Fatty acid binding to serum albumin: Molecular simulation approaches

Background

Binding affinity for human serum albumin (HSA) is one of the most important factors affecting the distribution and free blood concentration of many ligands. The effect of fatty acids (FAs) on HSA-ligand binding has long been studied. Since the elucidation of the 3-dimensional structure of HSA, molecular simulation approaches have been applied to studies of the structure–function relationship of HSA–FA binding.

Scope of review

We review current insights into the effects of FA binding on HSA, focusing on the biophysical insights obtained using molecular simulation approaches such as docking, molecular dynamics (MD), and binding free energy calculations.

Major conclusions

Possible conformational changes on binding of FA molecules to HSA have been observed through MD simulations. High- and low-affinity FA-binding sites on HSA have been identified based on binding free energy calculations. The relationship between the warfarin binding affinity of HSA and FA molecules has been clarified based on the results of simulations of multi-site FA binding that cannot be experimentally observed.

General significance

Molecular simulation approaches have great potentials to provide detailed biophysical insights into HSA as well as the effects of the binding of FAs or other ligands to HSA. This article is part of a Special Issue entitled Serum Albumin.     

Kinetics of stearic acid transfer between human serum albumin and sterically stabilized liposomes

The kinetics of the transfer of stearic acids between human serum albumin (HSA) and long circulating sterically stabilised liposomes (SSL) composed of dipalmitoylphosphatidylcholine (DPPC) and of submicellar content of the polymer-lipid poly(ethylene glycol:2000)-dipalmitoylphosphatidylethanolamine (PEG:2000-DPPE) have been studied by fluorescence spectroscopy. The study exploits the fact that HSA has a single tryptophan (Trp) residue and that the intrinsic Trp-emission intensity is quenched by the presence of doxyl spin-labelled stearic acids (SASL). Protein/lipid dispersions are considered in which SASL molecules are inserted either in the protein or in the SSL, and the transfer of SASL between the protein and SSL is conveniently monitored by the time variation of the inherent Trp-fluorescence intensity of HSA. It was found that the transfer of fatty acids between HSA and SSL depends on the type of donor and acceptor matrix, on the temperature (i.e., on the physical state of the lipid bilayers) and on the grafting density of the PEG-lipids at the lipid/protein interface. In the absence of polymer-lipids, the rate of transfer increases with temperature in both directions of transfer, and it is higher for the passage from DPPC bilayers to HSA. The presence of polymer-lipids reduces the rate of transfer both in the mushroom and in the brush regime of the polymer chains, especially at low grafting density and for lipid membranes in the fluid phase.     

Physicohemical characterization of the freezing behavior of mannitol-human serum albumin formulations

The goal of the study was to analyze the impact of human serum albumin (HSA) quality (stabilized or nonstabilized HSA), the addition of NaCl, and the HSA stabilizers Naoctanoate and Na-N-acetyltryptophanate on the freezing behavior of mannitol-HSA formulations. The focus was on crystallization, Tg' (glass transition temperature of the maximally freeze-concentrated phase), and Tc (collapse temperature). Differential scanning calorimetry (DSC), cryomicroscopy, and low-temperature x-ray powder diffraction (LTXRD) were used to study the frozen state. In mannitol-HSA formulations. mannitol crystallization was inhibited and Tg' lowered to a greater extent by stabilized HSA (containing Na-octanoate, Na-N-aceyltraptophanate, and NaCl) than by unstabilized HSA. Detailed DSC and LTXRD studies showed that in the concentrations used for stabilizing HSA, NaCl led to changes in the freezing behavior, an effect that was less pronounced for the other stabilizers. NaCl further lowered the Tc, which was determined by cryomicroscopy. As the freezing behavior governs the lyophilization process, the changes have to be taken into consideration for the development of a lyophilization cycle, to avoid collapse and instabilities.     

Physicochemical characterization of the freezing behavior of mannitol-human serum albumin formulations

The goal of the study was to analyze the impact of human serum albumin (HSA) quality (stabilized or nonstabilized HSA), the addition of NaCl, and the HSA stabilizers Na-octanoate and Na-N-acetyltryptophanate on the freezing behavior of mannitol-HSA formulations. The focus was on crystallization, Tg' (glass transition temperature of the maximally freeze-concentrated phase), and Tc (collapse temperature). Differential scanning calorimetry (DSC), cryomicroscopy, and low-temperature x-ray powder diffraction (LTXRD) were used to study the frozen state. In mannitol-HSA formulations, mannitol crystallization was inhibited and Tg' lowered to a greater extent by stabilized HSA (containing Na-octanoate, Na-N-acetyltryptophanate, and NaCl) than by unstabilized HSA. Detailed DSC and LTXRD studies showed that in the concentrations used for stabilizing HSA, NaCl led to changes in the freezing behavior, an effect that was less pronounced for the other stabilizers. NaCl further lowered the Tc, which was determined by cryomicroscopy. As the freezing behavior governs the lyophilization process, the changes have to be taken into consideration for the development of a lyophilization cycle, to avoid collapse and instabilities.     

