Familial dysalbuminemic byperthyroxinemia may result in altered warfarin pharmacokinetics

Two distinct genotypes that result in the amino acid substitutions R218P and R218H in subdomain 2A of human serum albumin (HSA) have been identified as the cause of familial dysalbuminemic hyperthyroxinemia (FDH). These substitutions increase the affinity of subdomain 2A for thyroxine by approximately 10-fold elevating plasma thyroxine levels in affected individuals. While many studies have examined the binding of thyroxine to FDH HSA, the binding of FDH HSA to drugs has not been widely investigated. The widely administered drug warfarin was selected as a model compound to study FDH HSA/drug interactions since it binds to subdomain 2A and its pharmacokinetics are dramatically influenced by HSA binding. Using two independent methods, fluorescence spectroscopy and equilibrium dialysis with radioactive warfarin, the binding of recombinant R218P, R218H, R218M and wild type HSA to warfarin was measured. Both methods showed an approximately 5-fold decrease in the affinity of R218P, R218H and R218M HSA for warfarin relative to wild type HSA. The Kd values determined by fluorescence spectroscopy for wild type, R218H, R218P and R218M HSA binding to warfarin were 1.35, 5.38, 5.61, and 8.34 microM, respectively. The values determined by equilibrium dialysis were 5.36, 29.5, 14.5, and 23.4 microM, respectively. Based on the above findings one would expect the free serum warfarin concentration in homozygous R218P and R218H FDH patients to be elevated about 5-fold, resulting in about a 5-fold reduction in the serum half-life of the drug.     

Analysis of tryptophan fluorescence lifetimes in a series of human serum albumin mutants with substitutions in subdomain 2 A

Tryptophan 214, the only tryptophan residue in human serum albumin, is located in the physiologically important subdomain 2A ligand binding site. In the present study the fluorescence lifetime of tryptophan 214 in the following human serum albumin (HSA) mutants with substitutions in subdomain 2A were determined: K195M, K199M, F211V, R218M, R218H, R218A, R222M, H242V, and R257M. An HSA mutant in which tryptophan was moved from subdomain 2A to subdomain 3A (W214L/Y411W) was also examined. Additionally, the fluorescence lifetime of tryptophan 214 in an HSA fragment consisting of subdomains 1A, 1B, and 2A (1A-1B-2A HSA) was determined. For those species expected to have the most dramatic changes in tryptophan microenvironment, W214L/Y411W and 1A-1B-2A HSA, clear changes in tryptophan lifetimes were observed. Significant changes were also seen for those species with mutations at position 218, which is next to tryptophan in the X-ray structure of HSA. However, significant changes were also observed for H242V and R257M, which contain substitutions at positions not immediately adjacent to tryptophan 214, highlighting the conformational flexibility of subdomain 2A.     

Probing the structure of the warfarin-binding site on human serum albumin using site-directed mutagenesis

The binding of warfarin to the following human serum albumin (HSA) mutants was examined: K195M, K199M, F211V, W214L, R218M, R222M, H242V, and R257M. Warfarin bound to human serum albumin (HSA) exhibits an intrinsic fluorescence that is approximately 10-fold greater than the corresponding signal for warfarin in aqueous solution. This property of the warfarin/HSA complex has been widely used to determine the dissociation constant for the interaction. In the present study, such a technique was used to show that specific substitutions in subdomain 2A altered the affinity of HSA for warfarin. The fluorescence of warfarin/mutant HSA complexes varied widely from the fluorescence of the warfarin/wild-type HSA complex at pH = 7.4, suggesting changes in the structure of the complex resulting from specific substitutions. The fluorescence of the warfarin/wild-type HSA complex increases about twofold as the pH is increased from 6.0 to 9.0 due to the neutral-to-base (N-B) transition, a conformational change that occurs in HSA as a function of pH. Changes in the fluorescence of warfarin/mutant HSA complexes as a function of pH suggests novel behavior for most HSA species examined. For the HSA mutants F211V and H242V, the midpoint of the N-B transition shifts from a wild-type pH of 7.8 to a pH value of 7.1-7.2.     

