We examined the reactivity of human serum albumin-bound bilirubin and its photoisomers as substrates for a direct bilirubin assay using bilirubin oxidase. The reduction of (EZ)-cyclobilirubin reached 100% 5 min after addition of the enzyme at any pH tested (3·5–7·4) in 0·1 mol/L phosphate buffer, whereas the reduction of (ZE)-bilirubin or (ZZ)-bilirubin reached 100% only below pH 4·5 or 5·5, respectively. (ZZ)-Bilirubin and its photoisomers did not react in citrate-lactate buffer at pH 3·7. The circular dichroism spectrum of (ZZ)-bilirubin in this buffer did not show a positive Cotton effect. These results indicate that a three-dimensional structure surrounding the reaction site of bilirubin is important for the reactivity with bilirubin oxidase.
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