Characterization of thyroxine-albumin binding using high-performance affinity chromatography II. Comparison of the binding of thyroxine,triiodothyronines and related compounds at the warfarin and indole sites of human serum albumin

High-performance affinity chromatography was used to examine the binding of thyroid hormones and related compounds at the warfarin and indole sites of human serum albumin (HSA). This was studied by continuously applying l-triiodothyronine (l-T₃), l-reverse triiodothyronine (l-rT₃) or structural analogs of these compounds to an immobilized HSA column while making injections of site-specific probe molecules (i.e. R-warfarin and l-tryptophan). The results were compared with those obtained previously for l-thyroxine (l-T₄). Equilibrium association constants and thermodynamic parameters measured by this approach showed good agreement with previous models reported for l-T₄ and l-T₃ at their high-affinity sites on HSA. This data confirmed that the phenol groups of l-T₄ and l-T₃ played a significant role in the binding of these compounds at the indole site. Work performed at the warfarin site and with other solutes (e.g. l-rT₃) indicated that additional factors, such as interactions through the thyronine backbone or terminal amine and carboxyl groups of these compounds, could also be involved in the binding of thyroid hormones to HSA.     

Development of tryptophan-modified human serum albumin columns for site-specific studies of drug–protein interactions by high-performance affinity chromatography

Human serum albumin (HSA) is one of the main proteins involved in the binding of drugs and small solutes in blood or serum. This study examined the changes in chromatographic properties that occur for immobilized HSA following the chemical modification of HSA's lone tryptophan residue (Trp-214). Trp-214 was reacted with o-nitrophenylsulfenyl chloride, followed by immobilization of the modified protein and normal HSA onto separate silica-based HPLC supports. The binding properties of the modified and normal HSA were then analyzed and compared by using frontal analysis and zonal elution experiments employing R/S-warfarin and l-tryptophan as probe compounds for the warfarin and indole binding regions of HSA. The modified HSA was found to have the same number of binding sites as normal HSA for R-warfarin and l-tryptophan but lower association equilibrium constants for these test solutes. Zonal elution studies with R- and S-warfarin on the modified HSA column demonstrated the importance of Trp-214 in determining the stereoselective binding of HSA for these agents. These studies also indicated that tryptophan modification can alter HSA-based separations for chiral solutes.     

Chromatographic analysis of acetohexamide binding to glycated human serum albumin

Acetohexamide is a drug used to treat type II diabetes and is tightly bound to the protein human serum albumin (HSA) in the circulation. It has been proposed that the binding of some drugs with HSA can be affected by the non-enzymatic glycation of this protein. This study used high-performance affinity chromatography to examine the changes in acetohexamide–HSA binding that take place as the glycation of HSA is increased. It was found in frontal analysis experiments that the binding of acetohexamide to glycated HSA could be described by a two-site model involving both strong and weak affinity interactions. The average association equilibrium constant (K_a) for the high affinity interactions was in the range of 1.2–2.0 × 10⁵ M⁻¹ and increased in moving from normal HSA to HSA with glycation levels that might be found in advanced diabetes. It was found through competition studies that acetohexamide was binding at both Sudlow sites I and II on the glycated HSA. The K_a for acetohexamide at Sudlow site I increased by 40% in going from normal HSA to minimally glycated HSA but then decreased back to near-normal values in going to more highly glycated HSA. At Sudlow site II, the K_a for acetohexamide first decreased by about 40% and then increased in going from normal HSA to minimally glycated HSA and more highly glycated HSA. This information demonstrates the importance of conducting both frontal analysis and site-specific binding studies in examining the effects of glycation on the interactions of a drug with HSA.     

