Structural features of the hydroxy- and keto-disubstituted bile salts: human serum albumin binding.
The binding of keto- and hydroxy bile salts to human serum albumin, the identity of the bile salts binding sites and the identification of the amino acids present in these sites were studied. The keto bile salts cholanate-3-one (C3), cholanate-3,6-dione (C3-6) and cholanate-3-hydroxy-6-one (KHC) were found to quench the native fluorescence emission of albumin. This suggested that the tryptophan residue of human albumin (residue 214) is accessible to the keto bile salts and not to the hydroxy parent compounds. The binding of the keto bile salts was characterized by a simple population of binding sites with Ka ranging from 22 x 10(4) M-1 for the mono keto bile salt (C3) down to 4 x 10(4) M-1 for the hydroxy-keto bile salt (KHC). The substitution of an oxo group at carbon C3 in C3-6 molecule for a hydroxy group (KHC) produce a significant decreasing of the interaction, suggesting that the hybridization state of the carbon at C3 in the steroid ring of the bile salt molecule is also an essential requirement for bile salts binding. It was found that bile salts are bound to the benzodiazepine binding site on human albumin (site II), producing a perturbation on site I, fatty acids and bilirubin binding site. The presence of only one substituent at C3 (oxo or OH) produce an important perturbation on the fatty acid binding sites, decreasing the polarity of the its microenvironment, while a little effect was observed for the dihydroxy and di-oxo-substituted BS, suggesting that the hydroxy substituents at C6, C7 and C12 do not interact in a significant manner with the fatty acid binding sites on HSA. The participation of specific amino acids in albumin-bile salt binding sites depends on the polar groups on the bile salt molecules as exemplified by the quantitatively different role of lysyl residues like those interacting with KHC, C3 and C3-6, and tyrosyl residue interacting with KHC. The following amino acids in human albumin were found to play a role in the bile salts-albumin interaction: lysyl 195 and 225, several arginyls, histidyl 146 and tyrosyl 411.
Farruggia
B
B
Departamento de Química-Física, Facultad de Ciencias Bioquímicas y Farmaceuticas, Consejo de Investigaciones de la U.N.R., Rosario, Argentina.
Garcia
F
F
Picó
G
G
D001647:Bile Acids and Salts / Q000737:chemistry / Q000378:metabolism*; D001663:Bilirubin / Q000737:chemistry; D001665:Binding Sites; D003619:Dansyl Compounds / Q000737:chemistry; D005453:Fluorescence; D005470:Fluorometry; D006801:Humans; D012709:Serum Albumin / Q000737:chemistry / Q000378:metabolism*; D013053:Spectrophotometry; D014212:Tretinoin / Q000737:chemistry
D001647:Bile Acids and Salts; D003619:Dansyl Compounds; D012709:Serum Albumin; C001026:5-dimethylaminonaphthalene-1-sulfonamide; D014212:Tretinoin; D001663:Bilirubin