title: Circular dichroism spectroscopy is a sensitive tool for investigation of bilirubin-enzyme interactions.
abstract: Noncovalent complex formation of unconjugated bilirubin with various enzymes has been demonstrated by measuring induced circular dichroism (ICD) peaks associated with the pigment VIS absorption band. Preferential binding of the P- or M-helical conformer of bilirubin to dehydrogenases, catalase, alkaline phosphatase, and α-chymotrypsin is responsible for the characteristic exciton CD couplet that undergoes remarkable changes upon the addition of enzymatic cofactors (NADH, AMP) and an inhibitor (acridine). Alterations of the ICD spectra refer to a direct binding competition between bilirubin and NADH for a common binding site on alcohol dehydrogenase and catalase, suggesting a potential mechanism for the inhibitory effect of BR reported on NAD(P)H dependent enzymes. NADH and bilirubin form a ternary complex with glutamate dehydrogenase indicated by peculiar CD spectral changes that are proposed to be generated by allosteric mechanism. α-chymotrypsin binds bilirubin in its catalytic site, as indicated by CD displacement experiments performed with the competitive inhibitor acridine. Surprisingly, the closely related trypsin does not induce any CD signal with bilirubin. Taking into consideration the clinically relevant but controversial and poorly understood areas of bilirubin biochemistry, the fast and simple CD spectroscopic approach presented here may help to unfold diverse physiological and pathophysiological roles of BR on a molecular level.
authors.affiliation: Department of Molecular Pharmacology, Institute of Biomolecular Chemistry, Chemical Research Center, H-1025 Budapest, Pusztaszeri út 59-67, Hungary. zsferi@chemres.hu
authors.forename: Ferenc
authors.initials: F
authors.lastname: Zsila
mesh_terms: D000249:Adenosine Monophosphate / Q000737:chemistry*; D000818:Animals; D001663:Bilirubin / Q000737:chemistry*; D002417:Cattle; D002942:Circular Dichroism; D004791:Enzyme Inhibitors / Q000737:chemistry*; D004798:Enzymes / Q000737:chemistry*; D006801:Humans; D009243:NAD / Q000737:chemistry*
chemical_list: D004791:Enzyme Inhibitors; D004798:Enzymes; D009243:NAD; D000249:Adenosine Monophosphate; D001663:Bilirubin