Study On The Enzyme-like Catalytic Mechanism Of Hemoglobin And Its Interaction With Active Nitrogen And Oxygen Species

Hemoglobin (Hb) is the major hemeprotein of red blood cells (RBCs) and is responsible the transport of oxygen to the tissues. Hb has oxidant activities, and it has been proved that Hb can be used as mimetic peroxidase. While, it is not clear about the reaction mechanism, especially the relationship of reaction intermediate, structure and antioxidant activities.In this work, we set out to confirm whether the product of H₂O₂ mediated oxidation of OPDA catalyzed by metHb is DAPN, the same as the product of OPDA catalyzed by HRP. The reaction intermediate was obtained in aqueous-organic two phases and the absorption peak at 710 nm was confirmed to be that of the intermediate in relation to OPDA. The isolated product and intermediate were characterized by UV-Vis spectrometry, IR spectrometry and HPLC-tandem mass spectrometry. The results showed that the product is 2,3-diaminophenazine, the molecular weight of the intermediate is 212 and a conceivable structure of the intermediate was suggested. Combining the catalyzed reaction mechanism of peroxidase and our experimental results, a conceivable oxidation reaction mechanism of OPDA and H₂O₂ using metHb as catalyst was proposed.The intrinsic fluorescence emission maxima of Hb was investigated in relation to peroxidase property of Hb. The peroxidase activity of Hb was based on its catalyticactivity for oxidation of OPDA by H2O2. Hb was treated in the condition (temperature, ethanol and salt) that tetramer-dimer equilibrium of Hb is shifted to the dimer state and its fluorescence spectrum was measured. Experimental results revealed that the activity and stability of Hb as mimetic peroxidase was closely relative to the hydrophobic environment of active center of Hb, and when Hb (Fe") converted into met Hb (Fe"'), its activity was 1.6 times as much as that of Hb.The effect of the metal ion in the active center of Hb on its enzyme activity was studied. Manganese protoporphyrin IX was recombined with apohemoglobin to prepare artificial Hb containing the manganese porphyrin in place of protoheme or iron protoporphyrin IX. UV-vis spectra and SDS-PAGE electrophoresis were used to confirm manganese (III) protoporphyrin IX and its apohemoprotein complexes (Mn-Hb) compared with those of the corresponding high spin iron (III) compounds. Kinetic studies were carried out with Mn-Hb and Hb to investigate their peroxidase and catalase activity. The experimental results showed that the peroxidase activity of Mn-Hb was 70% of that of Hb, but the catalase activity of Mn-Hb was about 1.9 folds of that of Hb.A carbodiimide method was used to label the human IgG using Hb as the mimetic enzyme of HRP. The immuo-conjugate of Hb with human IgG remained 95% of the catalytic activity of Hb. A new competing type immunoanalytical system was estabilished using Hb as the labeling enzyme, OPDA as the hydrogen donor, and the goat anti-human IgG as the analytical model. The concentration of human IgG had a good linear relationship with the absorbance of the system in the concentration range of 15-1000ng/mL. The linear regression equation A=0.297-1.286E-4[IgG](ng/mL), The correlation coefficient was 0.9992 (n=8) and the determination limit was 15 ng/mL.Furthermore, a Hb/poly-o-phenylenediamine(POPDA) membrane electrode was prepared by electropolymerization of OPDA on platinum electrode. The electrode obtained exhibits significantly well biology catalytic activity and shows kinetic characteristics of the typical enzyme-catalyzed reaction. The transfer rate constant ofthe immobilized Hb is 51.9% of that of Hb. and it remains the 91.2% activity of the primary after conserved at 4°C for 50 days. The influence of the electrode preparing condition on the performance of the electrode was studied and the reaction kinetic catalyzed by the Hb on the electrode was analyzed. The oxyreduction potential of Hb on the electrode are respectively 0.3V and -0.2V. The catalytic activity of Hb under the electrode potential was discussed and a electron transfer model between the membrane and the electrode was put forward. From these we can conclud that the activities of Hb can be controlled by methods of electrochemistry, and it was an interesting problem for discussion.Superoxide anion and NO can react to form the highly oxidizing species peroxynitrite(ONOO⁾ which can react directly with Hb even in the presence of physiological concentration CO2. The ONOCT-mediated oxidation damage to the heme of oxyHb under conditions expected in blood was studied. Results showed that 8-10 mol ONOO" was needed to quickly and completely convert 1 mol oxyhemoglobin(oxyHb) to methemoglobin(metHb). 1 mmol/L CO2 caused a small decrease in the ability of ONOO" to oxidize oxyHb, and ONOO -promoted conversion of oxyHb to metHb increased when pH decreased from 8.0 to 6.0. Relatively lower temperature in blood condition will inhibit this reaction in some degree. We postulate that ONOO" can mediate oxidation damage to the heme, and caused heme loss from the hydrophobic cavity of Hb when its concentration exceeded 90 mol/L. These results indicate that ONOO" can convert of oxyHb to metHb under the conditions expected in blood, and this reaction was regulated by CO2 concentration, reaction time, temperature and pH value.The reactions of nitrite (NO2") and peroxynitrite (ONOO") with organic molecules as well as with Hb were examined and the potential interference with the detection of H2O2 and Hb was investigated. ONOO" at low concentrations (35-140 uM) induced a concentration-dependent oxidation of OPDA and guaiacol, and this process can be improved by the addition of Hb with a concentration-dependent manner. This enhancing effect of Hb was possibly due to the formation of highlyreactive species as ferrylHb during the reaction of ONOO and Hb. NO2 can also oxidized the aromatic amines OPDA, but its efficiency is much lower than that of ONOO^. A 300-fold excess of NO2 over H2O2 can inhibited the oxidation of Pyrogallol Red mediated by H2O2 and Hb, which was due in part to the reaction of NO2 with Hb ferryl species compound I and compound II and the phenoxyl radical. These data suggest that ONOO" and NO2 can interfere with the detection of H2O2. The overestimation or underestimation of the H2O2 detected is dependent upon the organic molecule utilized for detection and by the relative rate of NO2, superoxide, and ONOO" generation.