A dynamic model for bilirubin binding to human serum albumin

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.     

Interaction of prostaglandins and fatty acids with native and acetaldehyde-alkylated proteins

Using intrinsic and probe fluorescence, microcalorimetry and isotopic methods, the interactions of prostaglandins (PG) E2 and F2 alpha and some fatty acids with native and alkylated proteins (human serum albumin (HSA) and rat liver plasma membrane PG receptors), were studied. The fatty acid and PG interactions with human serum albumin (HSA) resulted in effective quenching of fluorescence of the probe, 1.8-anilinonaphthalene sulfonate (ANS), bound to the protein. Fatty acids competed with ANS for the binding sites; the efficiency of this process increased with an increase in the number of double bonds in the fatty acid molecule. PG induced a weaker fluorescence quenching of HSA-bound ANS and stabilized the protein molecule in a lesser degree compared to fatty acids. The sites of PG E2 and F2 alpha binding did not overlap with the sites of fatty acid binding on the HSA molecule. Nonenzymatic alkylation of HSA by acetaldehyde resulted in the abnormalities of binding sites for fatty acids and PG. Modification of the plasma membrane proteins with acetaldehyde sharply diminished the density of PG E2 binding sites without changing the association constants. Alkylation did not interfere with the parameters of PG F2 alpha binding to liver membrane proteins.     

Crystal structure and biological implications of a bacterial albumin binding module in complex with human serum albumin

Many bactericide species express surface proteins that interact with human serum albumin (HSA). Protein PAB from the anaerobic bacterium Finegoldia magna (formerly Peptostreptococcus magnus) represents one of these proteins. Protein PAB contains a domain of 53 amino acid residues known as the GA module. GA homologs are also found in protein G of group C and G streptococci. Here we report the crystal structure of HSA in complex with the GA module of protein PAB. The model of the complex was refined to a resolution of 2.7 A and reveals a novel binding epitope located in domain II of the albumin molecule. The GA module is composed of a left-handed three-helix bundle, and residues from the second helix and the loops surrounding it were found to be involved in HSA binding. Furthermore, the presence of HSA-bound fatty acids seems to influence HSA-GA complex formation. F. magna has a much more restricted host specificity compared with C and G streptococci, which is also reflected in the binding of different animal albumins by proteins PAB and G. The structure of the HSA-GA complex offers a molecular explanation to this unusually clear example of bacterial adaptation.     

Expression of a human serum albumin fragment (consisting of subdomains IA, IB, and IIA) and a study of its properties

Site-directed mutagenesis and a yeast expression system were used to synthesize a human serum albumin (HSA) fragment (amino acids 1-297). The HSA fragment (half HSA) was evaluated with a number of biophysical techniques and found to be similar to the corresponding region in wild-type HSA. Specifically, the circular dichroism spectra of half HSA and wild-type HSA were superimposable, indicating that the highly alpha-helical secondary structure of wild-type HSA is preserved in half HSA. Additionally, half HSA was partially reactive with a polyclonal antibody against authentic HSA. Half HSA, which contains subdomain IIA, had an affinity for thyroxine and several thyroxine analogs, similar to that observed previously for wild-type HSA. This study suggests that the production of recombinant HSA fragments will be useful for the study of HSA ligand interactions.     

Helix 6 of subdomain III A of human serum albumin is the region primarily photolabeled by ketoprofen, an arylpropionic acid NSAID containing a benzophenone moiety.

