title: Direct electron transfer enhancement of covalently bound tyrosinase to glassy carbon via Woodward's reagent K.
abstract: This work describes the reaction mechanism for chemical modification of tyrosinase by Woodward's Reagent K and its covalent attachment to a glassy carbon electrode. The spectrophotometric studies revealed that the modification does not cause a significant structural change to tyrosinase. The direct electrochemistry of modified enzyme was achieved after immobilization on an oxidatively activated glassy carbon electrode. The enzyme film exhibited a pair of well-defined quasi-revesible voltammetric peaks corresponding to the Cu (II)/Cu (I) redox couple located in the active site of tyrosinase. The formal potential of immobilized enzyme was measured to be 90mV (vs. Ag/AgCl) in phosphate buffer solution at pH 7.0. The charge-transfer coefficient and apparent heterogeneous electron transfer rate constant were estimated to be 0.5 and 0.9±0.06s(-1), respectively. Finally, the electrochemical behavior of the immobilized enzyme in the presence of caffeic acid and L-3,4-dihydroxyphenylalanine as substrates was investigated. The amperometric study of biosensor toward L-3,4-dihydroxyphenylalanine resulted a linear response in the concentration range from 1.66×10(-6) to 8.5×10(-5)M with detection limit of 9.0×10(-5)M and sensitivity of 135mAμM(-1)cm(-2).
authors.affiliation: Institute of Biochemistry and Biophysics, University of Tehran, Iran. faridnouri@ibb.ut.ac.ir
authors.forename: Hassan
authors.initials: H
authors.lastname: Faridnouri
authors.forename: Hedayatollah
authors.initials: H
authors.lastname: Ghourchian
authors.forename: Seddigheh
authors.initials: S
authors.lastname: Hashemnia
mesh_terms: D000363:Agaricales / Q000201:enzymology; D015374:Biosensing Techniques / Q000379:methods*; D002244:Carbon / Q000737:chemistry*; D004295:Dihydroxyphenylalanine / Q000032:analysis* / Q000378:metabolism; D004566:Electrodes; D004579:Electron Transport; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D007555:Isoxazoles / Q000737:chemistry*; D057230:Limit of Detection; D014442:Monophenol Monooxygenase / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction
chemical_list: D004800:Enzymes, Immobilized; D007555:Isoxazoles; C009686:N-ethyl-5-phenylisoxazolium-3'-sulfonate; D004295:Dihydroxyphenylalanine; D002244:Carbon; D014442:Monophenol Monooxygenase