Acetylcholinesterase And Coumarin 460 Biological Conjugates Pass Perceptual And Molecular Docking

Bioconjugation involves the linking of two or more molecules to form a novel complex having the combined properties of its individual components. The technology of bioconjugation has affected nearly every discipline in the life sciences. The application of the available crosslinking reactions and reagent systems for creating novel conjugates with peculiar activities has made possible the assay of minute quantities of sub-stances, the in vivo targeting of molecules, and the modulation of specific biological processes.Molecular docking is to study the interaction between the ligand and the receptor and plays a important role in rational drug design. Its basic idea is to firstly feature receptor surface and find the receptor active site, then pocket the receptor into the activity of the substrate, searching conformational by geometric matching, and then evaluat of the appropriateness of matching by scoring function.Bioconjugate formed by acetylcholinesterase (AChE) and coumarin 460 was studied in this paper, and the method of detection of organophosphates was applied by using this bioconjugate; The molecular docking method was see to imitate the combining of acetylcholinesterase (AChE) and its ligand, and illustrate the interaction mechanism of acetylcholinesterase (AChE) and its ligand in theory. This thesis consises of three chapters:In chapter 1, the hazards of organophosphates was introduced and the current research activity on detection of organophosphates was reviewed. Finally, the aim of this paper was outlined.In chapter 2, AChE-coumarin 460 bioconjugate was fabricated and its characteristics were studied. In pH=7.5 phosphate buffer, AChE and coumarin 460 can form bioconjugate by electrostatic interaction.The fluorescence intensity of AChE-coumarin 460 bioconjugate was related linearly to concentration of paraoxon, thus, a method for the detection of organophosphates was established. According to the change of fluorescence intensity, the distance between Coumarin 460 and tryptophan residues of the AChE was calculated. The binding constant of AChE-coumarin 460 as well as the binding constant of paraxon with coumarin460-AChE bioconjugate were also calculated.The distance between coumarin 460 and tryptophan residues was 2.6 1nm (<7nm), proving that FRET can occur between AChE and coumarin 460. The binding constant of AChE with coumarin 460 was 7.936×103, the binding constant of coumarin 460-AChE with paraxon was 5.12×106, as the interaction between AChE and coumarin460 is electrostatic interaction, paraxon is covalently bound to AChE. Under the optimized conditions, the response of the bioconjugate fabricated was related linearly to concentration of paraxon in the range from 0.3μg·L-1-20μg·L-1, with a detection limit of 0.09μg·L-1.In chapter 3, molecular docking software GOLD was used to investigate the binding mode of coumarin 460. The conformation model indicated that phenyl ring of coumarin 460 interacts with peripheral anion sites of AChE via a classic parallelπ-πaccumulation and hydrogen bonding was formed between protonize nitrogen atom and Ser124. Paraxon interacts with the hydrophobic site Phe330 of AChE via cationic-πeffect and hydrogen bonding is simultaneously formed between oxygen atom and Tyr130 and Gly117. The fitness scores were 46.50 and 53.83, respectively.