Modification of glucose oxidase by the covalent attachment of a tetrathiafulvalene derivative

4-[(3'-Carbohydroxypropyl)thio]-5-(methylthio)tetrathiafulvalene was synthesized and used as a mediator for the oxidation of glucose oxidase both free in solution and after covalent attachment to the enzyme through carbodiimide coupling to amine residues in the protein, The modified enzyme was characterized by isoelectric focusing gel electrophoresis and found to have a higher pi than the native enzyme, Electrochemical studies show that the singly oxidized tetrathiafulvalene derivative attached to the enzyme can act as a mediator for the direct reoxidation of the enzyme at electrode surfaces, whereas the doubly oxidized dication is not a mediator. Similar results are found for mediation by the tetrathiafulvalene derivative in solution, The application of the modified enzyme in membrane enzyme electrodes was investigated, and the response were analyzed to give kinetic information about the modified enzyme kinetics, Our studies show that the modified enzyme has good stability on storage in the absence of glucose but is less stable during continuous operation in a glucose sensor, This appears to arise from reactions between the tetrathiafulvanene groups attached to the modified enzyme and traces of hydrogen peroxide generated by the enzymatic reaction of glucose with oxygen present in the solution.