Methods for covalently immobilizing glucose oxidase in polypyrrole are investigated. The enzyme was chemically modified with pyrrole using one of three different reactive side chains found in the protein. The reactions involve carbodiimide coupling to either lysyl or carboxyl residues on the enzyme and Schiff base reaction of the carbohydrate moiety. O ptimal coupling was achieved with the carbodiimide reaction, 15-20 mol of pyrrole/mol of enzyme compared with 6 mol of pyrrole/mol of enzyme for the Schiff base method. The pyrrole-substituted enzymes were electrochemically active, showing that the pyrrole moieties were oxidizable. Electropolymerized enzyme films deposited from solutions of free pyrrole and amounts of native or pyrrole-modified enzyme of equivalent activity resulted in covalently immobilized enzymes showing both higher enzyme activities and amperometric glucose responses than polypyrrole-entrapped native enzyme. The apparent Michaelis constant (KM′) and pH optimum of the modified enzymes electrodes correlated with that of the native enzyme electrode. Enzyme films generated from carbodiimide-modified enzymes were 6-fold more stable to thermal denaturation than native enzyme electrode. © 1993, American Chemical Society. All rights reserved.
