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...

The familial dysalbuminemic hyperthyroxinemia (FDH) phenotype results from a natural human serum albumin (HSA) mutant, with histidine instead of arginine at amino acid position 218. This mutation results in an enhanced affinity for thyroxine. In our earlier study, site-directed mutagenesis and a yeast protein expression system were used to synthesize FDH HSA and several other HSA mutants. Measurement of the binding of these HSA mutants to thyroxine and several thyroxine analogs using equilibrium dialysis and quenching of tryptophan 214 fluorescence allowed us to propose a preliminary model of thyroxine binding to the 2A subdomain of wild type and FDH HSA. In this study, we have produced several other HSA mutants. By comparing the binding affinity of these mutants for thyroxine and tetraiodothyroacetic acid to the binding affinity of other mutants, we were able to suggest a new model for thyroxine binding to the 2A subdomain of HSA. We found that the substitution of arginine at position 218 with alanine increased the binding affinity for thyroxine by 2 orders of magnitude relative to the binding affinity of wild type HSA for thyroxine. A more accurate understanding of the mechanism of thyroxine binding to HSA has allowed us to define an important structural characteristic of subdomain 2A, one of the two principal binding sites on HSA for small hydrophobic ligands.

No abstract

Ethanol effects on warfarin binding to human serum albumin (HSA) have been studied by equilibrium dialysis and fluorescence methods at pH 7.4 in phosphate-buffered saline at 37 degrees C. In the presence of various amounts of ethanol fluorescence intensity of bound warfarin decreased significantly but this intensity reduction was not solely from displacement of bound warfarin from HSA. By comparing fluorescence and equilibrium dialysis data we concluded that fluorescence intensity reduction of warfarin was mainly the result of changes in the surrounding environment of the warfarin binding site by ethanol interaction with HSA and that displacement of bound warfarin was not significant compared to the fluorescence intensity changes. The dissociation constant of warfarin binding to HSA decreased with an increasing amount of ethanol. From the changes in fluorescence intensity upon warfarin binding to HSA with the presence of ethanol ranging from 0 to 5.0% the following dissociation constants (Kd) were determined: 0% ethanol 5.39 +/- 0.2 microM, 0.1% ethanol 5.86 +/- 0.1 microM, 0.3% ethanol 5.83 +/- 0.2 microM, 0.5% ethanol 6.76 +/- 0.1 microM, 1% ethanol 7.01 +/- 0.1 microM, 3% ethanol 9.9 +/- 0.7 microM, 5% ethanol 13.01 +/- 0.1 microM. From the equilibrium dialysis with the same ranges of ethanol presence the following Kd values were obtained: 0% ethanol 6. 62 +/- 1.6 microM, 0.1% ethanol 6.81 +/- 1.1 microM, 0.3% ethanol 8. 26 +/- 2.5 microM, 0.5% ethanol 8.86 +/- 1.9 microM, 1% ethanol 11. 01 +/- 4.2 microM, 3% ethanol 20.75 +/- 2.4 microM, 5% ethanol 21.67 +/- 2.2 microM. The results suggest that warfarin bound to HSA was displaced by ethanol. These data indicate that ethanol influence on warfarin binding to HSA may alter the pharmacokinetics of warfarin.

Ethanol effects on warfarin binding to human serum albumin (HSA) have been studied by equilibrium dialysis and fluorescence methods at pH 7.4 in phosphate-buffered saline at 37°C. In the presence of various amounts of ethanol fluorescence intensity of bound warfarin decreased significantly but this intensity reduction was not solely from displacement of bound warfarin from HSA. By comparing fluorescence and equilibrium dialysis data we concluded that fluorescence intensity reduction of warfarin was mainly the result of changes in the surrounding environment of the warfarin binding site by ethanol interaction with HSA and that displacement of bound warfarin was not significant compared to the fluorescence intensity changes. The dissociation constant of warfarin binding to HSA decreased with an increasing amount of ethanol. From the changes in fluorescence intensity upon warfarin binding to HSA with the presence of ethanol ranging from 0 to 5.0% the following dissociation constants (Kd) were determined: 0% ethanol 5.39 ± 0.2 μM, 0.1% ethanol 5.86 ± 0.1 μM, 0.3% ethanol 5.83 ± 0.2 μM, 0.5% ethanol 6.76 ± 0.1 μM, 1% ethanol 7.01 ± 0.1 μM, 3% ethanol 9.9 ± 0.7 μM, 5% ethanol 13.01 ± 0.1 μM. From the equilibrium dialysis with the same ranges of ethanol presence the following Kd values were obtained: 0% ethanol 6.62 ± 1.6 μM, 0.1% ethanol 6.81 ± 1.1 μM, 0.3% ethanol 8.26 ± 2.5 μM, 0.5% ethanol 8.86 ± 1.9 μM, 1% ethanol 11.01 ± 4.2 μM, 3% ethanol 20.75 ± 2.4 μM, 5% ethanol 21.67 ± 2.2 μM. The results suggest that warfarin bound to HSA was displaced by ethanol. These data indicate that ethanol influence on warfarin binding to HSA may alter the pharmacokinetics of warfarin.