Monitoring catalytic reaction of bilirubin oxidase and determination of bilirubin and bilirubin oxidase activity by capillary electrophoresis.

Abstract

Micellar electrokinetic chromatography (MEKC) has been exploited as an analytical method for the separation as well as for the determination of serum bilirubin fractions (alpha-, beta-, gamma- and delta-fraction), the activity of bilirubin oxidase (BOD), and for monitoring the catalytic reaction of BOD. The samples were followed by detection at 450 nm. The average limit of detection was in the 5.0 microM concentration range. The relative standard deviation (RSD) of migration times and peak area of bilirubin was less than 0.25% and 3.0%, respectively. This allowed us complete separation of the four bilirubin fractions (alpha-, beta-, gamma- and delta-fraction) and monitoring of the oxidation progress and the assay of the activity of bilirubin oxidase. The MEKC method is a convenient technique for studying the catalytic reaction of bilirubin oxidase at different pH and assaying the relationship between the four bilirubin fractions and diseases.