Evidence that the principal CoII-binding site in human serum albumin is not at the N-terminus: implication on the albumin cobalt binding test for detecting myocardial ischemia

Human serum albumin (HSA) is the most abundant protein in the blood plasma and is involved in the transport of metal ions. Four metal-binding sites with different specificities have been described in HSA: (i) the N-terminal site provided by Asp1, Ala2, and His3, (ii) the site at the reduced Cys34, (iii) site A, including His67 as a ligand, and (iv) the nonlocalized site B. HSA can bind CoII, and HSA was proposed to be involved in CoII transport. Recently, binding of CoII to HSA has attracted much interest due to the so-called albumin cobalt binding (ACB) test approved by the Food and Drug Administration for evaluation of myocardial ischemia. Although the binding of CoII to HSA is important, the binding of CoII to HSA is not well-characterized. Here the binding of CoII to HSA was studied under anaerobic conditions to prevent CoII oxidation. Electronic absorption, EPR, and NMR spectroscopies indicate three specific and well-separated binding sites for CoII in HSA. CoII ions in all three sites are in a high-spin state and coordinated in a distorted octahedral geometry. Competition experiments with CdII (known to bind to sites A and B) and CuII (known to bind to the N-terminal site) were used to identify the sites of binding of CoII to HSA. They revealed that the first two equivalents of CoII bind to sites A and B. Only the third may be bound to the N-terminal site. The repercussions of these results on the understanding of the ACB test and hence the myocardial ischemia are discussed.     

Reactive oxygen species damaged human serum albumin in patients with type 1 diabetes mellitus: biochemical and immunological studies

The role of hydroxyl radical (.OH) damaged human serum albumin (HSA) in type 1 diabetes has been investigated in the present study. Hydroxyl radical induced modification on HSA has been studied by UV absorption spectroscopy, ANS fluorescence and carbonyl estimation. Hydroxyl radical modified HSA was found to be highly immunogenic in rabbits as compared to native HSA. The binding characteristics of circulating autoantibodies in type 1 diabetes patients against native and modified HSA were assessed. Diabetes patients (n=31) were examined by direct binding ELISA and the results were compared with healthy age-matched controls (n=22). High degree of specific binding by 54.8% of patients sera towards .OH modified HSA, in comparison to its native analogue (p<0.05) was observed. Sera from those type 1 diabetes patients having smoking history, high aging with high degree of disease showed substantially stronger binding to .OH modified HSA over native HSA in particular. Normal human sera showed negligible binding with either antigen. Competitive inhibition ELISA reiterates the direct binding results. Gel retardation assay further substantiated the enhanced recognition of modified HSA by circulating autoantibodies in diabetes patients. The increase in total serum protein carbonyl levels in the diabetes patients was largely due to an increase in oxidized albumin. HSA of diabetes mellitus patients (DM-HSA) and normal subjects (normal-HSA) were purified on a Sephacryl S-200 HR column. Spectroscopic analysis confirmed that the DM-HSA samples contained higher levels of carbonyls than normal-HSA (p<0.001). DM-HSA was conformationally altered, with more exposure of its hydrophobic regions. Collectively, the oxidation of plasma proteins, especially HSA, might enhance oxidative stress in type 1 diabetes mellitus patients.     

Characterization of the Co(2+) and Ni(2+) binding amino-acid residues of the N-terminus of human albumin. An insight into the mechanism of a new assay for myocardial ischemia.

Patients suffering from myocardial ischemia reportedly exhibit reduced in vitro binding of exogenous Co(2+) to the N-terminal of human serum albumin (HSA). The purpose of our investigation was to simulate changes in the N-terminus of HSA that may account for these ischemia-induced modifications to the cobalt binding site. HPLC, LC-MS and (1)H NMR analyses have shown that the N-terminal region of HSA Asp-Ala-His-Lys binds the transition metals Co(2+) and Ni(2+). Synthetic peptides with the first 2-12 amino acids of the HSA sequence demonstrated that the first three amino acids, Asp-Ala-His, are essential for strong binding of cobalt. Modification of the N-terminus peptide of HSA by way of N-acetylation or the deletion of one or more amino acid resulted in no binding of cobalt. Because the degradation of the susceptible, specific transition metal binding site of HSA may account for the decreased cobalt binding observed during ischemic events, an assay that detects this reduced binding could be useful in the diagnosis of ischemia.     