The effect of medium on functional and structural properties of serum albumins. II. The effect of temperature on the N-form of human serum albumin

In order to investigate effects of temperature in the physiological range (from 10 to 50 degrees C) on structural, physical and functional properties of the N-form of human serum albumin (HSA), the temperature dependences of fluorescence parameters of Trp-214 residue of HSA and of the specifically bound dye ANS, as well as of association constants of ANS binding in the primary and secondary binding sites on HSA molecule were measured. The temperature-induced changes of these properties of HSA are essentially dependent on pH (7.0 or 5,6) and ionic strength (0.001-0.008 or 0.2 M NaCl). At pH 7.0 and 0.2 M NaCl the environment of Trp-214 remained invariant at temperature changes between 10 and 50 degrees C. On the other hand, the affinity to ANS of a primary binding site doubled and that of secondary ones halved. These affinity changes seem to be due, are least partly, to the heating-induced dissociation of Cl-ions, which are inhibitors of the primary dye binding. By lowering pH (to 5.6) and ionic strength the temperature-induced changes in the Trp-214 environment were observed. The changes are interpreted as indole group transition into the buried region, inaccesible to water (the "closing" of a structural slit). The affinity of secondary binding sites of ANS was halved.     

Specific volume and compressibility of human serum albumin-polyanion complexes

The ultrasound velocimetry, densitometry, and differential scanning calorimetry have been used to study the formation of the complexes between human serum albumin (HSA) and polyanions heparin (HEP) and/or dextran sulfate (DS). The values of the ultrasound velocity and specific volume allowed us to determine the specific adiabatic compressibility, phi(K)/beta(0), which reflects the degree of volume compressibility of the complexes. We showed that in the presence of HEP and DS the adiabatic compressibility of HSA decreases with increasing concentration of polyanions. HEP more strongly interacts with HSA than DS. pH of electrolyte in the range 4.7-8.5 weakly affects the adiabatic compressibility. Changes of compressibility of HSA can be caused by increase of the hydration due to the formation of the HSA-polyanion complexes and due to partial unfolding of HSA. The HSA-polyanion interaction resulted in decrease of phase transition temperature of the protein. This evidences about protein destabilization in the presence of polyanions.     

The human serum albumin gene: structure of a unique locus

The entire gene for human serum albumin (HSA) has been isolated from a genomic DNA library, carried in the lambda Charon 4A vector. Six independent isolates have been found to hybridize to a cloned HSA cDNA probe, and all six clones share restriction site sequence homology in the overlapping portion of their DNA. These results seem to indicate that the albumin gene is single-copy, or unique, within the human haploid genome. Measuring from the "CAP" site to the "poly(A)" addition site, albumin gene comprises 16.5 kb of DNA.     

A Comparative Study of Interaction of Tetracycline with Several Proteins Using Time Resolved Anisotropy,Phosphorescence, Docking and FRET

A comparative study of the interaction of an antibiotic Tetracycline hydrochloride (TC) with two albumins, Human serum albumin (HSA) and Bovine serum albumin (BSA) along with Escherichia Coli Alkaline Phosphatase (AP) has been presented exploiting the enhanced emission and anisotropy of the bound drug. The association constant at 298 K is found to be two orders of magnitude lower in BSA/HSA compared to that in AP with number of binding site being one in each case. Fluorescence resonance energy transfer (FRET) and molecular docking studies have been employed for the systems containing HSA and BSA to find out the particular tryptophan (Trp) residue and the other residues in the proteins involved in the binding process. Rotational correlation time (θ_c) of the bound TC obtained from time resolved anisotropy of TC in all the protein-TC complexes has been compared to understand the binding mechanism. Low temperature (77 K) phosphorescence (LTP) spectra of Trp residues in the free proteins (HSA/BSA) and in the complexes of HSA/BSA have been used to specify the role of Trp residues in FRET and in the binding process. The results have been compared with those obtained for the complex of AP with TC. The photophysical behaviour (viz., emission maximum, quantum yield, lifetime and θ_c) of TC in various protic and aprotic polar solvents has been determined to address the nature of the microenvironment of TC in the protein-drug complexes.     