It is well known that the subdomain III A (site II) of human serum albumin (HSA) binds a variety of endogenous and exogenous substances. However, the nature of the microenvironment of the binding site remains unclear. Ketoprofen (KP), an arylpropionic acid NSAID which contains a benzophenone moiety, was used as a photoaffinity labeling agent to label the binding region. Subsequent CNBr cleavage of the photolabeled HSA revealed that the 11.6 kDa and 9.4 kDa fragments contained most of the incorporated radioactivity. Competition experiments showed that the 11.6 kDa fragment contains the common binding region for site II ligands. This fragment was redigested with Achromobacter lyticus protease I (AP-I) and the amino acid sequence of the photolabeled peptide was determined to be XCTESLVNRR, which corresponds to the sequence 476C-485K of HSA. The complete amino acid sequence of the corresponding AP-I digested HSA peptide encompasses residues 476 to 499, which form helices 5 and 6 of subdomain III A. The HSA-Myr X-ray crystallography data showed that helix 5 is involved to the least extent in ligand binding. A docking model provided further support that helix 6 represents the photolabeled region of KP.     

Olive phenols efficiently inhibit the oxidation of serum albumin-bound linoleic acid and butyrylcholine esterase

Background

Olive phenols are widely consumed in the Mediterranean diet and can be detected in human plasma. Here, the capacity of olive phenols and plasma metabolites to inhibit lipid and protein oxidations is investigated in two plasma models.

Methods

The accumulation of lipid oxidation products issued from the oxidation of linoleic acid bound to human serum albumin (HSA) by AAPH-derived peroxyl radicals is evaluated in the presence and absence of phenolic antioxidants. Phenol binding to HSA is addressed by quenching of the Trp214 fluorescence and displacement of probes (quercetin, dansylsarcosine and dansylamide). Next, the esterase activity of HSA-bound butyrylcholine esterase (BChE) is used as a marker of protein oxidative degradation.

Results

Hydroxytyrosol, oleuropein, caffeic and chlorogenic acids inhibit lipid peroxidation as well as HSA-bound BChE as efficiently as the potent flavonol quercetin. Hydroxycinnamic derivatives bind noncompetitively HSA subdomain IIA whereas no clear site could be identified for hydroxytyrosol derivatives.

General significance

In both models, olive phenols and their metabolites are much more efficient inhibitors of lipid and protein oxidations compared to vitamins C and E. Low postprandial concentrations of olive phenols may help to preserve the integrity of functional proteins and delay the appearance of toxic lipid oxidation products.     

Oxidation of Arg-410 promotes the elimination of human serum albumin

The effect of the oxidation of amino acid residues on albumin on its in vivo elimination was investigated using mutants and oxidized HSAs. The single-residue mutants (H146A, K199A, W214A, R218H, R410A, Y411A) and oxidized HSAs were produced by the recombinant DNA techniques and incubation with a metal ion-catalyzed oxidation (MCO) system for 12, 24, 48 or 72 h. Pharmacokinetics were evaluated in mice after labeling with 111In. Structural and functional properties were examined by several spectroscopic techniques. Time-dependent increase in carbonyl group content resulted in increase in the liver clearance of oxidized HSAs. Slight decreases in alpha-helical content as the result of oxidation was induced by the increases in accessible hydrophobic areas and the net negative charge on the HSA molecule. No significant change in the pharmacokinetics and structural properties was observed for the W214A, R218H and Y411A mutants, but the properties for the H146A, K199A and R410A mutants were affected (extent of effect: R410A > K199A > H146A). The liver clearance of these proteins is closely correlated to hydrophobicity (r = 0.929, P < 0.01) and the net charge of the proteins (r=0.930, P < 0.01). The rate of elimination of HSA is closely related to the hydrophobicity and net charge of the molecule. Further, the R410A mutants had a short half-life and structure similar to oxidized HSA after oxidation. Therefore, the modification of Arg-410 via oxidative stress may promote the elimination of HSA.     

