Spectroscopic and Molecular Modeling Studies on the Interaction Between a Fluorine-Containing Triazole Derivative and Human Serum Albumin

The interaction between 4-(4-fluorobenzylideneamino)-5-propyl-4H-1,2,4-triazole-3-thiol (FBTZ) and human serum albumin (HSA) under simulative physiological conditions was investigated by fluorescence, UV–vis absorption and circular dichroism (CD) spectroscopy as well as molecular modeling method. Fluorescence spectroscopic data showed that the fluorescence quenching of HSA was a result of the formation of FBTZ–HSA complex. According to the modified Stern–Volmer equation, the effective quenching constants (K _a) of FBTZ to HSA were obtained at three different temperatures. The enthalpy change (ΔH) and entropy change (ΔS) were calculated on the basis of van′t Hoff equation, and the results showed that hydrogen-bonding and van der Waals forces were the dominant intermolecular forces to stabilize the complex. Site marker competitive replacement experiments demonstrated that the binding of FBTZ to HSA primarily took place in sub-domain IIA (Sudlow’s site I). The binding distance (r) between FBTZ and the tryptophan residue of HSA was estimated according to the theory of fluorescence resonance energy transfer. The conformational investigation showed that the presence of FBTZ induced some changes of secondary structure of HSA. Molecular modeling study further confirmed the binding mode obtained by experimental study.     

Human serum albumin coordinates Cu(II) at its N-terminal binding site with 1 pM affinity

The conditional stability constant at pH 7.4 for Cu(II) binding at the N-terminal site (NTS) of human serum albumin (HSA) was determined directly by competitive UV–vis spectroscopy titrations using nitrilotriacetic acid (NTA) as the competitor in 100 mM NaCl and 100 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (Hepes). The log K _NTS^c value of 12.0 ± 0.1 was determined for HSA dissolved in 100 mM NaCl. A false log log K _NTS^c value of 11.4 ± 0.1 was obtained in the 100 mM Hepes buffer, owing to the formation of a ternary Cu(NTA)(Hepes) complex. The impact of the picomolar affinity of HSA for Cu(II) on the availability of these ions in neurodegenerative disorders is briefly discussed.     

Contrast agents for magnetic resonance angiographic applications: 1H and 17O NMR relaxometric investigations on two gadolinium(III) DTPA-like chelates endowed with high binding affinity to human serum albumin

N,N′,N″,N‴ -pentaacetic acid) bearing different substituents for binding to human serum albumin (HSA) are compared. In spite of the structural differences of the recognition synthon and of the residual electric charge, the two chelates display an analogous binding affinity for the serum protein. Upon formation of the adducts with HSA, the exchange rates of the coordinated water appear slowed down by an amount corresponding to ca. 50% of the rates found for the free complexes. The relaxivity of [Gd(BOM)₃DTPA (H₂O)]^2 − is significantly higher than that of MS-325 either in the free complex or in the macromolecular adduct. Finally, the effect of pH on the stability of the HSA adducts and on the values of their relaxivities has been investigated. Received: 11 June 1999 / Accepted: 15 September 1999     

<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> Phosphoenolpyruvate Carboxykinase: The Relevance of Glu299 and Leu460 for Nucleotide Binding

A homology model of Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase (ATP + oxaloacetate ⇄ ADP + PEP + CO₂) in complex with its substrates shows that the isobutyl group of Leu460 is in close proximity to the adenine ring of the nucleotide, while the carboxyl group of Glu299 is within hydrogen-bonding distance of the ribose 2′OH. The Leu460Ala mutation caused three-fold and seven-fold increases in the K _m for ADPMn⁻ and ATPMn²⁻, respectively, while the Glu299Ala mutation had no effect. Binding studies showed losses of approximately 2 kcal mol⁻¹ in the nucleotide binding affinity due to the Leu460Ala mutation and no effect for the Glu299Ala mutation. PEP carboxykinase utilized 2′deoxyADP and 2′deoxyATP as substrates with kinetic and equilibrium dissociation constants very similar to those of ADP and ATP, respectively. These results show that the hydrophobic interaction between Leu460 and the adenine ring of the nucleotide significantly contributed to the nucleotide affinity of the enzyme. The 2′deoxy nucleotide studies and the lack of an effect of the Glu299Ala mutation in nucleotide binding suggest that the possible hydrogen bond contributed by Glu299 and the ribose 2′OH group may not be relevant for nucleotide binding.     

Parenteral Delivery of HPβCD: Effects on Drug-HSA Binding

It is thought that cyclodextrins, such as 2-hydroxypropyl-β-cyclodextrin (HPβCD), will at high concentration affect pharmacokinetics of drugs through competitive binding with plasma proteins. Albumin is the major component of plasma proteins responsible for plasma protein binding. The purpose of this study was to evaluate in vitro the competitive binding of drugs between human serum albumin (HSA) and HPβCD in isotonic pH 7.4 phosphate buffer saline solution (PBS) at ambient temperature. Eight model drugs were selected based on their physicochemical properties and ability to form complexes with HSA and HPβCD. The drug/HPβCD stability constants (K _1:1) were determined by the phase-solubility method and HSA/HPβCD competitive binding determined by an equilibrium dialysis method. Protein binding of drugs that are both strongly protein bound and have high affinity to HPβCD (i.e., have high K _1:1 value) is most likely to be affected by parenterally administered HPβCD. However, this in vitro study indicates that even for those drugs single parenteral dose of HPβCD has to be as high as 70 g to have detectable effect on their protein binding. Weakly protein bound drugs and drugs with low affinity towards HPβCD are insensitive to the cyclodextrin presence regardless their lipophilic properties.     

