A Dynamic Model for Bilirubin Binding to Human Serum Albumin

Abstract

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.The binding of bilirubin, a toxic metabolite of heme, to human serum albumin (HSA) 1 has been studied extensively for many years. Early medical interest in the bilirubin-HSA interaction arose when it became clear to physicians that prolonged high blood concentrations of bilirubin, which often occur in premature infants, could result in bilirubin encephalopathy (1-5). In this condition, significant amounts of bilirubin, which is toxic to all tissues, partitions from the blood to neuronal tissue causing irreversible brain damage. The prolonged high blood concentrations of bilirubin in premature infants results from underdevelopment of the liver, the organ responsible for conversion of bilirubin to a soluble form and its excretion into the bile. HSA binds bilirubin (K d ϭ 10 Ϫ7 -10 Ϫ8 M) at a high affinity site and acts as a buffer preventing the transfer of bilirubin from blood to the tissues, thus playing a critical role in impairing the development of bilirubin encephalopathy. In total, other studies on the pathology of bilirubin encephalopathy in premature infants have highlighted the importance of HSA as a bilirubin transport molecule in normal neonates (who experience a transient hyperbilirubinemia after birth) and in normal adults. These findings provided the motivation for many years of study on the bilirubin-HSA binding mechanism, making it one of the most studied of the HSA-ligand interactions (6 -11).Although some studies have suggested that lower affinity binding components (K d ϭ 10 Ϫ6 -10 Ϫ3 M) contribute to HSAbilirubin binding, most studies have primarily attempted to locate the high affinity binding site and to identify amino acid residues involved in the high affinity binding process. A number of studies th...

Practical Aspects of the Ligand-Binding and Enzymatic Properties of Human Serum Albumin.

“…(i) The fact that ligands with very different chemical structures bind to the region with high affinity indicates that the site is adaptable. 1,9) Recently, this point of view has been supported by the results of Petersen et al, 11) who examined the binding of bilirubin to eight different single-residue mutants of rHSA. (ii) Many examples of mutual interactions between bound ligands have been observed.…”

“…Bilirubin has been reported to bind to a dynamic and flexible site in subdomain IIA. 11) In contrast, the primary binding site for hemin is located in domain I. 50) Several pieces of evidence suggest that eicosanoid binding occurs at site I 1) and that L-thyroxine appears to bind to site II.…”

Species‐dependent stereoselective drug binding to albumin: A circular dichroism study

“…8,14,20,21,36 Bilirubin exists in aqueous solution as an isoenergetic mixture of two conformational isomers of P-and M-helicity. [36][37][38] The appearance of a bisegnate CD spectrum when bilirubin binds to HSA has been interpreted as HSA bound bilirubin adopting a prevailing conformation of P helicity. (Fig.…”

Structural basis of albumin–thyroxine interactions and familial dysalbuminemic hyperthyroxinemia

“…Quenching of the fluorescent emission of W214 was used to determine K d value for T 4 binding to recombinant HSA essentially as described (6,30). In experiments to assay the effect of myristate on T 4 binding to HSA, 30 M myristate was used with 1 M HSA to ensure that all myristate sites on HSA were occupied by the ligand.…”