Albumin-bound quercetin repairs vitamin E oxidized by apolipoprotein radicals in native HDL3 and LDL

In the minor fraction of HDL3 containing alpha-tocopherol (alphaTocOH), selective one-electron oxidation of Trp and Tyr residues of apolipoproteins A-I and A-II by *Br2- radical-anions produces the corresponding semioxidized species, TyrO* and *Trp. Repair of TyrO* by endogenous alphaTocOH generates the alpha-tocopheroxyl radical (alphaTocO*). Fast spectroscopic studies show that two populations representing 80% of alphaTocO* initially formed are repaired over several seconds with rate constants of 3.0 x 10(6) and 1.5 x 10(5) M-1 s-1 by quercetin bound to human serum albumin (HSA) at physiologically relevant concentration. Formation of HSA-bound quercetin radicals (*Qb) is observed. In the major fraction of HDL3 particles lacking alphaTocOH, TyrO* and *Trp are repaired by free and HSA-bound quercetin. In LDL particles which all contain alphaTocOH, alphaTocO* radicals are formed in the millisecond time scale by repair of TyrO* radicals produced in apolipoprotein B. Then, 75% of initial alphaTocO* are repaired over seconds by HSA-bound quercetin (rate constant: 2.0 x 10(6) M-1 s-1). HSA-bound quercetin can also repair *Trp radicals. In O2-saturated solutions, the fraction of alphaTocO* radicals (more than 50%) not repaired by superoxide radical-anions can be repaired by HSA-bound quercetin with formation of *Qb but to a much lesser extent in LDL than in HDL.     

Improvement of radioimmunoassays for prostaglandins in bovine blood plasma and their application to monitor reproductive functions

Efficient RIA procedures are required for determination of prostaglandins (PGF(2alpha), PGE(2), PGI(2) and their metabolites) in bovine blood plasma to elucidate their significance in reproductive endocrinology. A new rapid efficient prepurification was developed using commercial octadecyl silicagel cartridges. Prepurification is especially necessary for the determination of 13,14-dihydro-15-keto-PGE(2) (PGEM). After prepurification, PGEM was first converted into the more stable 13,14-dihydro-15-keto-PGA(2) (PGAM) and measured in a RIA-system for PGAM. For PGF(2alpha), 13,14-dihydro-15-keto-PGF(2alpha) (PGFM), PGE(2) and 6-keto-PGF(1alpha) direct tests using 50 mul plasma per tube were elaborated. The validity of the tests was monitored by high performance liquid chromatography radioimmunoassay (HPLC RIA ). Infusion studies using PGF(2alpha) and PGE(2) showed that about 10% of these hormones remained unmetabolized after the first passage through the lungs. The biological half life of the metabolites PGFM and PGEM in bovines was estimated to be 4 min. Thus, PGFM and PGEM measurements in the peripheral circulation reflect even short-term secretory changes of PGF(2alpha) and PGE(2). During the infusion of PGF(2alpha) the levels of progesterone decreased but were not affected by PGE(2). Both prostaglandins caused increased oxytocin secretion. In the cow peripartum first PGEM elevations were measured 5 to 8 d ante partum, whereas PGFM increased 1 to 2 d ante partum. Then both prostaglandins increased simultaneously until parturition. In the postpartal phase PGFM was higher than PGEM, and both prostaglandins remained elevated for several days. Prostacyclin levels remained unchanged during the peripartal period.     

Flavonoid-serum albumin complexation: determination of binding constants and binding sites by fluorescence spectroscopy

After a meal rich in plant products, dietary flavonols can be detected in plasma as serum albumin-bound conjugates. Flavonol-albumin binding is expected to modulate the bioavailability of flavonols. In this work, the binding of structurally different flavonoids to human and bovine serum albumins is investigated by fluorescence spectroscopy using three methods: the quenching of the albumin fluorescence, the enhancement of the flavonoid fluorescence, the quenching of the fluorescence of the quercetin-albumin complex by a second flavonoid. The latter method is extended to probes whose high-affinity binding sites are known to be located in one of the two major subdomains (warfarin and dansyl-L-asparagine for subdomain IIA, ibuprofen and diazepam for subdomain IIIA). Overall, flavonoids display moderate affinities for albumins (binding constants in the range 1-15 x 10(4) M(-1)), flavones and flavonols being most tightly bound. Glycosidation and sulfation could lower the affinity to albumin by one order of magnitude depending on the conjugation site. Despite multiple binding of both quercetin and site probes, it can be proposed that the binding of flavonols primarily takes place in subdomain IIA. Significant differences in affinity and binding location are observed for the highly homologous HSA and BSA.     