Spectroscopic and thermodynamic determination of three distinct binding sites for Co(II) ions in human serum albumin

Human serum albumin (HSA) is the most abundant protein of blood serum, involved in the transport of metal ions, including Co(II). Using circular dichroism spectroscopic titrations we characterized three distinct Co(II) binding sites in HSA. Applying Cu(II), Ni(II) and Cd(II) ions as competitors we determined that these sites are identical with three binding sites known for other metal ions. We ordered these sites according to their binding affinities as cadmium site B (CdB) > multi-metal binding site (MBS) > N-terminal binding site (NTS). Using isothermal titration calorimetry (ITC) we confirmed the presence of these three binding sites and determined their conditional binding constants at pH 7.4 as 9 ± 5, 1.1 ± 0.5, and 0.9 ± 0.3 × 10⁴ M⁻¹, respectively. The impact of these results on the albumin cobalt binding (ACB) clinical assay for myocardial ischemia is discussed.     

A competitive inhibition ELISA as a probe for studying the refolding of human serum albumin

The refolding of iodoacetic acid-blocked human serum albumin (HSA) was studied using a modified competitive inhibition ELISA. A maximum of 89% native activity was detected 24 hours after initiating refolding using an albumin concentration of 600 micrograms/mL. The presence of both monomer and polymer HSA was studied using native polyacrylamide gel electrophoresis of thiol-blocked HSA samples. Monomer HSA was not detected until 2.5 hours after initiating refolding. Fractionated polymer and monomer HSA from a sample trapped at 72 hours after initiating refolding was determined to have 40% and 87% native activity respectively. Both polymer and monomer HSA fractions contribute to the overall immunological activity detected by the ELISA, at various times. The ELISA assay was able to detect the changing HSA conformation associated with refolding of totally reduced HSA.     

Effect of albumin conformation on the binding of ciprofloxacin to human serum albumin: a novel approach directly assigning binding site

Human serum albumin (HSA) is known to exist as N (pH approximately 7), B (pH approximately 9), and F (pH approximately 3.5) isomeric forms and an equilibrium intermediate state (I) accumulate in the urea induced unfolding pathway of HSA around 4.8-5.2 M urea concentrations. These states displayed characteristic structure and functions. To elucidate the ciprofloxacin (CFX) binding behavior of HSA, the binding of ciprofloxacin with these conformational states of human serum albumin (HSA) has been investigated by fluorescence spectroscopy. The binding constant (K) for N, B, F, and I conformation of HSA were 6.92 x 10(5), 3.87 x 10(5), 4.06 x 10(5), and 2.7 x 10(5) M(-1) and the number of binding sites (n) were 1.26,1.21, 1.16, and 1.19, respectively. The standard free energy changes (DeltaGbinding(0)) of interaction were found to be -33.3 (N isomer), -31.8 (B isomer), -32 (F isomer), and -30.0 kJ mol(-1) respectively. By using unfolding pathway of HSA, domain II of HSA has been assigned to possess binding site of ciprofloxacin. Plausible correlation between stability of CFX-N and CFX-B complexes and drug distribution have been discussed. At plasma concentration of HSA fraction of free CFX, which contributes potential to its rate of transport across cell membrane, was found to be approximately 80% more for B isomers compared to N isomers of HSA. The conformational changes in two physiologically important isomers of HSA (N and B isomers) upon ciprofloxacin binding were evaluated by measuring far, near-UV CD, and fluorescence properties of the CFX-HSA complex.     