The interaction of zinc, nickel and cadmium with serum albumin and histidine-rich glycoprotein assessed by equilibrium dialysis and immunoadsorbent chromatography

Human serum albumin (HSA) has been shown to bind 2–3 mol of Zn²⁺, Ni²⁺, or Cd²⁺ per mole of protein with apparent dissociation constants (K_d) in the range of 10 μm. Rabbit histidine-rich glycoprotein (HRG) binds 13, 9, and 6 mol of Zn²⁺, Ni²⁺, and Cd²⁺ per mole of protein, respectively, with apparent K_ds also near 10 μm. However, the binding of metals by HRG exhibits positive cooperativity, so that the apparent K_ds may underestimate HRGs true affinity for metal ions. The relative affinities of HSA and HRG for metal ions were found to be Zn²⁺ > Ni²⁺ > Cd²⁺. In addition, histidine (a serum metal chelator) affected the binding of Ni²⁺ by both proteins but not that of Zn²⁺ or Cd²⁺. At physiological concentrations of HSA (250 μm), HRG (2.5 μm), and histidine (100 μm), HRG bound 36% of the Zn²⁺, 9% of the Ni²⁺, and 13% of the Cd²⁺ at a total metal concentration of 25 μm. Under the same conditions HSA held 37% of the Zn²⁺, 14% of the Ni²⁺, and 56% of the Cd²⁺. Thus, HSA appears to have a lower intrinsic affinity for the three metals than HRG but would be expected to bind a higher proportion of these metals in serum. A specific immunoadsorbent column was prepared and used to study the metal binding by HRG in serum directly. Both ⁶⁵Zn²⁺ and ⁶³Ni²⁺ were associated with HRG in aliquots of rabbit serum after incubation with the corresponding metal ion. This evidence indicates that HRG must be considered as a metal binding component of serum.     

Improved chromatographic performance of a modified human albumin based stationary phase

Derivatization of the free cys₃₄ in human serum albumin (HSA) anchored to a silica matrix has been performed by in situ reaction with ethacrynic acid. This modification, which is reported to occur under physiological conditions, gives rise in practice to a new column with different binding properties with respect to the column based on the native protein. Significant differences were observed in the binding of drugs known to bind to site I, (R)-(S)-warfarin and phenylbutazone, and to site II, 1,4-benzodiazepin-2-ones and nonsteroidal anti-inflammatory agents. In particular, the chromatographic retentions markedly decreased for most of the drugs, and, in the case of chiral compounds, significant differences were often observed in the behavior of the two enantiomers, with higher values of enantioselectivity obtained for some of the examined compounds. Furthermore, the noncovalent binding of ethacrynic acid to the protein modifies the binding properties of the albumin. Chirality 9:335–340, 1997. © 1997 Wiley-Liss, Inc.     

Evaluation of blood-brain barrier penetration and examination of binding to human serum albumin of 7-O-arylpiperazinylcoumarins as potential antipsychotic agents

The delivery of drugs to the brain is complicated by the multiple factors including low blood–brain barrier (BBB) passive permeability, active BBB efflux systems, and plasma protein binding. Thus, a detailed understanding of the transport of the new potent substances through the membranes is vitally important and their physico-chemical characteristics should be analyzed at first. This work presents an evaluation of drug likeness of eight 7-O-arylpiperazinylcoumarin derivatives with high affinity towards serotoninergic receptors 5-HT_1A and 5-HT_2A with particular analysis of the requirements for the CNS chemotherapeutics. The binding constants to human serum albumin (HSA) were determined at physiological pH using fluorescence spectroscopy, and then their mode of action was explained by analysis of theoretical HSA complexes. Dynamic simulation of systems allowed for reliable evaluation of the interaction strength. The analyzed coumarins were able to pass BBB, and they present good drug likeness properties. They showed high affinities to HSA (log K_Q = 5.3–6.0 which corresponds to −8.12 to −7.15 kcalmol⁻¹ of Gibbs free energy). The changes of the emission intensity upon binding to HSA were scrutinized showing the different mode of action for 4-phenylpiperazinylcoumarins. The values of computed Gibbs free energy and determined on the basis of experimentally obtained binding constants log K_Q coincide suggesting a good quality of the theoretical model. Overall the 8-acetyl-7-O-arylpiperazinyl-4-methylcoumarin derivatives represent valuable lead compounds to be further tested in various preclinical assays as a possible chemotherapeutics against CNS diseases. Studied coumarins can be metabolized by cytochrome P450 to aldehydes and hydroxy derivatives. The existence of other binding sites inside HSA than Sudlow’s site 1 was postulated. The longer aliphatic linker between coumarin and piperazine moieties favored binding to HSA in other than Sudlow site 1 pocket.     

