysis of cobalt porphyrins on peroxynitrite decomposition and tyrosine nitration were investigated by kinetics experiments and time-resolved UV-Vis spectrum. The QSAR (quantitative relationship between structure and catalytic activity) of cobalt porphyrins for peroxynitrite decomposition was investigated by combining the catalytic activities with EHOMO of cobalt porphyrins and ELUMO of intermediates. The para-substituted cobalt porphyrins were concluded to catalyze ONOO- decomposition through the formation of high valent intermediates.The ortho-substituted cobalt porphyrins had no catalysis capability for peroxynitrite decomposition. Time-resolved spectrum showed that there was no intermediate formed during ortho-substituted cobalt porphyrins reacting with peroxynitrite. The relationships between catalytic activities and the charges on cobalt, EHOMO of cobalt porphyrins, ELUMO of intermediates were studied, respectively. With the increasing electron-withdrawing capabilities of the para-substituents, there were more charges on cobalt ions. The EHOMO of cobalt porphyrins and ELUMO of intermediates were lower when the electron-withdrawing capabilities of the para-substituents were stronger. These consequences were all helpful to enhance the catalytic activities of cobalts porphyrins for perixynitrite decomposition. Cobalt porphyrins had the ability to catalyze tyrosine nitration by peroxynitrite, which was similar to cobalt salts.At last, the synergistic effect of vitamin C (Vc) and cobalt compounds was inves- tigated. Spectrum and kinetics experiment results showed that vitamin C, as a reductant, had significant effect on the catalysis of cobalt salts and cobalt porphyrins on the reactions of peroxynitrite through reducing the intermediates. Cobalt salts or cobalt porphyrins inhibited the nitration of tyrosine in the presence of Vc, and the catalytic activities of cobalt compounds for peroxynitrite decomposition were enhanced sharply. Therefore, when coupled with Vc, cobalt salts and cobalt porphyrins served as peroxynitrite scavengers to protect tyrosine and other biomolecules from the nitration induced by peroxynitrite.