Unique,pH-dependent biphasic band shape of the visible circular dichroism of curcumin-serum albumin complex

Interaction between the plant derived polyphenolic type curcumin molecule having anticarcinogenic, antiinflammatory, and antioxidant activities, and human serum albumin was studied at different pH values by circular dichroism (CD) and electronic absorption spectroscopy. The weak, induced CD spectrum of curcumin-HSA complex measured at pH 7.4 in the visible spectral region shows striking changes upon alkalization; CD spectra collected between pH 7.7 and 9.3 exhibit characteristic, oppositely signed CD band pair according to the visible absorption band of HSA-bound curcumin. At 0.3 curcumin/HSA molar ratio, typical molar CD values are Delta epsilon (496.6nm)+40M(-1)cm(-1) and Delta epsilon (426.8nm)-40M(-1)cm(-1), respectively (pH 9.0, t=37 degrees C). The induced optical activity is attributed to a bent, right-handed chiral conformation of the HSA-bound curcumin molecule within which intramolecular exciton coupling occurs between the electric dipole transition moments of the dissymmetrically juxtaposed feruloyl chromophores. Deprotonation of phenolic OH group(s) of curcumin seems to be the reason leading to the conformational alteration of HSA-bound curcumin.     

Spectroscopic and computational exploration of hypoxanthine riboside interacting with plasma albumin

Hypoxanthine riboside (HXR) is a nucleoside essential for wobble base pairs to translate the genetic code. In this work, an absorption and luminescence study showed that HXR and human serum albumin (HSA) formed a new complex through hydrogen bonds and van der Waals forces at ground state. Fluorescence probe experiments indicated that HXR entered the first subdomain of domain II in HSA and was fixed by amino acid residues in site I defined by Sudlow, and after competing with a known site marker. The recognition interaction featured negative ΔH^?, ΔS^? and ΔG^? thermodynamic parameters. Fluorescence and circular dichroism spectra described the polarity of residues and α‐helix and β‐strand content changed because of HXR binding. The most rational structure for the HXR–HSA complex was recommended by the molecular docking method, in which the binding location, molecular orientation, adjacent amino acid residues, and hydrogen bonds were included. In addition, the influence of β‐cyclodextrin and some essential metal ions on the balance of the HSA–HXR system interaction was measured. The study gained comprehensive information on the transportation mechanism for HXR in blood, and was of great significance in understanding the theory of HXR biotransformation and in discussing its clinical in vivo half‐life.     

Bilirubin binding properties of pigeon serum albumin and its comparison with human serum albumin

Binding of bilirubin (BR) to pigeon serum albumin (PgSA) was studied by absorption, fluorescence and CD spectroscopy and results were compared with those obtained with human serum albumin (HSA). PgSA was found to be structurally similar to HSA as judged by near- and far-UV CD spectra. However, PgSA lacks tryptophan. Binding of BR to PgSA showed relatively weaker interaction compared to HSA in terms of binding affinity, induced red shift in the absorption spectrum of BR and CD spectral characteristics of BR-albumin complexes. Photoirradiation results of BR-albumin complexes also showed PgSA-bound BR more labile compared to HSA-bound BR.     

Interaction of ergosterol with bovine serum albumin and human serum albumin by spectroscopic analysis

This study was designed to examine the interactions of ergosterol with bovine serum albumin (BSA) and human serum albumin (HSA) under physiological conditions with the drug concentrations in the range of 2.99-105.88?μM and the concentration of proteins was fixed at 5.0?μM. The analysis of emission spectra quenching at different temperatures revealed that the quenching mechanism of HSA/BSA by ergosterol was the static quenching. The number of binding sites n and the binding constants K were obtained at various temperatures. The distance r between ergosterol and HSA/BSA was evaluated according to F?ster non-radioactive energy transfer theory. The results of synchronous fluorescence, 3D fluorescence, FT-IR, CD and UV-Vis absorption spectra showed that the conformations of HSA/BSA altered in the presence of ergosterol. The thermodynamic parameters, free energy change (ΔG), enthalpy change (ΔH) and entropy change (ΔS) for BSA-ergosterol and HSA-ergosterol systems were calculated by the van't Hoff equation and discussed. Besides, with the aid of three site markers (for example, phenylbutazone, ibuprofen and digitoxin), we have reported that ergosterol primarily binds to the tryptophan residues of BSA/HSA within site I (subdomain II A).     