Interaction of VO ion with human serum transferrin and albumin

The complexation of VO²⁺ ion with the high molecular mass components of the blood serum, human serum transferrin (hTf) and albumin (HSA), has been re-examined using EPR spectroscopy. In the case of transferrin, the results confirm those previously obtained, showing that VO²⁺ ion occupies three different binding sites, A, B₁ and B₂, distinguishable in the X-band anisotropic spectrum recorded in D₂O. With albumin the results show that a dinuclear complex (VO)₂^dHSA is formed in equimolar aqueous solutions or with an excess of protein; in the presence of an excess of VO²⁺, the multinuclear complex (VO)_x^mHSA is the prevalent species, where x = 5-6 indicates the equivalents of metal ion coordinated by HSA. The structure of the dinuclear species is discussed and the donor atoms involved in the metal coordination are proposed on the basis of the measured EPR parameters. Two different binding modes of albumin can be distinguished varying the pH, with only one species being present at the physiological value. The results show that the previously named “strong” site is not the N-terminal copper binding site, and some hypothesis on the metal coordination is discussed, with the ⁵¹V A_z values for the proposed donor sets obtained by DFT (density functional theory) calculations. Finally, preliminary results obtained in the ternary system VO²⁺/hTf/HSA are shown in order to determine the different binding strength of the two proteins. Due to the low VO²⁺ concentration used, the recording of the EPR spectra through the repeated acquisition of the weak signals is essential to obtain a good signal to noise ratio in these systems.     

Study the interactions between human serum albumin and two antifungal drugs: Fluconazole and its analogue DTP

Binding affinities of fluconazole and its analogue 2-(2,4-dichlorophenyl)-1,3-di(1H-1,2,4-triazol-yl)-2-propanol (DTP) to human serum albumin (HSA) were investigated under approximately human physiological conditions. The obtained result indicated that HSA could generate fluorescent quenching by fluconazole and DTP because of the formation of non-fluorescent ground-state complexes. Binding parameters calculated from the Stern–Volmer and the Scatchard equations showed that fluconazole and DTP bind to HSA with binding affinities of the order 10⁴ L/mol. The thermodynamic parameters revealed that the binding was characterized by negative enthalpy and positive entropy changes, suggesting that the binding reaction was exothermic. Hydrogen bonds and hydrophobic interaction were found to be the predominant intermolecular forces stabilizing the drug–protein. The effect of metal ions on the binding constants of fluconazole–HSA complex suggested that the presence of Mg²⁺ and Zn²⁺ ions could decrease the free drug level and extend the half-life in the systematic circulation. Docking experiments revealed that fluconazole and DTP binds in HSA mainly by hydrophobic interaction with the possibility of hydrogen bonds formation between the drugs and the residues Arg 222, Lys 199 and Lys 195 in HSA.     

Interactions of arene-Ru(II)-chloroquine complexes of known antimalarial and antitumor activity with human serum albumin (HSA) and transferrin

The interactions of π-arene-Ru(II)-chloroquine complexes with human serum albumin (HSA), apotransferrin and holotransferrin have been studied by circular dichroism (CD) and UV-Visible spectroscopies, together with isothermal titration calorimetry (ITC). The data for [Ru(η⁶-p-cymene)(CQ)(H₂O)Cl]PF₆ (1), [Ru(η⁶-benzene)(CQ)(H₂O)Cl]PF₆ (2), [Ru(η⁶-p-cymene)(CQ)(H₂O)₂][PF₆]₂ (3), [Ru(η⁶-p-cymene)(CQ)(en)][PF₆]₂ (4), [Ru(η⁶-p-cymene)(η⁶-CQDP)][BF₄]₂ (5) (CQ: chloroquine; DP: diphosphate; en: ethylenediamine), in comparison with CQDP and [Ru(η⁶-p-cymene)(en)Cl][PF₆] (6) as controls demonstrate that 1, 2, 3, and 5, which contain exchangeable ligands, bind to HSA and to apotransferrin in a covalent manner. The interaction did not affect the α-helical content in apotransferrin but resulted in a loss of this type of structure in HSA. The binding was reversed in both cases by a decrease in pH and in the case of the Ru-HSA adducts, also by addition of chelating agents. A weaker interaction between complexes 4 and 6 and HSA was measured by ITC but was not detectable spectroscopically. No interactions were observed for complexes 4 and 6 with apotransferrin or for CQDP with either protein. The combined results suggest that the arene-Ru(II)-chloroquine complexes, known to be active against resistant malaria and several lines of cancer cells, also display a good transport behavior that makes them good candidates for drug development.     