Influence of cis-[PtCl2(Me2SO)2] on the thermal denaturation of albumin

The interaction of cis-[PtCl2(Me2SO)2] with human serum albumin (HSA) and the sensitivity of the complex towards the thermal denaturation depending on the duration of incubation have been studied by absorption and fluorescence spectroscopy methods. Optimum conditions for cis-[PtCl2(Me2SO)2] binding to HSA have been determined. The results have been compared with the data obtained for HAS-cisplatin complex. It has been found that binding of HSA to cis-[PtCl2(Me2SO)2] does not result in significant structural changes of the protein.     

Using bound fatty acids to disclose the functional structure of serum albumin

Background

Serum albumin is a major transport protein in mammals and is known to have at least seven binding sites for long-chain fatty acids (FAs).

Scope of review

We have devised a new electron paramagnetic resonance (EPR) spectroscopic approach to gain information on the functional structure of serum albumin in solution in a “coarse-grained” manner from the ligands' point of view. Our approach is based on using spin labeled (paramagnetic) stearic acids self-assembled with albumin and subsequent nanoscale distance measurements between the FAs using double electron–electron resonance spectroscopy (DEER). Simple continuous wave (CW) EPR spectroscopy, which allows for quantification of bound ligands, complements our studies.

Major conclusions

Based on DEER nanoscale distance measurements, the functional solution structure of human serum albumin (HSA) has remarkably been found to have a much more symmetric distribution of entry points to the FA binding sites than expected from the crystal structure, indicating increased surface flexibility and plasticity for HSA in solution.In contrast, for bovine serum albumin (BSA), the entry point topology is in good agreement with that expected from the crystal structure of HSA. Changes in the solution structures between albumins can hence be revealed and extended to more albumins to detect functional differences at the nanoscale.Going beyond fundamental structural studies, our research platform is also excellently suited for general studies of protein–solvent interactions, temperature effects and ligand binding.

General significance

We discuss how our research platform helps illuminate protein dynamics and function and can be used to characterize albumin-based hybrid materials. This article is part of a Special Issue entitled Serum Albumin.     

Preparation and characterization of paramagnetic polychelates and their protein conjugates

The gadolinium complexes of poly-L-lysine-poly(diethylenetriamine-N,N,N',N",N"-pentaacetic acid) (Gd-PL-DTPA) and poly-L-lysine-poly(1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetr aacetic acid) (Gd-PL-DOTA) and their conjugates with human serum albumin (HSA) have been prepared and characterized. Poly-L-lysine (PL, degree of polymerization approximately 100) was N-acylated with a mixed anhydride of the chelating ligand (DTPA or DOTA). Sixty to ninety chelating groups per molecule of PL could be attached in this way. Following purification of the polychelate by size-exclusion chromatography, the gadolinium complexes were prepared by standard methods and conjugated to HSA with heterobifunctional cross-linking reagents. The molar relaxities of these macromolecular species were 2-3-fold higher than those of the corresponding monomeric metal complexes [( Gd(DTPA)] and [Gd(DOTA)]). The conjugation conditions were optimized to produce conjugates containing 60-90 metal centers per molecule of HSA (ca. one polychelate per protein).     

Automated detection of covalent adducts to human serum albumin by immunoaffinity chromatography, on-line solution phase digestion and liquid chromatography-mass spectrometry

A generic method for the detection of covalent adducts to the cysteine-34 residue of human serum albumin (HSA) has been developed, based on an on-line combination of immunoaffinity chromatography for selective sample pre-treatment, solution phase digestion, liquid chromatography and tandem mass spectrometry. Selective anti-HSA antibodies immobilized on agarose were used for sample pre-concentration and purification of albumin from the chemically produced alkylated HSA. After elution, HSA and HSA adducts are mixed with pronase and directed to a reaction capillary kept at a digestion temperature of 70 degrees C. The digestion products were trapped on-line on a C18 SPE cartridge. The peptides were separated on a reversed-phase column using a gradient of organic modifier and subsequently detected using tandem mass spectrometry. Modified albumin samples consisted of synthetically alkylated HSA by the reactive metabolite of acetaminophen, N-acetyl-p-benzoquinoneimine (NAPQI), and using the alkylating agent 1-chloro-2,4-dinitrobenzene (CDNB) as reference. The resulting mixture of alkylated versus non-modified albumin has been applied to the on-line system, and alkylation of HSA is revealed by the detection of the modified marker tetra-peptide glutamine-cysteine-proline-phenylalanine (QCPF) adducts NAPQI-QCPF and CDNB-QCPF. Detection of alkylated species was enabled by the use of data comparison algorithms to distinguish between unmodified and modified HSA samples. The in-solution digestion proved to be a useful tool for enabling fast (less than 2 min) and reproducible on-line digestion of HSA. A detection limit of 1.5 micromol/L of modified HSA could be obtained by applying 10 microL of NAPQI-HSA sample.     

