A dynamic model for bilirubin binding to human serum albumin.
Site-directed mutagenesis of human serum albumin was used to study the role of various amino acid residues in bilirubin binding. A comparison of thermodynamic, proteolytic, and x-ray crystallographic data from previous studies allowed a small number of amino acid residues in subdomain 2A to be selected as targets for substitution. The following recombinant human serum albumin species were synthesized in the yeast species Pichia pastoris: K195M, K199M, F211V, W214L, R218M, R222M, H242V, R257M, and wild type human serum albumin. The affinity of bilirubin was measured by two independent methods and found to be similar for all human serum albumin species. Examination of the absorption and circular dichroism spectra of bilirubin bound to its high affinity site revealed dramatic differences between the conformations of bilirubin bound to the above human serum albumin species. The absorption and circular dichroism spectra of bilirubin bound to the above human serum albumin species in aqueous solutions saturated with chloroform were also examined. The effect of certain amino acid substitutions on the conformation of bound bilirubin was altered by the addition of chloroform. In total, the present study suggests a dynamic, unusually flexible high affinity binding site for bilirubin on human serum albumin.
Petersen
C E
CE
Department of Biochemistry and Biophysics, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii 96822, USA.
Ha
C E
CE
Harohalli
K
K
Feix
J B
JB
Bhagavan
N V
NV
D019943:Amino Acid Substitution; D001663:Bilirubin / Q000097:blood* / Q000737:chemistry*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D010843:Pichia; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012709:Serum Albumin / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D011994:Recombinant Proteins; D012709:Serum Albumin; D001663:Bilirubin