Characterization of the interaction between human serum albumin and morin

The interaction of morin with human serum albumin (HSA) has been investigated by using fluorescence, UV absorption and Fourier transform infrared spectroscopic approaches for the first time. Fluorescence data revealed the presence of a specific binding site on HSA for morin, and the binding affinity was 1.13+/-0.11x10(-5) L Mol(-1) in the physiological condition. The intrinsic fluorescence of morin was conspicuously enhanced in the presence of HSA due to excited-state proton transfer. The binding ability of morin to protein decreased with the increase of the buffer pH from 6.4 to 8.4, which signified that the level of protonation of the hydroxyl groups played an important role during the drug-protein binding process. From the UV absorption spectra of morin in various pH medium, the dissociation behaviors of the hydroxyl groups on the drug molecule were assigned. The second derivative UV absorption spectra of morin after interacting with HSA were used to elucidate the binding mode of morin to protein. The obvious red shift of the UV absorption band I of morin upon binding to HSA further confirmed the formation of HSA-morin complex, and this property was also utilized to estimate the binding constant. The interaction between morin and HSA induced an obvious reduction of the protein alpha-helix and beta-sheet structures.     

Histidine146 of human serum albumin plays a prominent role at the interface of subdomains IA and IIA in allosteric ligand binding

Fatty acids are endogenous ligands of human serum albumin (HSA) that induce conformational changes and participate in allosteric ligand binding to HSA. In a previous study, we showed that, when myristate (MYR) is present, the binding of [(14) C]ketoprofen (KP) to subdomain IA of HSA was increased, indicating that, when MYR binds to HSA, a new binding site in formed in that region. Meanwhile, an N-B transition has been reported to increase the binding of ligands at alkaline pH when the status of albumin is the B-conformer. Six histidine single mutants of HSA, H9A, H39A, H67A, H105A, H128A and H146A were produced and photolabeled with [(14) C]KP at pH 6.5, 7.4 and 8.2 and the role of each histidine in causing the N-B transition induced allosteric ligand binding was examined. Cyanogen bromide cleavage of the photolabeled native HSA showed that subdomain IA was the site of the allosteric binding of KP at pH 8.2. From the photolabeling results, H146 was found to play a prominent role whilst H128 played little or no role in the allosteric binding. However, the remaining 4 mutants did not show a clear photolabeling pattern that was similar to either native HSA or H146A and, as a result, no firm conclusions can be made. An additional histidine mutant, H146I, was produced to confirm the results for H146A. A similar experiment using H146I showed that a benzene ring-like structure at position 146 is required for the allosteric ligand binding to occur.     

The binding of pyridoxal 5'-phosphate to human serum albumin

Most of the pyridoxal 5'-phosphate (PLP) in plasma is bound to protein, primarily albumin. Binding to protein is probably important in transporting PLP in the circulation and in regulating its metabolism. The binding of PLP to human serum albumin (HSA) was studied using absorption spectral analysis, equilibrium dialysis, and inhibition studies. The kinetics of the changes in the spectrum of PLP when mixed with an equimolar concentration of HSA at pH 7.4 followed a model for two-step consecutive binding with rate constants of 7.72 mM-1 min-1 and 0.088 min-1. The resulting PLP-HSA complex had absorption peaks at 338 and 414 nm and was reduced by potassium borohydride. The 414-nm peak is probably due to a protonated aldimine formed between PLP and HSA. The binding of PLP to bovine serum albumin (BSA) at equimolar concentrations at pH 7.4 occurred at about 10% the rate of its binding to HSA. The final PLP-BSA complex absorbed maximally at 334 nm and did not appear to be reduced with borohydride. Equilibrium dialysis of PLP and HSA indicated that there were more than one class of binding sites of HSA for PLP. There was one high affinity site with a dissociation constant of 8.7 microM and two or more other sites with dissociation constants of 90 microM or greater. PLP binding to HSA was inhibited by pyridoxal and 4-pyridoxic acid. It was not inhibited appreciably by inorganic phosphate or phosphorylated compounds. The binding of PLP to BSA was inhibited more than its binding to HSA by several compounds containing anionic groups. It is concluded that PLP binds differently to HSA than it does to BSA.     

