The Study Of Self-assembled Artificial Peroxidase Based On Nano-micelle And Cytochrome C

The design, imitation, and modification of proteins have recently become an active area of research in biochemistry and biophysics with the aim of indicatting the relationship between the structure and function of the natural proteins. These researches not only revealed the relationship between the structure and function, deeper understanding the mechanism and process of the chain of enzymatic reaction, but also can be applied to practical applications as artificial enzymes. However, the artificial peroxidase is one of the most important part in the field.This study is based on the principle of micelle enzyme model, used Cytochrome c(Cyt c) as the active center, selected sodium decyl sulfate(SDeS), sodium dodecyl sulfate(SDS) and sodium N-lauroyl sarcosine(SLS) as three anionic surfactant, dodecyl trimethyl ammonium chloride(DTAC), dodecyl trimethyl ammonium bromide(DTAB) and hexadecyl trimethyl ammonium cromide(CTAB) as three cationic surfactant for a total of six kinds of surfactants nano-micelle, to build the self-assembly nano-micelle artificial peroxidase. The research methods such as the UV–Vis spectrometry, circular dichroism spectrum and electrochemistry methods were utilized for the enzymes structure change, catalytic conditions, enzymatic dynamics, resistance and electrochemical mechanism and other additional characterization of the six kinds of nano-micelle artificial peroxidases.Results show that the structure of Cyt c has changed obviously. After adding surfactants, the degree of exposure and the micro-environmental hydrophobicities of heme were improved, leading to enhanced enzyme activity. More intensive research, nano-micelle artificial peroxidases of anion and cation surfactant were represented by SDeS and CTAB respectively, the percentages of antiparallel and beta-turn that the secondary structure may enhance enzyme catalytic activity and of spatial symmetry have increased significantly, the proportion of all secondary structures was changed small according to the different condition, or even invariant.In the test of the artificial peroxidase catalysis condition, the optimum pH of the anionic surfactant nano-micelle artificial peroxidases were pH 10, of which, the SLS was both pH 5 and pH 10. The catalytic activity was remain unchanged beyond critical micelle concentration. However, the optimum pH of the cationic surfactants nano-micelle artificial peroxidases were pH 8. The catalytic activity presented a plateau beyond the critical micelle concentration, what’s more, with the increase of surfactant concentration the catalytic activity declined gradually. The test of enzyme dynamics showed that all the six kind artificial peroxidases presented high peroxidase catalytic activity and efficiency, the kinetics parameters varied with the change of the cytochrome c concentration. The most outstanding artificial peroxidase is SDeS nano-micelle artificial peroxidase, Km was calculated to 6.747 μmol/L, the catalytic efficiency was 0.0861 μM-1s-1, this was 119% higher than the natural horseradish peroxidase. In the resistance test of high concentration of hydrogen peroxide, the ki of all artificial peroxidases were higher than natural enzymes, showed that the resistance of artificial peroxidases to hydrogen peroxide were stronger. The ki of cationic surfactant nano-micelle artificial peroxidases were higher than the anionic, their structural changes also can prove this.Electrochemical study chosed SDeS-Cytc nano-micelle artificial peroxidase and hydroxyl fullerenes(HFs) to make nano composite film modified glassy carbon electrode for testing. Results showed that the artificial peroxidase has direct electron transport with electrode, the ks was 14.3±0.1 s-1. Electric catalytic test were used to investigate the response of modified electrode to the hydrogen peroxide, the linear response range was 5 μmol/L-1400 μmol/L, the detection limit was 5 μmol/L, Kmapp was 0.57 ± 0.06 μmol/L, which may provide inspiration for the developing of the third generation biosensors and the new hydrogen peroxide biosensors.