Characterization and kinetic studies of a novel dye prepared from the oxidation of a water-soluble 1,1′-dimethylferrocene-2-hydroxypropyl-β-cyclodextrin complex using immobilized bilirubin oxidase

Bilirubin oxidase, a commercially available copper-containing enzyme isolated from Myrothecium verrucaria, was observed to oxidize a yellow 1,1′-dimethylferrocene-2-hydroxypropyl-β-cyclodextrin inclusion complex to a stable blue dye known as 1,1′-dimethylferricinium at pH 5-7. During the oxidation process, there was a noticeable increase in pH as a result of the consumption of both H⁺ and oxygen, and the molar ratio between 1,1′-dimethylferrocene and oxygen was established as 4:1. The enzyme was covalently immobilized onto either aminopropyl or arylamine glass beads to form an immobilized enzyme reactor which could be used for the repeated preparation of the blue dye. For bilirubin oxidase immobilized onto the aminopropyl beads, the reaction was much more efficient and was completed within 60-90 min, corresponding to a conversion yield of 84%. The blue dye was reduced instantaneously by ascorbic acid or uric acid to its original reduced form and was insensitive to a wide pH variation from 2 to 11. Application of the blue dye as a colorimetric assay reagent for glucose using β-d-glucose oxidase, hypoxanthine using xanthine oxidase, and lactate using lactate oxidase was successfully demonstrated. Kinetic studies revealed that the Michaelis-Menten constant for the blue dye with respect to glucose oxidase was noticeably higher than that of lactate oxidase or xanthine oxidase.