Investigation of the molecular interactions of triclocarban with human serum albumin using multispectroscopies and molecular modeling

Triclocarban (TCC), as a broad spectrum antibacterial agent widely used in personal care products, has recently been recognized as environmental pollutant with the potential of adversely affecting wildlife and human health. However, the behavior of TCC in blood circulatory system and the potential toxicity of TCC at the molecular level have been poorly investigated. In this study, the effect of TCC on human serum albumin (HSA) and the binding mechanism of TCC to HSA were examined using spectroscopic techniques and molecular modeling methods. The fluorescence results suggested that the fluorescence of HSA was quenched by TCC through a static quenching mechanism and nonradiation energy transfer, and TCC was bound to HSA with moderately strong binding affinity via hydrophobic interaction based on the analysis of the thermodynamic parameters. The site marker competitive experiments revealed that TCC bound into subdomain IIA (site I) of HSA. In addition, the results obtained from the circular dichroism, Fourier transform infrared (FT-IR), 8-anilino-1-naphthalenesulfonic acid fluorescence, synchronous fluorescence, three-dimensional fluorescence spectra and dynamic light scattering suggested the change in the microenvironment and conformation of HSA during the binding reaction. Finally, the best binding mode of TCC and specific interaction of TCC with amino acid residues were determined using molecular docking and molecular dynamics simulations. In a word, the present studies can provide a way to help us well understand the transport, distribution and toxicity effect of TCC when it diffused in the human body.

Communicated by Ramaswamy H. Sarma     

Interaction of Cu(II) 3,5-diisopropylsalicylate with human serum albumin - an evaluation of spectroscopic data

The copper(II) complex of 3,5-diisopropylsalicylate is a lipophilic water-insoluble binuclear complex, Cu(II) (3,5-DIPS) , that has attracted interest because of a wide range of pharmacological activities. This study was undertaken to examine bonding interactions between the complex and human serum albumin (HSA) to help elucidate the mode of transport of the complex in vivo. Electron paramagnetic resonance, numerical magnetic resonance and UV-visible absorption spectroscopic studies were performed using 200 M aqueous solutions (pH 7.5) of HSA to which had been added up to three molar equivalents of CuCl , CuSO , or Cu(II) (3,5-DIPS). Both EPR and UV-visible spectra demonstrated the presence of more than one copper bonding site on HSA, and proton NMR spectra showed that the 3,5-DIPS ligand is also bonded to HSA. These results indicate that there is no observable direct coordination of the ligand to copper in the presence of HSA, and that the majority of the copper and 3,5-DIPS bond to HSA at separate sites. Addition of solid Cu(II) (3,5-DIPS) to HSA at pH 7.5 similarly resulted in spectra that suggest that there are no ternary Cu(II)(3,5-DIPS), Cu(II)(3,5-DIPS) , or Cu(II) (3,5-DIPS) complexes formed with HSA. It is concluded that any ternary complexes formed in the presence of HSA are below the spectroscopic detection limits and represent less than 5% of the total copper. © Rapid Science 1998.     

Assignment of 117 genes from HSA5 to the porcine IMpRH map and generation of a dense human-pig comparative map

Twenty-two and eight significant quantitative trait loci for economically important traits have been located on porcine chromosomes (SSC) 2q and SSC16 respectively, both of which have been shown to correspond to human chromosome 5 (HSA5) by chromosome painting. To provide a comprehensive comparative map for efficient selection of candidate genes, we assigned 117 genes from HSA5 using a porcine radiation hybrid (IMpRH) panel. Sixty-six genes were assigned to SSC2 and 48 to SSC16. One gene was suggested to link to SSC2 markers and another to SSC6. One gene did not link to any gene, expressed sequence tag or marker in the map, including those in the present investigation. This study demonstrated the following: (1) SSC2q21-q28 corresponds to the region ranging from 74.0 to 148.2 Mb on HSA5q13-q32 and the region from 176.0 to 179.3 Mb on HSA5q35; (2) SSC16 corresponds to the region from 1.4 to 68.7 Mb on HSA5p-q13 and to the region from 150.4 to 169.1 Mb on HSA5q32-q35 and (3) the conserved synteny between HSA5 and SSC2q21-q28 is interrupted by at least two sites and the synteny between HSA5 and SSC16 is also interrupted by at least two sites.     

