Study On The Effect Of Organic Media And Self-assembly Nanostructure On The Enzyme-like Activity Of DhHP-6

Mimicking enzymes are synthesized by artificial methods according to the nature enzyme chemistry structure,characteristic,principle and function of enzyme in vivo.Natural enzymes are a kind of protein with catalytic activity.They have the characteristics of strong specificity,high catalytic efficiency,and mild reaction conditions and so on.However,natural enzymes are susceptible to a variety of factors,such as physical factors,chemical factors.Hence,the nature enzyme usually would not be used in the industrial production.Mimic enzymes can solve the above shortcomings of natural enzymes.Mimicking enzyme is a new research field developed in 1960 s.At present,there are several main aspects in the research of the mimicking enzyme: the character of the mimicking enzyme,the simulation of the metal cofactor of the enzyme,the mode of action of the mimicking enzyme,the functional base of the mimicking enzyme,the function of the mimetic enzyme and the substrate,etc.Deuterohemin?Dh?is a kind of synthetic iron porphyrin compound.The difference between Dh and natural heme is that it lacks two vinyl groups?which are easily oxidized?,so that the structure of the Dh is more stable in the oxidized substrate.Many studies have shown that Dh could oxidize organic substrates in the presence of hydrogen peroxide.Although the activity is not high,the results suggest that Dh can simulate the peroxidase activity.Dh have a metal center which can perform the peroxidase activity,but the lack of an additional ligand results in a very low activity of peroxidase.Deuterohemin-?-Ala-His-Thr-Val-Glu-Lys?Dh HP-6?is an artificial synthesized heme-containing peroxidase mimic composed of six amino acid residues and one iron porphyrin.We synthesized a short length peptide of six amino acid residues?Ala-His-Thr-Val-Glu-Lys?which was referred to the residues?Cys-His-Thr-Val-Glu?of MP-11,a mimic peroxidase degradated from cytochrome c.This short length peptide modified on the propionic acid side chain of Dh.In order to study the influence of histidine,we replaced the histidine with alanine,a new peptide synthesis?Ala-Ala-Thr-Val-Glu-Lys?,and also the peptide linked with the Dh carboxyl group?Dh AP-6?.By using UV spectra,we analyzed the structure of two kinds of Dh-peptide.We found that the peroxidase activity of Dh HP-6 was much higher than that of Dh AP-6.Therefore,the coordination of histidine played a key role in the activity of the Dh peroxidase.Then,reaction conditions of the Dh HP-6 were optimized.Under the optimum conditions,the kinetics of Dh HP-6 were compared to horseradish peroxidase which showed a better substrate affinity.The structure of nature enzyme is affected in organic solvents.The organic solvent will deprive the surface of the enzyme molecule which leads to decreased enzyme activity.The organic solvent will directly affect the enzyme,by destroying the hydrogen bond and hydrophobic bond configuration function,which will inhibit the enzyme activity or inactivate enzymes.Due to the simple structure of Dh HP-6 and the lack of the three-dimensional structure of protein,the activity of Dh HP-6 was also shown to be very good in organic solvents.We found that the peroxidase activity of Dh HP-6 was related to the polarity of organic solvent.In 15% methanol solution,the activity of Dh HP-6 was the highest which showed 15 times higher than that of HRP.A facile method for hybridizing deuterohemin-peptide?Dh HP-6?with copper phosphate to form deuterohemin-peptide-inorganic hybrid flowers?Dh HP-6-Cu₃?PO₄?₂?by self-assembly was proposed.The Dh HP-6-Cu₃?PO₄?₂ flowers have been characterized by scanning electron microscope,fourier transform infrared and solid state UV–Vis.In the assembly process,the Dh HP-6 peptides were fixed on Cu₃?PO₄?₂ through the coordination of the end amino acid Lys6 with copper centers.The rearrangement of Lys6 prevented Dh HP-6 aggregation.Hence,Dh HP-6-Cu₃?PO₄?₂ flowers exhibited nearly 300% enhancement of peroxidase-like activity in comparison with free Dh HP-6 in solution.Meanwhile,the Dh HP-6-Cu₃?PO₄?₂ flowers showed stronger affinity towards 3,3,5,5-tetramethylbenzidine?TMB?and H₂O₂ than free Dh HP-6 and horseradish peroxidase.In addition,EPR results provided direct evidences for the mechanisms of Dh HP-6-Cu₃?PO₄?₂ flower growth and activity enhancement.Furthermore,the flowers presented excellent reusability and storage stability.We tried to apply Dh HP-6-Cu₃?PO₄?₂ in phenol detection and found that it had good LOD and sensitivity.Moreover,there was no significant loss of activity for reusing and storage.Finally,we studied the reaction mechanism of Dh HP-6 oxidation of phenol by theoretical calculation.In the process of oxidation of phenol,there were two state reaction mode?TSR?,and the energy and structure of the two spin states were very close.The analysis of electronic structure showed that the activation of O-H bond was a PCET mechanism.The electron was transferred from phenol to the porphyrin ring and the hydrogen proton was transferred to the metal oxygen.Furthermore the electron of the metal center did not change.The difference between the doublet and quartet spin-state was that the former was ? electron transfer,and the latter was ? electron transfer.