A new ditopic GdIII complex functionalized with an adamantyl moiety as a versatile building block for the preparation of supramolecular assemblies

A dimeric GdAAZTA-like complex (AAZTA is 6-amino-6-methylperhydro-1,4-diazepinetetraacetic acid) bearing an adamantyl group (Gd₂ L1) able to form strong supramolecular adducts with specific hosts such as β-cyclodextrin (β-CD), poly-β-CD, and human serum albumin (HSA) is reported. The relaxometric properties of Gd₂ L1 were investigated in aqueous solution by measuring the ¹H relaxivity as a function of pH, temperature, and magnetic field strength. The relaxivity of Gd₂ L1 (per Gd atom) at 40 MHz and 298 K is 17.6 mM^?1 s^?1, a value that remains almost constant at higher fields owing to the great compactness and rigidity of the bimetallic chelate, resulting in an ideal value for the rotational correlation time for high-field MRI applications (1.5–3.0 T). The noncovalent interaction of Gd₂ L1 with β-CD, poly-β-CD, and HSA and the relaxometric properties of the resulting host–guest adducts were investigated using ¹H relaxometric methods. Relaxivity enhancements of 29 and 108 % were found for Gd₂ L1–β-CD and Gd₂ L1–poly-β-CD, respectively. Binding of Gd₂ L1 to HSA (K _A = 1.2 × 10⁴ M^?1) results in a remarkable relaxivity of 41.4 mM^?1 s^?1 for the bound form (+248 %). The relaxivity is only limited by the local rotation of the complex within the binding site, which decreases on passing from Gd₂ L1–β-CD to Gd₂ L1–HSA. Finally, the applicability of Gd₂ L1 as tumor-targeting agent through passive accumulation of the HSA-bound adduct was evaluated via acquisition of magnetic resonance images at 1 T of B16-tumor-bearing mice. These experiments indicate a considerable signal enhancement (+160 %) in tumor after 60 min from the injection and a very low hepatic accumulation.     

The investigation of structural and thermosensitive properties of new phosphazene derivative bearing glycol and aminoalcohol

Three isomers (nongeminal cis-2,4,6 (2); nongeminal trans-2,4,6 (3); geminal 2,2,4 (4)) were isolated from the reaction of hexachlorocyclotriphosphazatriene (trimer) (1) with diethylene glycol monobutyl ether (DEGBE). The substitution reactions of cis-tris isomer (2) with 3-amino-1-propanol were investigated under different solutions conditions to provide amphiphilic phosphazene (5). All of compounds were characterized by using elemental analysis, ³¹P NMR and mass spectroscopies. Thermosensitive properties of compound 5 were studied. The compound 5 is soluble in both water and organic media. This indicates that compound 5 is an amphiphilic molecule. Concentration-dependent LCST (Lower Critical Solution Temperature) behavior of 5 was measured in water. Compound 5 exhibited a reversible and thermosensitive phase transition in aqueous medium, from soluble to insoluble states. Compound 5 showed LCST at 37 °C (for 7 wt.% concentration) which is near to body temperature.     

Analysis of the structure and dynamics of human serum albumin

A computational approach to designing a peptide-based ligand for the purification of human serum albumin (HSA) was undertaken using molecular docking and molecular dynamics (MD) simulation. A three-step procedure was performed to design a specific ligand for HSA. Based on the candidate pocket structure of HSA (warfarin binding site), a peptide library was built. These peptides were then docked into the pocket of HSA using the GOLD program. The GOLDscore values were used to determine the affinity of peptides for HSA. Consequently, the dipeptide Trp–Trp, which shows a high GOLDscore value, was selected and linked to a spacer arm of Lys[CO(CH₂)₅NH] on the surface of ECH-lysine sepharose 4 gel. For further evaluation, the Autodock Vina program was used to dock the linked compound into the pocket of HSA. The docking simulation was performed to obtain a first guess of the binding structure of the spacer–Trp–Trp–HSA complex and subsequently analyzed by MD simulations to assess the reliability of the docking results. These MD simulations indicated that the ligand–HSA complex remains stable, and water molecules can bridge between the ligand and the protein by hydrogen bonds. Finally, absorption spectroscopic studies were performed to illustrate the appropriateness of the binding affinity of the designed ligand toward HSA. These studies demonstrate that the designed dipeptide can bind preferentially to the warfarin binding site. Graphical Abstract