Direct observation of covalent adducts with Cys34 of human serum albumin using mass spectrometry

The interactions of the unpaired thiol residue (Cys34) of human serum albumin (HSA) with low-molecular-weight thiols and an Au(I)-based antiarthritic drug have been examined using electrospray ionization mass spectrometry. Early measurements of the amount of HSA containing Cys34 as the free thiol suggested that up to 30% of circulating HSA bound cysteine as a mixed disulfide. It has also been suggested that reaction of HSA with cysteine, occurs only on handling and storage of plasma. In our experiments, there were three components of HSA in freshly collected plasma from normal volunteers, HSA, HSA+cysteine, and HSA+glucose in the ratio approximately 50:25:25. We addressed this controversy by using iodoacetamide to block the free thiol of HSA in fresh plasma, preventing its reaction with plasma cysteine. When iodoacetamide was injected into a vacutaner tube as blood was collected, the HSA was modified by iodoacetamide, with 20-30% present as the mixed disulfide with cysteine (HSA+cys). These data provide strong evidence that 20-30% of HSA in normal plasma contains one bound cysteine. Reaction of HSA with [Au(S(2)O(3))(2)](3-) resulted in formation of the adducts HSA+Au(S(2)O(3)) and HSA+Au. Reaction of HSA with iodoacetamide prior to treatment with [Au(S(2)O(3))(2)](3-) blocked the formation of gold adducts.     

Oxidative modification of albumin in the parenchymal lung tissue of current smokers with chronic obstructive pulmonary disease

Background

There is accumulating evidence that oxidative stress plays an important role in the pathophysiology of chronic obstructive pulmonary disease (COPD). One current hypothesis is that the increased oxidant burden in these patients is not adequately counterbalanced by the lung antioxidant systems.

Objective

To determine the levels of oxidised human serum albumin (HSA) in COPD lung explants and the effect of oxidation on HSA degradation using an ex vivo lung explant model.

Methods

Parenchymal lung tissue was obtained from 38 patients (15F/23M) undergoing lung resection and stratified by smoking history and disease using the GOLD guidelines and the lower limit of normal for FEV₁/FVC ratio. Lung tissue was homogenised and analysed by ELISA for total levels of HSA and carbonylated HSA. To determine oxidised HSA degradation lung tissue explants were incubated with either 200 μg/ml HSA or oxidised HSA and supernatants collected at 1, 2, 4, 6, and 24 h and analysed for HSA using ELISA and immunoblot.

Results

When stratified by disease, lung tissue from GOLD II (median = 38.2 μg/ml) and GOLD I (median = 48.4 μg/ml) patients had lower levels of HSA compared to patients with normal lung function (median = 71.9 μg/ml, P < 0.05). In addition the number of carbonyl residues, which is a measure of oxidation was elevated in GOLD I and II tissue compared to individuals with normal lung function (P < 0.05). When analysing smoking status current smokers had lower levels of HSA (median = 43.3 μg/ml, P < 0.05) compared to ex smokers (median = 71.9 μg/ml) and non-smokers (median = 71.2 μg/ml) and significantly greater number of carbonyl residues per HSA molecule (P < 0.05). When incubated with either HSA or oxidised HSA lung tissue explants rapidly degraded the oxidised HSA but not unmodified HSA (P < 0.05).

Conclusion

We report on a reliable methodology for measuring levels of oxidised HSA in human lung tissue and cell culture supernatant. We propose that differences in the levels of oxidised HSA within lung tissue from COPD patients and current smokers provides further evidence for an oxidant/antioxidant imbalance and has important biological implications for the disease.

