Ability Of Human Serum Albumin Research Articles

Effects of ultraviolet radiation and reactive oxygen species on the structure and ligand-binding ability of human serum albumin for norfloxacin

To explore the influence of ultraviolet radiation (UV) and reactive oxygen species (ROS) on the structure and ligand-binding ability of human serum albumin (HSA) for norfloxacin (NFLX), HSA was treated with UV (HSAUV) and UV/H2O2 (HSAUV/H2O2), respectively. The structure difference of HSA, HSAUV and HSAUV/H2O2 were investigated by Fourier transform infrared, fluorescence (i.e., emission fluorescence, synchronous fluorescence and fluorescence lifetime) and circular dichroism spectroscopy. The intermolecular interactions of HSA, HSAUV and HSAUV/H2O2 with NFLX were studied at pH 7.4 and temperatures 291, 310, and 318 K, using fluorescence emission and synchronous fluorescence spectroscopy, and combined with molecular docking. The results suggested that UV and ROS could destroy the α-helix of HSA and induce the loose of HSA structure, and the average fluorescence lifetimes of HSA decreased from 6.12 ns to 3.76 ns (HSAUV) and 3.40 ns (HSAUV/H2O2). The conformational changes of HSA caused by UV and ROS allowed NFLX to get access to more potential binding sites and promoted the binding affinity of HSA for NFLX, while the temperature stability of the HSA-NFLX complex significantly decreased. The results are useful and valuable for evaluating the protein damage caused by UV and ROS in organisms.

Influence of mutations caused by radiation exposure on the bilirubin binding sites of human serum albumin

In this article we analyze the bilirubin binding sites of human serum albumin from the point of view of the secondary structure instability, as well as the effect of amino acid substitutions caused by radiation exposure on the ability of albumin to bind bilirubin-IX-alpha. Based on calculations of binding energy and inhibition constants of bilirubin-albumin complexes before and after the amino acid substitutions, it was found that amino acid substitutions have different effects on the ability of human serum albumin to bind bilirubin. Amino acid substitutions Asp269-Gly269 (Nagasaki-1), Glu354-Lys354 (Hiroshima-1), Asp375-Asn375 (Nagasaki-2) reduce the binding free energy of bilirubin with human serum albumin, and the amino acid substitutions His3-Gln3 (Nagasaki-3) and Glu382-Lys382 (Hiroshima-2) increase it during molecular docking with the corresponding areas of the protein surface. The inhibition constants are significantly higher than with known binding sites. In general, mutations caused by radiation exposure cannot effect on bilirubin binding sites of human serum albumin, since the amino acid residues that are replaced do not interact with the amino acid residues from the binding sites (Leu115, Arg117, Phe134, Tyr138, Ile142, Phe149, Phe157, Tyr161, Arg186, Lys190, Lys240, Arg222). All amino acid residues from known binding sites are located in stable elements of the secondary structure of human serum albumin.The data obtained are important for understanding the impact of radiation exposure on the development of bilirubin encephalopathy in the population of the Chernobyl region and Japan.

Selective binding of pyrene in subdomain IB of human serum albumin: Combining energy transfer spectroscopy and molecular modelling to understand protein binding flexibility

The ability of human serum albumin (HSA) to bind medium-sized hydrophobic molecules is important for the distribution, metabolism, and efficacy of many drugs. Herein, the interaction between pyrene, a hydrophobic fluorescent probe, and HSA was thoroughly investigated using steady-state and time-resolved fluorescence techniques, ligand docking, and molecular dynamics (MD) simulations. A slight quenching of the fluorescence signal from Trp214 (the sole tryptophan residue in the protein) in the presence of pyrene was used to determine the ligand binding site in the protein, using Förster's resonance energy transfer (FRET) theory. The estimated FRET apparent distance between pyrene and Trp214 was 27Å, which was closely reproduced by the docking analysis (29Å) and MD simulation (32Å). The highest affinity site for pyrene was found to be in subdomain IB from the docking results. The calculated equilibrium structure of the complex using MD simulation shows that the ligand is largely stabilized by hydrophobic interaction with Phe165, Phe127, and the nonpolar moieties of Tyr138 and Tyr161. The fluorescence vibronic peak ratio I1/I3 of bound pyrene inside HSA indicates the presence of polar effect in the local environment of pyrene which is less than that of free pyrene in buffer. This was clarified by the MD simulation results in which an average of 5.7 water molecules were found within 0.5nm of pyrene in the binding site. Comparing the fluorescence signals and lifetimes of pyrene inside HSA to that free in buffer, the high tendency of pyrene to form dimer was almost completely suppressed inside HSA, indicating a high selectivity of the binding pocket toward pyrene monomer. The current results emphasize the ability of HSA, as a major carrier of several drugs and ligands in blood, to bind hydrophobic molecules in cavities other than subdomain IIA which is known to bind most hydrophobic drugs. This ability stems from the nature of the amino acids forming the binding sites of the protein that can easily adapt their shape to accommodate a variety of molecular structures.