Three-step computational approach to the design of a dipeptide ligand for human serum albumin purification exploiting structure-based docking and molecular dynamics simulation     

Probing the Binding Sites of Antibiotic Drugs Doxorubicin and N-(trifluoroacetyl) Doxorubicin with Human and Bovine Serum Albumins

We located the binding sites of doxorubicin (DOX) and N-(trifluoroacetyl) doxorubicin (FDOX) with bovine serum albumin (BSA) and human serum albumins (HSA) at physiological conditions, using constant protein concentration and various drug contents. FTIR, CD and fluorescence spectroscopic methods as well as molecular modeling were used to analyse drug binding sites, the binding constant and the effect of drug complexation on BSA and HSA stability and conformations. Structural analysis showed that doxorubicin and N-(trifluoroacetyl) doxorubicin bind strongly to BSA and HSA via hydrophilic and hydrophobic contacts with overall binding constants of K _DOX-BSA = 7.8 (±0.7)×10³ M⁻¹, K _FDOX-BSA = 4.8 (±0.5)×10³ M⁻¹ and K _DOX-HSA = 1.1 (±0.3)×10⁴ M⁻¹, K _FDOX-HSA = 8.3 (±0.6)×10³ M⁻¹. The number of bound drug molecules per protein is 1.5 (DOX-BSA), 1.3 (FDOX-BSA) 1.5 (DOX-HSA), 0.9 (FDOX-HSA) in these drug-protein complexes. Docking studies showed the participation of several amino acids in drug-protein complexation, which stabilized by H-bonding systems. The order of drug-protein binding is DOX-HSA > FDOX-HSA > DOX-BSA > FDOX>BSA. Drug complexation alters protein conformation by a major reduction of α-helix from 63% (free BSA) to 47–44% (drug-complex) and 57% (free HSA) to 51–40% (drug-complex) inducing a partial protein destabilization. Doxorubicin and its derivative can be transported by BSA and HSA in vitro.     

Study on the interaction of a copper(II) complex containing the artificial sweetener aspartame with human serum albumin

A copper(II) complex containing aspartame (APM) as ligand, Cu(APM)₂Cl₂·2H₂O, was synthesized and characterized. In vitro binding interaction of this complex with human serum albumin (HSA) was studied at physiological pH. Binding studies of this complex with HSA are useful for understanding the Cu(APM)₂Cl₂·2H₂O–HSA interaction mechanism and providing guidance for the application and design of new and more efficient artificial sweeteners drive. The interaction was investigated by spectrophotometric, spectrofluorometric, competition experiment and circular dichroism. Hyperchromicity observed in UV absorption band of Cu(APM)₂Cl₂·2H₂O. A strong fluorescence quenching reaction of HSA to Cu(APM)₂Cl₂·2H₂O was observed and the binding constant (K_f) and corresponding numbers of binding sites (n) were calculated at different temperatures. Thermodynamic parameters, enthalpy change (?H) and entropy change (?S) were calculated to be ?458.67 kJ mol^?1 and ?1,339 J mol^?1 K^?1 respectively. According to the van’t Hoff equation, the reaction is predominantly enthalpically driven. In conformity with experimental results, we suggest that Cu(APM)₂Cl₂·2H₂O interacts with HSA. In comparison with previous study, it is found that the Cu(II) complex binds stronger than aspartame.     

Characterisation of the binding of digitoxin and acetyldigitoxin to human serum albumin by high-performance affinity chromatography

Zonal elution and high-performance affinity chromatography were used to examine interactions of the drugs digitoxin and acetyldigitoxin with the protein human serum albumin (HSA). This was done by injecting small amounts of digitoxin and acetyldigitoxin onto an immobilized HSA column in the presence of mobile phases that contained various concentrations of digitoxin, acetyldigitoxin or other solutes as competing agents. A fixed concentration of β-cyclodextrin was also present in the mobile phase as a solubilising agent. It was found that digitoxin and acetyldigitoxin each had strong interactions at a single common binding site on HSA, but with slightly different equilibrium constants for this region. Neither compound showed any competition with warfarin or L-tryptophan, which were used as probes for binding at the warfarin-azapropazone and indole-benzodiazepine sites of HSA. These results confirmed the presence of a separate binding region on HSA for digitoxin-related compounds.     

