Deflavo xanthine oxidase was prepared by removing the flavin adenine dinucleotide (FAD) group from the native enzyme using 2 M CaCl,. Unlike the native enzyme, the resulting deflavo xanthine oxidase no longer reacted with dioxygen for the oxidation of hypoxanthine. However, its capability for oxidizing hypoxanthine remained intact since deflavo xanthine oxidase reacted with l,l'-dimethylferricinium through its molybdenum and iron reactive groups. Using cyclic voltammetry, the second order homogeneous rate constant, k,, of the reaction between the reduced enzyme and the mediator for native and deflavo enzymes were determined to be (10.2 + 0.5) X lo4 M-' s-l and (9.6 f 0.5) X lo4 M-' s-l, respectively. The deflavo enzyme was effectively stabilized by 5 mM EDTA in Tris buffer, pH 8 at 277 K and retained about 80% of its activity after several days when stored in this medium. The deflavo enzyme was cross-linked by glutaraldehyde and retained onto the sensing area of a glassy carbon electrode by a porous Nylon membrane with a pore size of 5 pm. The limiting catalytic current attained a maximum value around an enzyme loading of about 0.12 nmol/cm'.
The k, value was estimated to be 1.64 X lo4 M-' s-l which was six-fold lower than the value obtained for the homogeneous enzyme system. The biosensor with immobilized deflavo enzyme showed high operational and storage stabilities with a detection limit of about 0.1 PM for hypoxanthine. No significant sensitivity loss was observed on the enzyme electrode after 150 repeated analyses during a 7.5-h operation. The deflavo enzyme biosensor retained 80% of its sensitivity when stored at 277 K after a month.