Effect of different glycation agents on Cu(II) binding to human serum albumin, studied by liquid chromatography, nitrogen microwave-plasma atomic-emission spectrometry, inductively-coupled-plasma mass spectrometry, and high-resolution molecular-mass spectrometry

The ability of human serum albumin to capture unbound copper under different clinical conditions is an important variable potentially affecting homeostasis of this element. Here, we propose a simple procedure based on size-exclusion chromatography with on-line UV and nitrogen microwave-plasma atomic-emission spectrometry (MP-AES) for quantitative evaluation of Cu(II) binding to HSA upon its glycation in vitro. The Cu-to-protein molar ratio for non-glycated albumin was 0.98 ± 0.09; for HSA modified with glyoxal (GO), methylglyoxal (MGO), oxoacetic acid (GA), and glucose (Glc), the ratios were 1.30 ± 0.22, 0.72 ± 0.14, 0.50 ± 0.06, and 0.95 ± 0.12, respectively. The results were confirmed by using ICP-MS as an alternative detection system. A reduced ability of glycated protein to coordinate Cu(II) was associated with alteration of the N-terminal metal-binding site during incubation with MGO and GA. In contrast, glycation with GO seemed to generate new binding sites as a result of tertiary structural changes in HSA. Capillary reversed-phase liquid chromatography with electrospray-ionization quadrupole-time-of-flight tandem mass spectrometry enabled detection and identification of Cu(II) coordinated to the N-terminal metal-binding site (Cu(II)-DAHK) in all tryptic digests analyzed. This is the first report confirming Cu(II)-DAHK species in HSA by means of high-resolution tandem mass spectrometry, and the first report on the use of MP-AES in combination with chromatographic separation.

Probing the interaction of human serum albumin with DPPH in the absence and presence of the eight antioxidants

Albumin represents a very abundant and important circulating antioxidant in plasma. DPPH radical is also called 2,2-diphenyl-1-picrylhydrazyl. It has been widely used for measuring the efficiency of antioxidants. In this paper, the ability of human serum albumin (HSA) to scavenge DPPH radical was investigated using UV–vis absorption spectra. The interaction between HSA and DPPH was investigated in the absence and presence of eight popular antioxidants using fluorescence spectroscopy. These results indicate the antioxidant activity of HSA against DPPH radical is similar to glutathione and the value of IC50 is 5.200×10−5molL−1. In addition, the fluorescence experiments indicate the quenching mechanism of HSA, by DPPH, is a static process. The quenching process of DPPH with HSA is easily affected by the eight antioxidants, however, they cannot change the quenching mechanism of DPPH with HSA. The binding of DPPH to HSA primarily takes place in subdomain IIA and exists two classes of binding sites with two different interaction behaviors. The decreased binding constants and the number of binding sites of DPPH with HSA by the introduction of the eight antioxidants may result from the competition of the eight antioxidants and DPPH binding to HSA. The binding of DPPH to HSA may induce the micro-environment of the lone Trp-214 from polar to slightly nonpolar.

Correspondence of Fatty Acid and Drug Binding Sites on Human Serum Albumin: A Two-Dimensional Nuclear Magnetic Resonance Study

The ability of human serum albumin (HSA) to bind fatty acids (FA) in multiple sites has been revealed by many studies. Here we detect and characterize nine individual binding sites by two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy of 18-[(13)C]-oleic acid (OA) complexed with HSA. We characterize site-specific FA binding by addition of (i) different FA molar ratios (from 1:1 to 4:1 OA:HSA) to observe the order of filling and occupancy of binding sites; (ii) methyl-β-cyclodextrin, as a FA acceptor, to observe the dissociation of FA; and (iii) drugs (with known binding sites in the crystal structure) to reveal the correspondence of three NMR peaks with sites in the crystal structure. At 1:1 and 2:1 OA:HSA ratios, three sites were shown to fill sequentially. These high-affinity sites were well resolved from additional sites (one medium-affinity and five low-affinity) observed at 3:1 and 4:1 OA:HSA ratios. Methyl-β-cyclodextrin extracted OA from individual sites in the reverse order of filling. FA bound in three low-affinity sites were displaced by drugs shown to bind in crystalline HSA to FA sites 7 and 3 (Sudlow's drug sites I and II, respectively) and FA site 6. With this strategy, 2D NMR spectral analysis permits site-specific characterization of the binding of drugs and FA and provides a sensitive probe of the mutual effects of FA and ligand binding.