Interaction of anesthetic supplement thiopental with human serum albumin

Thiopental (TPL) is a commonly used barbiturate anesthetic. Its binding with human serum albumin (HSA) was studied to explore the anesthetic-induced protein dysfunction. The basic binding interaction was studied by UV-absorption and fluorescence spectroscopy. An increase in the binding affinity (K) and in the number of binding sites (n) with the increasing albumin concentration was observed. The interaction was conformation-dependent and the highest for the F isomer of HSA, which implicates its slow elimination. The mode of binding was characterized using various thermodynamic parameters. Domain II of HSA was found to possess a high affinity binding site for TPL. The effect of micro-metal ions on the binding affinity was also investigated. The molecular distance, r, between donor (HSA) and acceptor (TPL) was estimated by fluorescence resonance energy transfer (FRET). Correlation between the stability of the TPL-N and TPL-F complexes and drug distribution is discussed. The structural changes in the protein investigated by circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy reflect perturbation of the albumin molecule and provide an explanation for the heterogeneity of action of this anesthetic.     

Characteristics of a nickel–albumin binding assay for assessment of myocardial ischaemia

Background: The aim of this study was to describe a method to measure ischaemia-induced alterations of the binding capacity of serum albumin to exogenous nickel.

Methods: We measured the levels of cardiac troponin I (cTnI), serum albumin, ischaemia-modified albumin (IMA) measured by a cobalt–albumin binding assay (CABA), and a nickel–albumin binding assay (NABA) in the following groups: myocardial infarction (n?=?32) and non-ischaemic chest pain (n?=?64).

Results: IMA, cTnI and NABA levels were higher in the myocardial infarction group. NABA presented a higher ability to discriminate myocardial ischaemia than CABA.

Conclusions: Patients with myocardial infarction have reduced nickel binding to human serum albumin, and NABA may have an important role as an early marker of myocardial ischaemia.     

A combined spectroscopic and crystallographic approach to probing drug–human serum albumin interactions

The displacement of probes that bind selectively to subdomains IIA or IIIA on human serum albumin (HSA) by competing compounds has been followed using fluorescence spectroscopy, and has therefore been used to assign a primary binding site for these compounds in the presence and absence of fatty acids. The crystal structures have also been solved for three compounds: a matched pair of carboxylic acids whose binding strength to HSA unexpectedly decreased as the lipophilicity increased; and a highly bound sulphonamide that appeared not to displace the probes in the displacement assay. The crystallography results support the findings from the fluorescence displacement assay. The results indicate that drug binding to subdomain IB might also be important location for certain compounds.     

Mutational analysis of the interaction between albumin-binding domain from streptococcal protein G and human serum albumin

Streptococcal protein G (SpG) is a bacterial cell surface receptor exhibiting affinity to both human immunoglobulin (IgG) and human serum albumin (HSA). Interestingly, the serum albumin and immunoglobulin-binding activities have been shown to reside at functionally and structurally separated receptor domains. The binding domain of the HSA-binding part has been shown to be a 46-residue triple alpha-helical structure, but the binding site to HSA has not yet been determined. Here, we have investigated the precise binding region of this bacterial receptor by protein engineering applying an alanine-scanning procedure followed by binding studies by surface plasmon resonance (SPR). The secondary structure as well as the HSA binding of the resulting albumin-binding domain (ABD) variants were analyzed using circular dichroism (CD) and affinity blotting. The analysis shows that the HSA binding involves residues mainly in the second alpha-helix.     