Phenotypic characterization of the binding of tetracycline to human serum albumin

Because of the widely usage of the veterinary drug tetracycline (TC), its residue exist extensively in the environment (e.g., animal food, soils, surface water, and groundwater) and can enter human body, being potential harmful. Human serum albumin (HSA) is a major transporter for endogenous and exogenous compounds in vivo. The aim of this study was to examine the interaction of HSA with TC through spectroscopic and molecular modeling methods. The inner filter effect was eliminated to get accurate binding parameters. The site marker competition experiments revealed that TC binds to site II (subdomain IIIA) of HSA mainly through electrostatic interaction, illustrated by the calculated negative ΔH° and ΔS°. Furthermore, molecular docking was applied to define the specific binding sites, the results of which show that TC mainly interacts with the positively charged amino acid residues Arg 410 and Lys 414 predominately through electrostatic force, in accordance with the conclusion of thermodynamic analysis. The binding of TC can cause conformational and some microenvironmental changes of HSA, revealed by UV-visible absorption, synchronous fluorescence, and circular dichroism (CD) results. The accurate and full basic data in the work is beneficial to clarifying the binding mechanism of TC with HSA in vivo and understanding its effect on protein function during the blood transportation process.     

Chiral recognition of metalaxyl enantiomers by human serum albumin: evidence from molecular modeling and photophysical approach

Metalaxyl is an acylamine fungicide, belonging to the most widely known member of the amide group. This task is aimed to scrutinize binding region and spatial structural change of principal vector human serum albumin (HSA) complex with (R)-/(S)-metalaxyl by exploiting molecular modeling, steady-state and time-resolved fluorescence, and circular dichroism (CD) approaches. According to molecular modeling, (R)-metalaxyl is situated within subdomains IIA and IIIA and the affinity of site I with (R)-metalaxyl is greater than site II, whereas (S)-metalaxyl is only located at subdomain IIA and the affinity of (S)-metalaxyl with site I is superior compared with that with (R)-metalaxyl. This coincides with the competitive ligand binding, guanidine hydrochloride-induced unfolding of protein, and hydrophobic 8-anilino-1-naphthalenesulfonic acid experiments; the acting forces between (R)-/(S)-metalaxyl and HSA are hydrophobic, π-π interactions, and hydrogen bonds, as derived from molecular modeling. Fluorescence emission manifested that the complex of (R)-/(S)-metalaxyl to HSA is the formation of adduct with an affinity of 10(4) M(-1), which corroborates the time-resolved fluorescence that the static type was operated. Furthermore, the changes of far-UV CD spectra evidence the polypeptide chain of HSA partially unfolded after conjugation with (R)-/(S)-metalaxyl. Through this work, we envisage that it can offer central clues on the biodistribution, absorption, and bioaccumulation of (R)-/(S)-metalaxyl.     

Effect of ionizing radiation on the structure of human serum albumin. Effect of irradiation on the content of free amino groups and on the ability of HSA to be digested by proteinases

The effect of gamma-quanta on human serum albumin (HSA) solutions (1.6 mg/ml, borate buffer pH 7.45) in the air has been investigated. Using 2,4,6-trinitro-benzene-sulfonic acid it has been shown that 2-22 kGy radiation reduces the free amino groups content of HSA and increases its resistance to the hydrolytic effect of trypsin and pronase which is not influenced by the postirradiation exposure to heat. It is concluded that epsilon-NH2-groups of lysine residues are modified and firm cross-links are formed in HSA under the effect of radiation.