Glucose oxidase (GOX) or lactate oxidase (LOX) were immobilized in an osmium-based three-dimensional redox hydrogel that electrically connected the enzyme's redox centers to electrodes. The enzyme "wiring" hydrogel was formed by cross-linking poly(1-vinylimidazole) (PVI) complexed with Os-(4,4'-dimethylbpy)2Cl (termed PVI15-dmeOs) with poly(ethylene glycol) diglycidyl ether (peg). Glucose and lactate sensors exhibited typical limiting current densities of 250 and 500 microA/cm2, respectively. When the electrodes were poised at 200 mV (SCE), the currents resulting from electrooxidation of ascorbate, urate, acetaminophen, and L-cysteine were negligible. When a Nafion film was employed, the linear range was extended from 6 to 30 mM glucose and from 4 to 7 mM lactate. The redox potential of the gel-forming polymer was 95 mV (SCE). Glucose and lactate measurements performed in bovine calf serum correlated well with a substrate calibration in phosphate buffer.

Enzyme electrodes based on a redox hydrogel formed upon complexing water-soluble poly(1-vinylimidazole) (PVI) with [Os(bpy)2Cl]+ and cross-linked with water-soluble poly(ethylene glycol) diglycidyl ether (molecular weight 400, peg 400) are described. The properties of the electrodes depended on their polymers' osmium content, the extent of cross-linking, the pH, and the ionic strength in which they were used. The redox hydrogels' electron diffusion coefficients (De) increased with osmium content of their polymers. The De values were 1.5 x 10(-8), 1.3 x 10(-8), and 4.3 x 10(-9) cm2/s for PVI3-Os, PVI5-Os, and PVI10-Os, respectively, the subscripts indicating the number of monomer units per osmium redox center. De decreased with increasing ionic strength and increased upon protonation of the polymer. In glucose electrodes, made by incorporating into their films glucose oxidase (GOX) through covalent bonding in the cross-linking step, glucose was electrooxidized at > 150 mV (SCE). The characteristics of these electrodes depended on the GOX concentration, film thickness, O2 concentration, pH, NaCl concentration, and electrode potential. The steady-state glucose electrooxidation currents were independent of the polymers' osmium content in the studied (3-10 monomer units per osmium center) range. Electrodes containing 39% GOX reached steady-state glucose electrooxidation current densities of 400 microA/cm2 and, when made with thick gel films, were selective for glucose in the presence of physiological concentrations of ascorbate and acetaminophen.

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Redox polymers based on the poly(vinylpyridine) complex of [Os(bpy) 2 Cl] +/2+ were quaternized with methyl iodide, and the quaternized polymers were used to "wire" glucose oxidase. Quaternization enhanced both the rate of electron transport in cross-linked redox hydrogels containing glucose oxidase and the strength of the electrostatic complex formed between the polycationic redox polymer and the polyanionic glucose oxidase. Quaternization with methyl groups also decreased the number of pyridine rings available for cross-linking by the water soluble cross-linker poly(ethylene glycol) diglycidyl ether. The current densities of glucose electrooxidation increased with the degree of quaternization of the "wires" until one-third of the pyridine rings were quaternized, and the activation energies decreased until one-half of the rings were quaternized.

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The protease activity of hepatitis C virus nonstructural protein 3 (NS3) is essential for viral replication. ITMN-191, a macrocyclic inhibitor of the NS3 protease active site, promotes rapid, multilog viral load reductions in chronic HCV patients. Here, ITMN-191 is shown to be a potent inhibitor of NS3 with a two-step binding mechanism. Progress curves are consistent with the formation of an initial collision complex (EI) that isomerizes to a highly stable complex (EI*) from which ITMN-191 dissociates very slowly. K(i), the dissociation constant of EI, is 100 nM, and the rate constant for conversion of EI to EI* is 6.2 x 10(-2) s(-1). Binding experiments using protein fluorescence confirm this isomerization rate. From progress curve analysis, the rate constant for dissociation of ITMN-191 from the EI* complex is 3.8 x 10(-5) s(-1) with a calculated complex half-life of approximately 5 h and a true biochemical potency (K(i)*) of approximately 62 pM. Surface plasmon resonance studies and assessment of enzyme reactivation following dilution of the EI* complex confirm slow dissociation and suggest that the half-life may be considerably longer. Abrogation of the tight binding and slow dissociative properties of ITMN-191 is observed with proteases that carry the R155K or D168A substitution, each of which is likely in drug resistant mutants. Slow dissociation is not observed with closely related macrocyclic inhibitors of NS3, suggesting that members of this class may display distinct binding kinetics.

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