Protocol for the assay of biliverdin by the recombinant protein HUG

Biliverdin is a secondary metabolite of heme catabolism. It is formed by the reaction catalyzed by heme oxygenase, which converts the heme group contained in proteins, such as hemoglobin, myoglobin, cytochromes, and catalase, to biliverdin, iron (II), and CO in equimolar amounts, consuming NADPH. Biliverdin is then reduced to bilirubin by biliverdin reductase (again by the simultaneous oxidation of NADPH), which together forms an intracellular redox couple. Heme oxygenase-1 is an inducible enzyme induced by hypoxic conditions, increased availability of heme, or proinflammatory mechanisms, such as LPS, UV radiation, etc. Moreover, both heme oxygenase-1 and biliverdin reductase play roles other than catalysis, by modulating specific metabolic pathways at the transcriptional level. We present here a protocol for the determination of biliverdin based on its enzymatic conversion to bilirubin, which specifically binds to the recombinant protein HUG, resulting in fluorescence emission. This method allows the measurement of bilirubin and biliverdin in nM concentrations.