Molecular Structure-Affinity Relationship of Bufadienolides and Human Serum Albumin In Vitro and Molecular Docking Analysis

The development of bufadienolides as anti-tumor agents is limited due to poor pharmacokinetic properties regarding drug half-lives and toxicity in vivo. These serious factors might be improved by increasing the drug/albumin-binding ratio. This study therefore investigated the relationship between the structural properties of nine bufadienolides and their affinities for human serum albumin (HSA) by a fluorescence spectroscopy-based analysis and molecular docking. Fluorescence quenching data showed that the interaction of each bufadienolide with HSA formed a non-fluorescent complex, while thermodynamic parameters revealed negative ΔS and ΔH values, corresponding to changes in enthalpy and entropy, respectively. The structural differences between the various bufadienolides markedly influenced their binding affinity for HSA. With the exception of a C = O bond at the C12 position that decreased the binding affinity for HSA, other polar groups tended to increase the affinity, especially a hydroxyl (OH) group at assorted bufadienolide sites. The rank order of binding affinities for drugs with tri-hydroxyl groups was as follows: 11-OH > 5-OH > 16-OH; in addition, 16-acetoxy (OAc), 10-aldehyde and 14-epoxy constituents notably enhanced the binding affinity. Among these groups, 11-OH and 16-acetyl were especially important for a seamless interaction between the bufadienolides and HSA. Furthermore, molecular docking analysis revealed that either an 11-OH or a 16-OAc group spatially close to a five-membered lactone ring significantly facilitated the anchoring of these compounds within site I of the HSA pocket via hydrogen bonding (H-bonding) with Tyr150 or Lys199, respectively. In summary, bufadienolide structure strongly affects binding with HSA, and 11-OH or 16-OAc groups improve the drug association with key amino acid residues. This information is valuable for the prospective development of bufadienolides with improved pharmacological profiles as novel anti-tumor drugs.     

Molecular Interaction Studies of Trimethoxy Flavone with Human Serum Albumin

Background

Human serum albumin (HSA) is the most abundant protein in blood plasma, having high affinity binding sites for several endogenous and exogenous compounds. Trimethoxy flavone (TMF) is a naturally occurring flavone isolated from Andrographis viscosula and used in the treatment of dyspepsia, influenza, malaria, respiratory functions and as an astringent and antidote for poisonous stings of some insects.

Methodology/Principal Findings

The main aim of the experiment was to examine the interaction between TMF and HSA at physiological conditions. Upon addition of TMF to HSA, the fluorescence emission was quenched and the binding constant of TMF with HSA was found to be K_TMF = 1.0±0.01×10³ M⁻¹, which corresponds to −5.4 kcal M⁻¹ of free energy. Micro-TOF Q mass spectrometry results showed a mass increase of from 66,513 Da (free HSA) to 66,823 Da (HAS +Drug), indicating the strong binding of TMF with HSA resulting in decrease of fluorescence. The HSA conformation was altered upon binding of TMF to HSA with decrease in α-helix and an increase in β-sheets and random coils suggesting partial unfolding of protein secondary structure. Molecular docking experiments found that TMF binds strongly with HSA at IIIA domain of hydrophobic pocket with hydrogen bond and hydrophobic interactions. Among which two hydrogen bonds are formed between O (19) of TMF to Arg 410, Tyr 411 and another one from O (7) of TMF to Asn 391, with bond distance of 2.1 Å, 3.6 Å and 2.6 Å, respectively.

Conclusions/Significance

In view of the evidence presented, it is imperative to assign a greater role of HSA''s as a carrier molecule for many drugs to understand the interactions of HSA with TMF will be pivotal in the design of new TMF-inspired drugs.     