     

The Role of Caprylate Ligand Ion on the Stabilization of Human Serum Albumin

Sodium caprylate was added to a pharmaceutical-grade human serum albumin (HSA) to stabilize the product. In this study we have aimed to establish how caprylate ligand protects HSA from thermal degradation. The fatty acid stabilizer was first removed from commercial HSA by charcoal treatment. Cleaned HSA was made to 10% w/v in pH 7.4 buffered solutions and doped with sodium caprylate in serial concentrations up to 0.16 mmol/g-protein. These solutions as well as a commercial HSA, human serum, and enriched-albumin fraction were subjected to differential scanning calorimetry (DSC) within the temperature range of 37–90°C at a 5.0°C/min scanning rate. The globular size of the cleaned HSA solutions was measured by dynamic light scattering. The denaturing temperatures for albumin with sodium caprylate and a commercial one were significantly higher than for albumin only. It was found that the protein globules of cleaned HSA were not as stable as that of the native one due to aggregation, and the caprylate ion may reduce the aggregation by enlarging the globules’ electrical double layer. A rational approximation of the Lumry-Eyring protein denaturation model was used to treat DSC denaturing endotherms. The system turned from irreversible dominant Scheme: to reversible dominant Scheme: with the increase in caprylate concentration from null to ~0.08 mmol/g-protein. It was postulated that the caprylate ligand may decrease the rate of reversible unfolding as it binds to the IIIA domain which is prone to reversible unfolding/refolding and causes further difficulty for irreversible denaturation which, in turn, HSA can be stabilized.KEY WORDS: differential scanning calorimetry, human serum albumin, Lumry-Eyring model, protein denaturation, sodium caprylate     

Efficient removal of albumin from human serum by monosize dye-affinity beads

Cibacron Blue F3GA was covalently attached onto monosize poly(glycidyl methacrylate) [poly(GMA)] beads for removal of human serum albumin (HSA) from human serum. Monosize poly(GMA) beads, 1.6 microm in diameter, were produced by dispersion polymerization. Cibacron Blue F3GA loading was 1.73 mol/g. HSA adsorption experiments were performed by stirred-batch adsorption. The non-specific adsorption of HSA was low (0.8 mg/g polymer). Dye attachment onto the monosize beads significantly increased the HSA adsorption (189.8 mg/g). The maximum HSA adsorption was observed at pH 5.0. With an increase of the aqueous phase concentration of sodium chloride, the adsorption capacity decreased drastically. The equilibrium adsorption of HSA significantly decreased with increasing temperature. The elution studies were performed by adding 0.1 M Tris/HCl buffer containing 0.5 M NaSCN to the HSA solutions in which adsorption equilibria had been reached. The elution results demonstrated that the adsorption of HSA to the adsorbent was reversible. The depletion efficiencies for HSA were above 87% for all studied concentrations. To test the efficiency of HSA removal from human serum, proteins in the serum and eluted portion were analyzed by two-dimensional gel electrophoresis. Eluted proteins include mainly albumin, and a small number of nonalbumin proteins such as apo-lipoprotein A1, sero-transferrin, haptoglobulin and alpha1-antitrypsin were bound by the dye-affinity beads. IgA was not identified in eluted fraction.     

Novel insights into the pleiotropic effects of human serum albumin in health and disease

Background

Human serum albumin is the principal protein in human serum. It participates in regulation of plasma oncotic pressure and transports endogenous and exogenous ligands such as thyroxine, free fatty acids, bilirubin, and various drugs. Therefore, studying its ligand binding mechanism is important in understanding many functions of the protein.

Scope of review

This review discusses the pleiotropic biochemical effects and their relevance to physiologic functions of albumin.

Major conclusions

Although HSA is traditionally recognized for its ligand transport and oncotic effects in human circulation, our studies have revealed its participation in several other important physiological functions. In some instances, it may function as a catalyst. Pleiotropic properties of HSA have been exploited by development of recombinant HSA and its mutants, and the use of these recombinant proteins in studies with various biochemical and biophysical techniques. These studies allowed us to obtain new insights on the diverse roles of HSA in human physiology. The following aspects of HSA were discussed in this review: 1) HSA and its mutants' role in thyroxine transport, 2) structural details of the ligand binding functions of HSA to ligands such as warfarin, digoxin, halothane anesthetics, nitric oxide, bilirubin, free fatty acids, etc, and 3) the formation of modified albumin during myocardial ischemia, its diagnostic significance, and HSA's role in cardiovascular disease.

General significance

The appreciation and understanding of structural details and new physiological roles has provided a renewed interest in HSA research. Specific structural information gained on various mechanisms of HSA–ligand interaction can be used to develop a model to better understand protein–drug interactions, aid in the development of new drugs with improved pharmacokinetic effects, and ultimately be used to improve the quality of healthcare. This article is part of a Special Issue entitled Serum Albumin.