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1. The spectrophotometric and steady-state kinetic properties of glucose oxidase (EC 1.1.3.4, from Aspergillus niger) that is covalently linked to porous glass beads have been examined. These properties have been compared with those of soluble glucose oxidase, for which the kinetic mechanism at pH5.5 and 25 degrees C has been established previously by a combination of conventional and rapid-reaction techniques to be the following: [Formula: see text] where E(o) and E(r) represent oxidized and reduced forms of the enzyme, respectively. 2. The ratio k(+4)/k(+2) is unchanged after insolubilization, and evidence is presented which suggests that the absolute magnitudes of k(+4) and k(+2) are unchanged. 3. The kinetic efficiency of the insolubilized enzyme is greatly enhanced because of a 14-fold increase in the apparent affinity of glucose for E(o). This effect is attributed either to the binding of glucose to the glass surface or to a change in enzyme structure imposed by the insolubilization process. 4. Only 6% of the insolubilized enzyme which can be reduced by glucose is catalytically active. It is shown by calculation and direct experimental evidence that this fraction of catalytically active enzyme is bound to the exterior bead surface. The remaining 94% of the enzyme is bound within the pore network and may be subject to severe substrate diffusion control.

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Galactose oxidase (E.C. 1.1.3.9) was covalently immobilized to chemically modified porous silica particles by reaction of the native enzyme with pendant benzoyl azide groups on the carrier. The enzyme loading on the carrier was 100-150 units per milliliter. The immobilized enzyme was incorporated into a hardware assembly suitable for the determination of galactose or lactose concentrations in complex biological fluids. The prototype instrument as described is suitable for continuous, on-line monitoring or discrete sample analysis. Reaction conditions can be readily provided which maintain global first order kinetics within the reactor and strict linearity of the procedure over a wide range of sample concentrations. Auto-inactivation of the immobilized enzyme can be prevented by K3Fe(CN)6 and long-term reactor stability can be achieved by the periodic application of the reagent to the enzyme reactor in situ.