Interactions between 1-benzoyl-4-p-chlorophenyl thiosemicarbazide and serum albumin: investigation by fluorescence spectroscopy

The interactions between 1-benzoyl-4-p-chlorphenyl thiosemicarbazide (BCPT) and bovine serum albumin (BSA) or human serum albumin (HSA) have been studied by fluorescence spectroscopy. By the analysis of fluorescence spectrum and fluorescence intensity, it was showed that BCPT has a strong ability to quench the intrinsic fluorescence of both bovine serum albumin and human serum albumin through a static quenching procedure. The binding constants of BCPT with BSA or HSA were determined at different temperatures based on the fluorescence quenching results. The binding sites were obtained and the binding force were suggested to be mainly hydrophobic. The effect of common ions on the binding constants was also investigated. A new fluorescence spectroscopy assay of the proteins is presented. The linear range is 5.36-67.0 microg mL(-1) with recovery of 101.1% for BSA, and the linear range is 8.28-144.9 microg mL(-1) with recovery of 102.6% for HSA. Determination of the proteins in bovine serum or in human serum by this method gives results which are very close to those obtained by using Coomassie Brilliant Blue G-250 colorimetry. A practical method was proposed for the determination of BCPT in human serum samples.     

Species-dependent stereoselective drug binding to albumin: a circular dichroism study

Drug binding to albumins from different mammalian species was investigated to disclose evidence of species-dependent stereoselectivity in drug-binding processes and affinities. This aspect is important for evaluating the reliability of extrapolating distribution data among species. The circular dichroism (CD) signal induced by drug binding to the albumins [human serum albumin (HSA), bovine serum albumin (BSA), rat serum albumin (RSA), and dog serum albumin (DSA)] were measured and analyzed. The binding of selected drugs and metabolites to HSA significantly differed from the binding to the other albumins in terms of affinity and conformation of the bound ligands. In particular, phenylbutazone, a marker of site one on HSA, showed a higher affinity for binding to BSA with respect to RSA, HSA, and DSA, respectively. In the case of diazepam, a marker of site two on HSA, the affinity decreased in order from HSA to DSA, RSA, and BSA. The induced CD spectra were similar in terms of energy and band signs, suggesting almost the same conformation for the bound drug to the different albumins. Stereoselectivity was high for the binding of ketoprofen to HSA and RSA. A different sign was observed for the CD spectra induced by the drug to the two albumins because of the prevalence of a different conformation of the bound drug. Interestingly, the same induced CD spectra were obtained using either the racemic form or the (S)-enantiomer. Finally, significant differences were observed in the affinity of bilirubin, being highest for BSA, then decreasing for RSA, HSA, and DSA. A more complex conformational equilibrium was observed for bound bilirubin.     

Deciphering the binding of carbendazim (fungicide) with human serum albumin: A multi-spectroscopic and molecular modelling studies

Carbendazim is a benzimidazole fungicide used to control the fungal invasion. However, its exposure might lead to potential health problems. The present study evaluates the interaction of carbendazim (CAR) with human serum albumin (HSA) which is an important drug carrier protein and plays a very crucial role in the transportation of small molecules. A number of biophysical techniques were employed to investigate the binding of CAR with HSA. The increased UV-absorption of HSA on titrating with CAR suggests the formation of HSA–CAR complex and it could be due to the exposure of aromatic residues. The fluorescence study confirmed that CAR quenches the fluorescence of HSA and showed the static mode of quenching. CAR (50 µM) quenches around 56.14% of the HSA fluorescence. The quenching constant, binding constant, number of binding site and free energy change was calculated by fluorescence quenching experiment. Competitive displacement assay showed Sudlow’s site I as the primary binding site of CAR on HSA. The synchronous fluorescence study revealed the perturbation in the microenvironment around tyrosine and tryptophan residues upon binding of CAR to HSA. The circular dichroism results suggested that the binding of CAR to HSA altered its secondary structure. Molecular docking experiment demonstrated the binding of CAR to Sudlow’s site I of HSA. Docking studies suggested that the hydrogen bonding, van der Waals and pi-alkyl are playing role in the interaction of CAR with HSA. The study confirmed the conformational changes within HSA upon binding of CAR.     

