The Three Recombinant Domains of Human Serum Albumin

Abstract

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-polyacryl- amide gel electrophoresis, isoelectric focusing, gel filtra- tion, 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 proper- ties toward hemin, warfarin, and diazepam, each of which represents a standard ligand for HSA, respec- tively, were investigated by the measurement of in- duced 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 pri- mary diazepam binding site to be on domain III. Fur- ther, secondary binding sites were found for hemin to be located on domains II and III, and for diazepam on do- main 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.