From A Protein To A Simulated Peroxidase:The Study On The Properties Of Quenched Cytochrome C

Horseradish peroxidase as the natural peroxidase representative is very widely applied inindustrial,medical and environmental pollution.The price of natural peroxidase is expensive,and the application environment is limited.At this time,the development and design of cytochrome c regulation as the simulation of peroxidase was a hot topic in the field of enzyme simulation,which can replace the natural enzyme in industrial production.In the previous studies,there have been a lot of reports on the construction of artificial peroxidases with cytochrome c?Cyt c?as the active center.However,there were only a few studies on how to improve the catalytic activity of Cyt c itself.In this study,it was found that the enzyme catalytic activity of Cyt c was greatly improved,which was of great significance to understand the peroxidase catalytic mechanism.In addition,for the first time,the improvement of the catalytic activity of the constructed quenching cytochrome c artificial enzyme was closely related to the structural changes of cytochrome c protein.The improvement of catalytic function was described from molecular structure.The catalytic activity of quenching Cyt c was indirectly characterized by oxidation of 2-methoxyphenol solution.The measurement of enzyme catalytic activity and spectral data were acquired at room temperature.The oxidation-reduction reaction with hydrogen peroxide?H₂O₂?was produced by using 2-methoxyphenol solution.Besides,the enzyme catalytic activity is measured using UV-Vis instrument.Definitely,in this work,it was found that the enzyme catalysis rate increased significantly after quenching and different quenching conditions had diverse effects on the catalytic rate.In this work,quenching Cyt c enzyme catalytic activity was decided under different temperature,different pH values,and different concentrations of Cyt c.In the end,the optimal conditions for quenching Cyt c was heating at temperature 85?,pH9.0 phosphate environment,and the suitable concentration of Cyt c 10?mol/L.Under the condition of the most appropriate treatment,quenching Cyt c was vulnerable to regulate into artificial peroxidase,and play a catalytic role.The catalytic function of Cyt c was enhanced after quenching,which was closely related to the change of Cyt c structure.UV-Vis is an instrument that was used to test the absorption of Cyt c ultraviolet spectra.The maximum absorption value of heme in407nm was decreased,and the exposure decreased.Protein absorption increased at 280nm.Hence,the enhancement of catalytic rate was associated with the change of Cyt c and heme exposure change.Quenching Cyt c?-helix percentage content drop,?folding and random curl content rose using CD spectra,protein spatial structure was changing.Fourier infrared spectroscopy?FTIR?showed that there was a difference in the structure before and after quenching Cyt c.The electrochemical properties of quenched Cyt c were further analyzed.A series of parameters of the electrochemical research of quenching Cyt c were obtained by the means of direct electrochemical analysis.The optimum pH value was 7.0,and the electron migration rate of electrochemical redox?k_s?was9.3s^-1.The density of active electroactive material on the electrode surface???was 9.4×10^-8 mol/cm^-2.In the detection substrate of hydrogen peroxide,the detection concentration was very easy to achieve the range of 5 to 3000?mol/L.The apparent Michaelis-Menten constant(K_m^app)was 1.67×10²?mol/L.It shows that the quenching Cyt c is sensitive to hydrogen peroxide detection and has a wide linear range.