Species differences in the interaction of serum albumin with antibiotics

Interaction of oxacillin, cloxacillin, dicloxacillin, phenoxymethylpenicillin, methicillin, nafcillin and benzylpenicillin with human serum albumin (HSA) was studied with flow microcalorimetry and differential scanning calorimetry. The measured thermodynamic parameters of complex formation between the penicillins and HSA were compared with similar characteristics of their binding to bovine serum albumin. It was shown that there were species differences between these two globular proteins in their interaction with the above antibiotics in relation to both the number of the biopolymer active sites and the nature of the molecular forces in the complex formation. The effect of the first bound molecule of oxacillin, cloxacillin, dicloxacillin, nafcillin, phenoxymethylpenicillin and benzylpenicillin on HSA conformation was observed. It was demonstrated that there was thermostabilization of HSA on its interaction with the above drugs with preserving cooperative nature of thermal denaturation of the complexes in relation to HSA melting.     

Interactions between antimalarial indolone-N-oxide derivatives and human serum albumin

The binding affinity of human serum albumin (HSA) to three antimalarial indolone-N-oxide derivatives, INODs, was investigated under simulated physiological conditions using fluorescence spectroscopy in combination with UV-vis absorption and circular dichroism (CD) spectroscopy. Analysis of fluorescence quenching data of HSA by these compounds at different temperatures using Stern-Volmer and Lineweaver-Burk methods revealed the formation of a ground state indolone-HSA complex with binding affinities of the order 10(4) M(-1). The thermodynamic parameters ΔG, ΔH, and ΔS, calculated at different temperatures, indicated that the binding reaction was endothermic and hydrophobic interactions play a major role in this association. The conformational changes of HSA were investigated qualitatively using synchronous fluorescence and quantitatively using CD. Site marker competitive experiments showed that the binding process took place primarily at site I (subdomain IIA) of HSA. The number of binding sites and the apparent binding constants were also studied in the presence of different ions.     

Analysis of the progesterone displacement of its human serum albumin binding site by beta-estradiol using biochromatographic approaches: effect of two salt modifiers

The mechanisms of (i) the binding of two sex-hormones (i.e. progesterone and beta-estradiol) to human serum albumin (HSA) and (ii) the progesterone displacement of its HSA binding cavity by beta-estradiol were studied by biochromatography using three different methods. In the first time, zonal elution method was used to prove the direct competition effect between the two sex-hormone. In the second time, the competition effect between beta-estradiol and progesterone to bound on the same HSA site was analysed by the competitive bi-Langmuir approach. Finally, the thermodynamic data of these two binding processes were studied. The Gibbs free energy value (Delta(approximately)G degrees) of the displacement equilibrium was negative demonstrating that beta-estradiol displaced progesterone of its HSA binding cavity. Moreover, the effect of two chloride modifiers (i.e. Na(+), Mg(2+)) on these two binding processes were analysed. Results showed that in the salt biological concentration ranges, the Mg(2+) cation enhanced strongly the bioavailable progesterone, whereas the Na(+) cation interacted slowly on the progesterone displacement of its HSA binding site by beta-estradiol. This study showed that it must be useful to carry out more in vivo test on the magnesium supplementation effect for women who suffer from estrogen dominance syndrome.     

Interaction of an organic selenium compound with human serum albumin

The interaction between 4,4'-diselenadibenzoic acid and human serum albumin (HSA) was investigated by fluorescence and absorption spectroscopy. The quenching mechanism of fluorescence of HSA by 4,4'-diselenadibenzoic acid was discussed. It is proved that the fluorescence quenching of HSA by 4,4'-diselenadibenzoic acid is a result of the formation of the HSA-4,4'-diselenadibenzoic acid complex. The binding sites number n, apparent corporation constant K, and corresponding thermodynamic parameters, deltaH(theta), deltaG(theta), and deltaS(theta) were calculated. Results indicate that the electrostatic interactions forces played major role in the reaction.