Enzymes bilirubin oxidase

Aizawa and Wang have reported123 that the copper-containing enzyme, bilirubin oxidase (BOX), catalyzes the oxidative polymerization of pyrrole to give thin films of PPy on substrates such as glass, plastic, or platinum plates. The BOX was first adsorbed onto the matrix support from an aqueous acetate buffer solution (pH 5.5), followed by incubation with the pyrrole monomer (0.2 M) in acetate buffer (pH 6) for several hours at room temperature. The deposited PPy film was reported to have similar properties to PPy made by conventional chemical or electrochemical methods. 

Figure 17.17 Schematic representation of a single-compartment glucose/02 enzyme fuel cell built from carbon fiber electrodes modified with Os -containing polymers that incorporate glucose oxidase at the anode and bilirubin oxidase at the cathode. The inset shows power density versus cell potential curves for this fuel cell operating in a quiescent solution in air at pH 7.2, 0.14 M NaCl, 20 mM phosphate, and 15 mM glucose. Parts of this figure are reprinted with permission from Mano et al. [2003]. Copyright (2003) American Chemical Society.

The alcohol tolerance of O2 reduction by bilirubin oxidase means that membraneless designs should be possible provided that the enzymes and mediators (if required) are immoblized at the electrodes. Minteer and co-workers have made use of NAD -dependent alcohol dehydrogenase enzymes trapped within a tetraaUcylammonium ion-exchanged Nafion film incorporating NAD+/NADH for oxidation of methanol or ethanol [Akers et al., 2005 Topcagic and Minteer, 2006]. The polymer is coated onto an electrode modified with polymethylene green, which acts as an electrocatalyst... 

Catalytic reduction of oxygen directly to water, while not as yet possible with traditional catalyst technology at neutral pH, is achieved with some biocatalysts, particularly by enzymes with multi-copper active sites such as the laccases, ceruloplasmins, ascorbate oxidase and bilirubin oxidases. The first report on the use of a biocatalyst... 

Different chemical environments surrounding the T1 copper result in different redox potentials. Fungal laccases demonstrate the highest potential, close to the equilibrium potential of oxygen reduction in their respective pH regions (see Table 1). Laccases, however, are anion sensitive, with deactivation involving dissociation of T2 copper from the active site of the enzyme. Alternative copper oxidases such as bilirubin oxidase and ceruloplasmin ° ... 

A similar polymer, composed of osmium complexed with bis-dichlorobipyridine, chloride, and PVI in a PVI—poly(acrylamide) copolymer (Table 2, compound 3), demonstrated a lower redox potential, 0.57 V vs SHE, at 37.5 °C in a nitrogen-saturated buffer, pH 5 109,156 adduct of this polymer with bilirubin oxidase, an oxygen-reducing enzyme, was immobilized on a carbon paper RDE and generated a current density exceeding 9 mA/cm at 4000 rpm in an O2-saturated PBS buffer, pH 7, 37.5 °C. Current decayed at a rate of 10% per day for 6 days on an RDE at 300 rpm. The performance characteristics of electrodes made with this polymer are compared to other reported results in Table 2. 

This electrode is unique in that the bilirubin oxidase is active at neutral pH, whereas the laccase cited above is not, even though the redox potential of laccase is somewhat higher. Additionally, the bilirubin oxidase is much less sensitive to high concentrations of other anions such as chloride and bromide, which deactivate laccase. It was shown that mutations of the coordination sphere of bilirubin oxidase led to an increased redox potential of the enzyme, which increased current density and reduced current decay to 5%/day over 6 days at 300 rpm. The latter improvement was attributed to improved electrostatic attraction between the enzyme and the redox polymer. An electrode made with high-purity bilirubin oxidase and this redox polymer has recently been shown to outperform a planar platinum electrode in terms of activation potential and current density of oxygen reduction. ... 

Monolayers and multilayers of redox enzymes (e.g., glucose oxidase [70], bilirubin oxidase [71]) have been organized on electrode surfaces using bifunctional reagents (producing covalent bonding between the layers) [70, 71] or using bioaffinity... 

Similar optical biosensors have been prepared for many other analytes. For example, a cholesterol optical biosensor has been devised based on fluorescence quenching of an oxygen-sensitive dye that is coupled to consumption of oxygen resulting from the enzyme-catalyzed oxidation of cholesterol by the enzyme cholesterol oxidase. Serum bilirubin has been detected using bilirubin oxidase, coimmobilized with a ruthenium dye, on an optical fiber.The bilirubin sensor was reported to exhibit a lower detection limit of iO Xmol/L, a linear range up to 30mmol/L, and a typical reproducibihty of 3% (CV), certainly adequate for clinical application. 

The use of highly specific enzymes was proposed to remove bilirubin from the bloodstream. More than 50 enzymes were tested, and an enzyme from Myrothecium verrucaria, bilirubin oxidase, which catalyzes the oxidation of bilirubin with Oz, was chosen. The initial product of the enzymatic reaction is biliverdin, which is much less toxic than bilirubin. The stability of bilirubin oxidase was greatly enhanced by immobilization At physiological temperature and pH, free bilirubin oxidase lost half of its activity in 12 h, whereas the half-life of the immobilized enzyme was 60 h. 

To determine the effectiveness of the immobilized enzyme in blood, a column containing 5 g of agarose with the active enzyme was tested. Control columns contained the same amount of either untreated agarose or agarose containing denatured bilirubin oxidase. For the experiments in vivo, Gunn rats on lipid-free or regular diets were used. For the experiments in vitro, a blood reservoir of human umbilical-cord blood was used. 

One major concern in developing an oral enzymatic therapy is the retention of enzyme activity with passage through the gastrointestinal tract, especially the acidic pH environment of the stomach. To be useful the enzyme must retain its activity to act in the near-neutral pH environment of the small intestine. Thus, immobilized bilirubin oxidase was... 

Results show that immobilized bilirubin oxidase incubated at pH 3.2 and 1.4 still retained greater that 90% of its original activity. In contrast, free soluble enzyme lost more than 80% of its activity at pH 3.2 and more than 95% of its activity at pH 1.4. These results indicate that the immobilized enzyme has a greatly increased stability that may survive the harshly acidic environment of the stomach. 

A number of oxidations and reductions have been carried out in microemulsions using enzymes such as cholesterol oxidase [64,108,109], bilirubin oxidase [110], horseradish peroxidase [111,112], and horse liver alcohol dehydrogenase (HLADH) [73,112-118]. It is noteworthy that cofactor-dependent enzymes also work well in different types of microemulsions. In fact, kinetic studies on the HLDAH-NADH (NADH stands for the reduced form of nicotinamide adenine dinucleotide) system showed that the presence of coenzyme was essential for the long-term stability of the enzyme in a microemulsion [113,115]. 

Bilirubin and bilirubin oxidase were used for cathodic oxygen reduction, while Ru(bpy)3 " /Ru(bpy)3 [Ru(bpy)3 + and Ru(bpy)3 are complex tris (bipyridine)ruthenium(III) and tris(bipyridine)ruthenium(II) cations] was the mediator redox system. In the electrodes, these enzymes were immobilized with Nafion solution treated with quaternary ammonium salts, and put on a support of carbonized cloth, serving as the current collector. The treated Nafion solution helped to maintain enzyme activity for a long time. 

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