The Study Of Spectroscopy And Electrochemistry Of Hemoglobin And Heme-structure

The study of heme redox protein or enzyme has been a hot in bioelectrochemistry field. Forexample, a lot of reports are about Hb, Mb, HRP, CAT, Cty c in the electrochemistry field. Direct electrontransfer between a redox protein and an electrode not only can understand the electrode-transfer mechanismand physiological mechanism in biological systems, but also can provide a foundation for fabricating athird-biosensor. The method and technique of artificial enzyme or protein mimics are very important forsolving the limitations as natural protein or enzyme and using for the analysis of biology andbioelectrochemistry in place of them.In the text, the hemoglobin was immobilized on glassy carbon electrode which was modified bypolymer nano composite functional materials. The direct electrochemical characteristic of hemoglobin wasstudied by cyclic voltammetry, and catalytic response of H₂O₂of modified electrode was tested by linearscanning and time current. The results show that the hemoglobin on electrode is surface control process; thehemoglobin was successfully immobilized on the surface of the electrode and can effectively achieve directelectron transfer. The redox reaction between hemoglobin and electrode is a single electron transfer process;the electron transfer rate （ks） is4.2（±0.2） s^-1. The electrode can maintain bio-catalytic activity for thereduction of H₂O₂by the current time testing. The detection range of H₂O₂is from0.05to1nM, the lowestdetection limit was0.05（±0.01nM）. The apparent Michaelis constant Kmappof hemoglobin is0.85（±0.1）nM. The detection of high-sensitivity, low detection limit for H₂O₂, which establish the foundation for thedevelopment of a third generation trace H₂O₂biosensor.In the text, based on the principle of the semi-synthetic enzyme preparation, the SDS with the internal structure of a hydrophobic was used as the external structure of enzymes, the heme and histidinewhich serve as the internal catalytic activity of the enzyme, artificial peroxidase was constructed by aself-assembled manner that is characterized by natural horseradish peroxidase （HRP）, and was simulated itscatalytic activity and applied to the study of bioelectrochemistry.First, artificial peroxidase with50mM SDS and10μM heme （heme）-50mM histidine （His）solution by self-assembly was built, the catalysis active, structure, mechanistic, suicide inactivation of APwere studied through spectroscopy. The results show that apparent catalytic activity of artificial peroxidase,heme and natural HRP was measured by the guaiacol oxidation method, observed artificial peroxidase haswell catalytic activity with the oxidation reaction of hydrogen peroxide and guaiacol, and artificialperoxidase showed a higher apparent activity at pH8.0; SDS micelles above the critical micelleconcentration （CMC） concentration was almost no effect for artificial peroxidase activity; artificialperoxidase activity was increased with His concentration increased within a certain concentrations. Thetests obtained that the artificial peroxidase has considerable catalytic efficiency, kinetic parameter （Km） ofartificial peroxidase was5.5μM, the catalytic rate constant of the artificial peroxidase was0.06s^-1, thecatalytic efficiency of artificial peroxidase was0.011μM^-1s^-1, catalytic efficiency of artificial peroxidasewas equal to15%of the natural HRP. The stability of the peroxidase was also tested in a high concentrationof H₂O₂, the data show that the reaction rate of artificial peroxidase was increased with the concentration ofH₂O₂increased. Artificial Peroxidase has resistance ability to the high concentration of H₂O₂in an acidicenvironment, but the reaction speed is relatively slow; artificial peroxidase resist suicidal inactivation of thesubstrate is relatively weak in an alkaline environment, but the enzyme reaction was fastest, which showthat low pH solution to contribute to a stable structure, the solution of high pH is conducive to theimprovement in activity. Furthermore, the electrochemical characteristics of the artificial peroxidase were studied by usingelectrochemical analysis. In the text, artificial peroxidase was immobilized on glassy carbon electrodewhich was modified by polymer nanocomposite. The results show that The artificial peroxidase on themodified electrode surface can effectively achieve direct electron transfer and which is immobilizationmechanism; the response of the modified electrode to H₂O₂was detected by linear scanning method. Theresults showed that artificial peroxidase was immobilized on the electrode surface which can maintain theirbiocatalytic activity and can be achieved apparent michaelis constant of the substrate hydrogen peroxidedetection, the Kmappof artificial peroxidase was measured to1.57mM, the research filed is extended for thebioelectrochemistry and provide a new ideas, new methods for the study of artificial peroxidase.