Resveratrol Binding to Human Serum Albumin

Abstract

Resveratrol (Res), a polyphenolic compound found largely in the skin of red grape and wine, exhibits a wide range of pharmaceutical properties and plays a role in prevention of human cardiovascular diseases [Pendurthi et al., Arterioscler. Thromb. Vasc. Biol. 19, 419–426 (1999)]. It shows a strong affinity towards protein binding and used as inhibitor for cyclo- oxygenase and ribonuclease reductase. The aim of this study was to examine the interaction of resveratrol with human serum albumin (HSA) in aqueous solution at physiological conditions, using a constant protein concentration (0.3 mM) and various pigment contents μM to mM). FTIR, UV-Visible, CD, and fluorescence spectroscopic methods were used to determine the resveratrol binding mode, the binding constant and the effects of pigment complexation on protein secondary structure.

Structural analysis showed that resveratrol bind non-specifically (H-bonding) via polypeptide polar groups with overall binding constant of K_Res = 2.56× 10⁵ M^?1. The protein secondary structure, analysed by CD spectroscopy, showed no major alterations at low resveratrol concentrations (0.125 mM), whereas at high pigment content (1 mM), major increase of α-helix from 57% (free HSA) to 62% and a decrease of β-sheet from 10% (free HSA) to 7% occurred in the resveratrol-HSA complexes. The results indicate a partial stabilization of protein secondary structure at high resveratrol content.     

Warfarin modulates the nitrite reductase activity of ferrous human serum heme–albumin

Human serum heme–albumin (HSA–heme–Fe) displays reactivity and spectroscopic properties similar to those of heme proteins. Here, the nitrite reductase activity of ferrous HSA–heme–Fe [HSA–heme–Fe(II)] is reported. The value of the second-order rate constant for the reduction of $ {\text{NO}}_{2}^{ - } $ to NO and the concomitant formation of nitrosylated HSA–heme–Fe(II) (i.e., k _on) is 1.3 M^?1 s^?1 at pH 7.4 and 20 °C. Values of k _on increase by about one order of magnitude for each pH unit decrease between pH 6.5 to 8.2, indicating that the reaction requires one proton. Warfarin inhibits the HSA–heme–Fe(II) reductase activity, highlighting the allosteric linkage between the heme binding site [also named the fatty acid (FA) binding site 1; FA1] and the drug-binding cleft FA2. The dissociation equilibrium constant for warfarin binding to HSA–heme–Fe(II) is (3.1 ± 0.4) × 10^?4 M at pH 7.4 and 20 °C. These results: (1) represent the first evidence for the $ {\text{NO}}_{2}^{ - } $ reductase activity of HSA–heme–Fe(II), (2) highlight the role of drugs (e.g., warfarin) in modulating HSA(–heme–Fe) functions, and (3) strongly support the view that HSA acts not only as a heme carrier but also displays transient heme-based reactivity.     

Supramolecular interaction of 6-shogaol,a therapeutic agent of Zingiber officinale with human serum albumin as elucidated by spectroscopic,calorimetric and molecular docking methods

Background6-Shogaol, one of the main bioactive constituents of Zingiber officinale has been shown to possess various therapeutic properties. Interaction of a therapeutic compound with plasma proteins greatly affects its pharmacokinetic and pharmacodynamic properties.PurposeThe present investigation was undertaken to characterize the interaction between 6-shogaol and the main in vivo transporter, human serum albumin (HSA).MethodsVarious binding characteristics of 6-shogaol–HSA interaction were studied using fluorescence spectroscopy. Thermal stability of 6-shogaol–HSA system was determined by circular dichroism (CD) and differential scanning calorimetric (DSC) techniques. Identification of the 6-shogaol binding site on HSA was made by competitive drug displacement and molecular docking experiments.ResultsFluorescence quench titration results revealed the association constant, K_a of 6-shogaol–HSA interaction as 6.29 ± 0.33 × 10⁴ M⁻¹ at 25 ºC. Values of the enthalpy change (−11.76 kJ mol⁻¹) and the entropy change (52.52 J mol⁻¹ K⁻¹), obtained for the binding reaction suggested involvement of hydrophobic and van der Waals forces along with hydrogen bonds in the complex formation. Higher thermal stability of HSA was noticed in the presence of 6-shogaol, as revealed by DSC and thermal denaturation profiles. Competitive ligand displacement experiments along with molecular docking results suggested the binding preference of 6-shogaol for Sudlow's site I of HSA.ConclusionAll these results suggest that 6-shogaol binds to Sudlow's site I of HSA through moderate binding affinity and involves hydrophobic and van der Waals forces along with hydrogen bonds.