CHARACTERIZATION AND KINETIC-STUDIES OF A NOVEL DYE PREPARED FROM THEOXIDATION OF A WATER-SOLUBLE IMETHYLFERROCENE-2-HYDROXYPROPYL-BETA-CYCLODEXTRIN COMPLEX USING IMMOBILIZED BILIRUBIN OXIDASE

Bilirubin oxidase, a commercially available copper containing enzyme i solated from Myrothecium verrucaria, was observed to oxidize a yellow imethylferrocene-2-hydroxypropyl-beta-cyclodextrin inclusion complex t o a stable blue dye known as 1,1'-dimethylferricinium at pH 5-7. Durin g the oxidation process, there was a noticeable increase in pH as a re sult of the consumption of both H+ and oxygen, and the molar ratio bet ween 1,1'-dimethylferrocene and oxygen was established as 4:1. The enz yme was covalently immobilized onto either aminopropyl or arylamine gl ass beads to form an immobilized enzyme reactor which could be used fo r the repeated preparation of the blue dye. For bilirubin oxidase immo bilized onto the aminopropyl beads, the reaction was much more efficie nt and was completed within 60-90 min, corresponding to a conversion y ield of 84%. The blue dye was reduced instantaneously by ascorbic acid or uric acid to its original reduced form and was insensitive to a wi de pH variation from 2 to 11. Application of the blue dye as a colorim etric assay reagent for glucose using beta-D-glucose oxidase, hypoxant hine using xanthine oxidase, and lactate using lactate oxidase was suc cessfully demonstrated. Kinetic studies revealed that the Michaelis-Me nten constant for the blue dye with respect to glucose oxidase was not iceably higher than that of lactate oxidase or xanthine oxidase.