Bilirubin oxidase, immobilized

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. 

In a second example, a small blood filter containing immobilized bilirubin oxidase to reduce serum bilirubin concentrations was developed for potential application in the treatment of neonatal jaundice. 

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. 

Chen JP, Wang HY (1998) Improved properties of bilirubin oxidase by entrapment in alginate-silicate sol-gel matrix. Biotechnol Tech 12(ll) 851-853 Chen Y, Kang ET, Neoh KG et al. (2000) Covalent immobilization of invertase onto the surface-modified polyaniline from graft copolymeiization with acrylic acid. Eur Polym J 36(10) 2095-2103... 

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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