The Study Of An Eiffcient Self-assembled Nano-structured Artificial Peroxidase

Based on the principal of semi synthase construction, we used SDS nano-micelle, which is aspecific structure with a hollow hydrophobic inner core, and horse heart cytochrome c which serve as theactive center to construct an artificial peroxidase enzyme and imitate the behavior of peroxidase. Toconstruct the artificial peroxidase, horse heart cytochrome c was solubilized in sodium dodecyl sulfatenano-micelle solution to make the self assembly process occur.The peroxidase activity, the change of secondary structures, the process of self assembly, enzymekinetics, suicide inactivation and electrochemical behavior were evaluated spectrophotometrically,thermally and electrochemistrically.First of all, using the UV-vis absorption spectroscopy, detected and compared the apparentperoxidase activity of cytochrome c, artificial peroxidase and native HRP, the experimental resultsdemonstrated that the activity of artificial peroxidase is much higher than native cytochrome c.In addition, according to changing the experimental conditions, including pH value of thesolutions, the concentration of cytochrome c and SDS nano micelle, sodium ionic strength, we observedthat the catalytic activity of cytochrome c in nano-micelle was enhanced when the pH value of sodiumdodecyl sulfate nano-micelle solution was around10.5. The study also indicated that guaiacol oxidationrates were linear with the concentration of cytochrome c between1and10μM, and the activity ofcytochrome c in nano-micelle is independent of SDS nano micelles concentration once it is above criticalmicelle concentration of SDS. Furthermore, in SDS nano-micelles the optimum buffer concentrationshifted toward higher buffer concentration, which depends on the pH value.Thirdly, using the UV-vis absorption spectroscopy and CD spectra, to detect the environmentalchange around amino acid residues of tyrosine, tryptophane and phenylalanine and the heme moiety,secondary structural change of artificial peroxidase and cytochrome c. The experimental results illustratethat the environmental hydrophobicities of heme moiety were enhanced when the pH value of solutionincreased, and this may serve as the reason trigger the high catalytic activity. According to the secondarystructures analysis, the percentages of β-turn and anti parallel are higher in the artificial peroxidase than in the native cytochrome c. The catalytic activity may enhance due to the increasing percentage of β-turn,because this structure can provide a more hydrophobic environment. And the enhanced anti parallelillustrate the structure of artificial enzyme is a more symmetrical structure than native cytochrome c.Fourthly, using the thermal analysis to detect the interaction between SDS nano micelle andcytochrome c and the process of self assembly. The experimental results demonstrated that116SDSmolecular bind to one cytochrome c molecular, which is different from the solutions only contain SDS nanomicelles. And surface tension analysis indicated that the CMC of artificial enzyme is less than5mM, whichis also less than SDS nano micelles. According to DLS analysis, we realized that the diameter of artificialenzyme is6.4nm..Then, the enzyme kinetics of artificial enzyme were obtained, including Michaelis-Menten (Km)and catalytic rate (Kcat) and catalytic efficiency of artificial peroxidase in pH10.5nano-micelle. Accordingto the experiments, we found that the catalytic efficiencies were changed due to the cytochrome cconcentration. The value of Km, Kcat and catalytic efficiency of different artificial peroxidases wereevaluated, the highest catalytic efficiency was0.0335μM-1s-1, which obtained from the0.1μM cytochromec, and its values of Km and Kcat were5.205μM,0.174s-1respectively. The suicide inactivation of artificialenzyme and HRP were detected, The results suggest that artificial enzyme can be used in higherconcentration of hydrogen peroxide, which is the HRP suicide inactivation concentration.Lastly, using nano polymeric material Nafion to immobilized the artificial enzyme onto theGlassy Carbon electrode. The cyclic voltammetry analysis was used to observe the electrochemicalcharacteristics of artificial enzyme. The experimental results suggested that the direct electron transportbetween artificial enzyme and Glassy Carbon electrode was realized, and this modified electrode can beused to detect the hydrogen peroxide. The Km value of artificial enzyme based on electrochemical analysiswas evaluated to5.28μM, which may be used as a kind of third generation biosensor.