Journal of Biological Chemistry
Volume 274, Issue 41, 8 October 1999, Pages 29303-29310
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PROTEIN CHEMISTRY AND STRUCTURE
The Three Recombinant Domains of Human Serum Albumin: STRUCTURAL CHARACTERIZATION AND LIGAND BINDING PROPERTIES*

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In an attempt to systematically dissect the ligand binding properties of human serum albumin (HSA), the gene segments encoding each of its three domains were defined based on their conserved homologous structural motifs and separately cloned into a secretion vector for Pichia pastoris. We were able to establish a generally applicable purification protocol based on Cibacron Blue affinity chromatography, suggesting that each of the three domains carries a binding site specific for this ligand. Proteins were characterized by SDS-polyacrylamide gel electrophoresis, isoelectric focusing, gel filtration, N-terminal sequencing, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, as well as near- and far-UV CD. In addition to the affinity chromatography ligand Cibacron Blue, binding properties toward hemin, warfarin, and diazepam, each of which represents a standard ligand for HSA, respectively, were investigated by the measurement of induced circular dichroism. Clear experimental evidence is provided here for the location of the primary hemin binding site to be on domain I of HSA, and for the primary diazepam binding site to be on domain III. Further, secondary binding sites were found for hemin to be located on domains II and III, and for diazepam on domain I. The warfarin binding site was located primarily on domain II, while on domain I, a secondary binding site and/or parts of the primary binding site were found.

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This work was supported by Fonds zur Förderung der wissenschaftlichen Forschung Grant P11280-MED, Jubiläumsfonds der Oesterreichischen Nationalbank Grant P6875, and EU Concerted Action BIO4-CT96-0389 “Phage Club.”The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.