α‐Tocopherol binding to human serum albumin

Given the ability of human serum albumin (HSA) to bind hydrophobic ligands, the binding mode of α-tocopherol, the most representative member of the vitamin E family, is reported. α-Tocopherol binds to HSA with Kd0 = (7.0 ± 3.0) × 10(-6) M (pH 7.2, 25.0°C). Competitive and allosteric modulation of α-tocopherol binding to full-length and truncated (Asp1-Glu382) HSA by endogenous and exogenous ligands suggests that it accommodates preferentially in the FA3-FA4 site. As HSA is taken up into cells, colocalizes with the α-tocopherol transfer protein, and contributes to ligand secretion via ABCA1, it might participate in the distribution of α-tocopherol between plasma, cells, and tissues.

( EZ)-Cyclobilirubin formation from bilirubin in complex with serum albumin derived from various species

We determined the specificity of photochemical changes of bilirubin in complex with serum albumin from various species. There were no general trends in the configurational photoisomerizations of ( ZE)-bilirubin/( ZZ)-bilirubin and ( EZ)-bilirubin/( ZZ)-bilirubin associated with the albumins from various species as compared to those associated with human serum albumin. The absorbance spectra of bilirubin in complex with albumins from various species differed, indicating that the three-dimensional structures of ( ZZ)-bilirubin bound to the various serum albumins, which are substrates of ( ZE)- and ( EZ)-bilirubin, differ among species. The rates of conversion of the ( EZ)-bilirubin isomer into the structural cyclobilirubin isomer were similar for the albumins of chicken, rat, rabbit, dog, bovine, and pig, and were significantly slower than the rate for human serum albumin. This suggests that the three-domain human albumin has evolved to allow ready conversion of ( EZ)-bilirubin to ( EZ)-cyclobilirubin. Cyclobilirubin formation in a bilirubin-alpha-fetoprotein solution was much lower than that in a bilirubin–human serum albumin solution. It is believed that the ability of human serum albumin to facilitate the photochemical change of bilirubin was evolutionarily selected in response to neonatal jaundice in humans.

Influence of non-enzymatic post-translation modifications on the ability of human serum albumin to bind iron: Implications for non-transferrin-bound iron speciation

Human serum albumin (HSA) is known as a low affinity iron binding protein and it has been proposed as a ligand for the non-transferrin-bound iron (NTBI) pool existing in the sera of iron-overload patients, but definitive evidence is still lacking. In this study, gel filtration linked to inductively coupled plasma mass spectrometry (GF-ICP-MS) was employed to assess iron speciation in solutions containing physiological concentrations of citrate and HSA at physiological values of pH and ionic strength. The influence of common non-enzymatic post-translation modifications on the ability of HSA to bind iron was also studied. HSA was found to bind between 60 and 20% of the available iron when iron concentration was varied over the common clinical range of NTBI concentrations (1 to 10 µM), thus proving to be a relevant ligand for NTBI speciation. Analysis of modified albumins showed that both glycation and oxidation increase the albumin iron binding capacity. The results presented indicate that non-enzymatic modifications of albumin, that are prevalent in diabetes and increased oxidative stress, may have a fundamental role in NTBI speciation. Experiments with the nitrilotriacetic acid (NTA) based assay for NTBI determination demonstrate the inability of NTA to quantitatively mobilize albumin-bound iron, especially after the albumin had undergone oxidation or glycation. This result would indicate that the low NTBI levels so far measured in diabetic sera may be understated and that different analytical techniques are required to determine NTBI levels in diabetic patients.

Protein-mediated boundary lubrication in arthroplasty

Wear of articulated surfaces can be a major lifetime-limiting factor in arthroplasty. In the natural joint, lubrication is effected by the body's natural synovial fluid. Following arthroplasty, and the subsequent reformation of the synovial membrane, a fluid of similar composition surrounds the artificial joint. Synovial fluid contains, among many other constituents, a substantial concentration of the readily adsorbing protein albumin. The ability of human serum albumin to act as a boundary lubricant in joint prostheses has been investigated using a pin-on-disc tribometer. Circular dichroism spectroscopy was employed to follow the temperature- and time-dependent conformational changes of human serum albumin in the model lubricant solution. Effects of protein conformation and polymer surface hydrophilicity on protein adsorption and the resulting friction in the boundary lubrication regime have been investigated. Unfolded proteins preferentially adsorb onto hydrophobic polymer surfaces, where they form a compact, passivating layer and increase sliding friction—an effect that can be largely suppressed by rendering the substrate more hydrophilic. A molecular model for protein-mediated boundary friction is proposed to consolidate the observations. The relevance of the results for in vivo performance and ex vivo hip-joint testing are discussed.

Interaction between human serum albumin and liposomes: a monolayer and liposome study

The penetration ability of human serum albumin in neutral monolayers of cholesterol, dipalmitoylphosphatidylcholine and mixtures at the same molar ratio, has been studied. The effect of the protein on unilamellar liposomes of cholesterol-di- palmitoylphosphatidylcholine has been determined by measurement of the amount of Carboxyfluorescein released from the liposomes. There is a relation between the penetration ability in such monolayers and the leakage effect on the liposomes.