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 selec...

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Bibliographic Details
Published in:The Journal of biological chemistry 2000-07, Vol.275 (28), p.20985-20995
Main Authors: Petersen, Charles E., Ha, Chung-Eun, Harohalli, Krishna, Feix, Jimmy B., Bhagavan, Nadhipuram V.
Format: Article
Language:English
Subjects:
Amino Acid Substitution
Bilirubin - blood
Bilirubin - chemistry
Binding Sites
Crystallography, X-Ray
Humans
Kinetics
Models, Chemical
Models, Molecular
Molecular Conformation
Mutagenesis, Site-Directed
Pichia
Recombinant Proteins - chemistry
Recombinant Proteins - metabolism
Serum Albumin - chemistry
Serum Albumin - metabolism
Spectrometry, Fluorescence
Thermodynamics
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Summary: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.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M001038200