Enzyme Activity Measurement for Bilirubin Oxidase

Creative Enzymes is competent to provide professional and superb services of enzyme activity measurement for bilirubin oxidase. Our unique service and passion for the best customer satisfaction are only exceeded by our excellent reputation in the marketplace.

Bilirubin oxidase (EC 1.3.3.5; BOX) is a copper-containing enzyme that catalyzes the oxidation of bilirubin, a product of heme catabolism, to biliverdin, with the concomitant reduction of O₂ to water. It is involved in porphyrin and chlorophyll metabolism. This enzyme was initially isolated from ascomycete plant pathogen Myrothecium verrucaria, and later it was also found in Bacillus pumilus, rat, and some other organisms. Bilirubin oxidase is a monomeric protein with a molecular mass of 60 kDa and the systematic name of this enzyme class is bilirubin:oxygen oxidoreductase, which is also called bilirubin oxidase M-1. Bilirubin oxidase is a member of the multicopper oxidase family, which contains one type I, one type II and two (one pair of) type III coppers. The type II and III coppers form a trinuclear center that reduces oxygen to water molecules, and the type I copper functions as the electron mediator from the substrate to the trinuclear center.

Bilirubin oxidase serves as a crucial enzyme in diagnostics, biotechnology, medical and energy production industry. Bilirubin, the substrate of bilirubin oxidase, consists of tetrapyrrole, which is the product of physiological heme degradation and the principal pigment of bile. Thus bilirubin oxidase can be used for diagnostic measurement of the bilirubin concentration in serum. Moreover, bilirubin oxidase is of considerate interest for low-temperature biofuel cell applications because it was reported to be one of the best enzymes for reactions in biofuel cell cathodes owing to its high reactivity at neutral pH.

Figure: The structure of bilirubin oxidase from Myrothecium verrucaria.
Reference: Kimihiko Mizutani et al. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2010 Jul 1; 66(Pt 7): 765-770.

Despite the multiple functions performed by bilirubin oxidase, the activity assay of this enzyme has been found to be complicated and difficult, probably because the loss of NAD⁺, NADP⁺, FAD and FMN during the purification process of this enzyme. Here at Creative Enzymes, we are able to prepare the holo-protein of sufficient purify and quantity, and perform spectrophotometric assays for bilirubin oxidase activities by following the absorbance at 440 nm. Creative Enzymes, equipped with the superb technology, professional knowledge, and the most advanced instruments, support the customers’ research in an excellent way. Overall, Creative Enzymes will always be your trustworthy partner.