title: The location of the high- and low-affinity bilirubin-binding sites on serum albumin: ligand-competition analysis investigated by circular dichroism.
abstract: The locations of three bilirubin (BR)-binding sites with different affinities were identified as subdomains IB, IIA and IIIA for five mammalian serum albumins (SAs): human (HSA), bovine (BSA), rat, (RSA), rabbit (RbSA) and sheep (SSA). The stereoselectivity of a high-affinity BR-binding site was identified in the BR/SA=1/1 system by circular dichroism (CD) spectroscopy, the sites with low affinity to BR were analyzed using difference CD. Site-specific ligand-competition experiments with ibuprofen (marker for subdomain IIIA) and hemin (marker for subdomain IB) did not reveal any changes for the BR/SA=1/1 system and showed a decrease of the bound BR at BR/SA=3/1. Both sites were identified as sites with low affinity to BR. The correlation between stereoselectivity and the arrangement of Arg-Lys residues indicated similarity between the BR-binding sites in subdomain IIIA for all of the SAs studied. Subdomain IB in HSA, BSA, SSA and RbSA has P-stereoselectivity while in RSA it has M-selectivity toward BR. A ligand-competition experiment with gossypol shows a decrease of the CD signal of bound BR for the BR/SA=1/1 system as well as for BR/SA=3/1. Subdomain IIA was assigned as a high-affinity BR-binding site. The P-stereoselectivity of this site in HSA (and RSA, RbSA) was caused by the right-hand localization of charged residues R257/R218-R222, whereas the left-hand orientation of R257/R218-R199 led to the M-stereoselectivity of the primary binding site in BSA (and SSA).
authors.affiliation: Department of Analytical Chemistry, Institute of Chemical Technology, Prague, Technická 5, 166 28 Prague 6 Czech Republic. Electronic address: iryna.goncharova@vscht.cz.
authors.forename: Iryna
authors.initials: I
authors.lastname: Goncharova
authors.forename: Sergey
authors.initials: S
authors.lastname: Orlov
authors.forename: Marie
authors.initials: M
authors.lastname: Urbanová
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D001663:Bilirubin / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002417:Cattle; D002942:Circular Dichroism; D006427:Hemin / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007052:Ibuprofen / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D008968:Molecular Conformation; D062105:Molecular Docking Simulation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D051381:Rats; D012709:Serum Albumin / Q000737:chemistry / Q000378:metabolism*; D012756:Sheep
chemical_list: D008024:Ligands; D012709:Serum Albumin; D006427:Hemin; D001663:Bilirubin; D007052:Ibuprofen
keywords: BR; BSA; Bilirubin; Binding site; CD; GS; HSA; High affinity; Ibf; Ligand-completion; Low affinity; PSB; RSA; RbSA; SA; SSA; Stereoselectivity; bilirubin; bovine serum albumin; circular dichroism; dCD; difference circular dichroism; gossypol; human serum albumin; ibuprofen; phosphate saline buffer; rabbit serum albumin; rat serum albumin; serum albumin; sheep serum albumin