Amplifying the fluorescence of bilirubin enables the real-time detection of heme oxygenase activity.
Heme oxygenases (HO) are the rate-limiting enzymes in the degradation of heme to equimolar amounts of antioxidant bile pigments, the signaling molecule carbon monoxide, and ferric iron. The inducible form HO-1 confers protection on cells and tissues that mediates beneficial effects in many diseases. Consequently, measurement of the enzymatic activity is vital in the investigation of the regulatory role of HO. Here we report that the fluorescence characteristics of bilirubin in complex with serum albumin can be used for the real-time detection of HO activity in enzymatic kinetics measurements. We characterized the enzymatic activity of a truncated human HO-1 and measured the HO activity for various cell types and organs, in either the basal naive or the HO-1-induced state. The bilirubin-dependent increase in fluorescence over time monitored by this assay facilitates a very fast, sensitive, and reliable measurement of HO activity. Our approach offers the basis for a highly sensitive high-throughput screening, which provides, inter alia, the opportunity to discover new therapeutic HO-1-inducing agents.
Klemz
Roman
R
Department of Anesthesiology and Intensive Care Medicine, Charité-Universitätsmedizin Berlin, Campus Virchow, 13353 Berlin, Germany. roman.klemz@charite.de
Mashreghi
Mir-Farzin
MF
Spies
Claudia
C
Volk
Hans-Dieter
HD
Kotsch
Katja
K
D000818:Animals; D001663:Bilirubin / Q000737:chemistry / Q000378:metabolism*; D005453:Fluorescence; D051547:Heme Oxygenase-1 / Q000235:genetics / Q000378:metabolism*; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D012680:Sensitivity and Specificity; D017384:Sequence Deletion; D012709:Serum Albumin / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D011994:Recombinant Proteins; D012709:Serum Albumin; C496541:HMOX1 protein, human; D051547:Heme Oxygenase-1; D001663:Bilirubin