The three recombinant domains of human serum albumin - Structural characterization and ligand binding properties

In an attempt to systematically dissect the ligand binding properties of hu man serum albumin (HSA), the gene segments encoding each of its three domai ns were defined based on their conserved homologous structural motifs and s eparately 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 ca rries a binding site specific for this ligand. Proteins were characterized by SDS-polyacrylamide gel electrophoresis, isoelectric focusing, gel filtra tion, N-terminal sequencing, and matrix-assisted laser desorption/ionizatio n time-of-flight mass spectrometry, as well as near- and far-UV CD, In addi tion to the affinity chromatography ligand Cibacron Blue, binding propertie s toward hemin, warfarin, and diazepam, each of which represents a standard ligand for HSA, respectively, were investigated by the measurement of indu ced circular dichroism, Clear experimental evidence is provided here for th e 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, seconda ry 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 primari ly on domain II, while on domain I, a secondary binding site and/or parts o f the primary binding site were found.