Effect of Human Serum Albumin on the Kinetics of 4-Methylumbelliferyl-β-D-<Emphasis Type="Italic">N-N</Emphasis>′-<Emphasis Type="Italic">N</Emphasis>″ Triacetylchitotrioside Hydrolysis Catalyzed by Hen Egg White Lysozyme

The effect of human serum albumin (HSA) addition on the rate of hydrolysis of the synthetic substrate 4-methylumbelliferyl-β-D-N-N′-N″ triacetylchitotrioside ((NAG)₃-MUF) catalyzed by hen egg white lysozyme has been measured in aqueous solution (citrate buffer 50 mM pH = 5.2 at 37 °C). The presence of HSA leads to a decrease in the rate of the process. The reaction follows a Michaelis–Menten mechanism under all the conditions employed. The catalytic rate constant decreases tenfold when the albumin concentration increases, while the Michaelis constant remains almost constant in the albumin concentration range employed. Ultracentrifugation experiments indicate that the main origin of the observed variation in the kinetic behavior is related to the existence of an HSA–lysozyme interaction. Interestingly, the dependence of the catalytic rate constant with albumin concentration parallels the decrease of the free enzyme concentration. We interpret these results in terms of the presence in the system of two enzyme populations; namely, the HSA associated enzyme which does not react and the free enzyme reacting as in the absence of albumin. Other factors such as association of the substrate to albumin or macromolecular crowding effects due to the presence of albumin are discarded. Theoretical modeling of the structure of the HSA–lysozyme complex shows that the Glu35 and Asp52 residues located in the active site of lysozyme are oriented toward the HSA surface. This conformation will inactivate lysozyme molecules bound to HSA.     

Gd(III) complexes as contrast agents for magnetic resonance imaging: a proton relaxation enhancement study of the interaction with human serum albumin

 The non-covalent interaction between human serum albumin (HSA) and DOTA-like Gd(III) complexes containing hydrophobic benzyloxymethyl (BOM) substituents has been thoroughly investigated by measuring the solvent proton relaxation rates of their aqueous solutions. The binding association constants (K _A) to HSA are directly related to the number of hydrophobic substituents present on the surface of the complexes. Furthermore, an estimation of ΔH° and ΔS° has been obtained by the temperature dependence of K _A. Assays performed with the competitor probes warfarin and ibuprofen established that the complexes interact with HSA through two nearly equivalent binding sites located in the subdomains IIA and IIIA of the protein. Strong relaxation enhancements, promoted by the formation of slowly tumbling paramagnetic adducts, have been measured at 20 MHz for complexes containing two and three hydrophobic substituents. The macromolecular adduct with the latter species has a relaxivity of 53.2±0.7 mM^–1 s^–1, which represents the highest value so far reported for a Gd(III) complex. The temperature dependence of the relaxivity for the paramagnetic adducts with HSA indicates long exchange lifetimes for the water molecules dipolarly interacting with the paramagnetic centre. This is likely to be related to the formation, upon hydrophobic interaction of the complexes with HSA, of a clathrate-like, second-coordination-sphere arrangement of water molecules. Besides affecting the dissociative pathway of the coordinated water molecule, this water arrangement may itself significantly contribute to enhancement of the bulk solvent relaxation rate. Received: 6 November 1995 / Accepted: 17 April 1996     

Interaction of human serum albumin with 10-hydroxycamptothecin: spectroscopic and molecular modeling studies

In this work, fluorescence spectroscopy in combination with circular dichroism spectroscopy and molecular modeling was employed to investigate the binding of 10-hydroxycamptothecin (HCPT) to human serum albumin (HSA) under simulative physiological conditions. The experiment results showed that the fluorescence quenching of HSA by HCPT was a result of the formation of HCPT–HSA complex. The corresponding association constants (K _a) between HCPT and HSA at four different temperatures were determined according to the modified Stern–Volmer equation. The results of thermodynamic parameters ΔG, ΔH, and ΔS indicated that hydrogen bonds and van der Waals forces played major roles for HCPT–HSA association. Site marker competitive displacement experiment indicated that the binding of HCPT to HSA primarily took place in sub-domain IIA (site I). Molecular docking study further confirmed the binding mode and the binding site obtained by fluorescence and site marker competitive experiments. The conformational investigation showed that the presence of HCPT decreased the α-helical content of HSA and induced the slight unfolding of the polypeptides of protein, which confirmed some micro-environmental and conformational changes of HSA molecules.     

Isoniazid and rifampicin inhibit allosterically heme binding to albumin and peroxynitrite isomerization by heme–albumin

Human serum heme–albumin (HSA-heme) displays globin-like properties. Here, the allosteric inhibition of ferric heme [heme-Fe(III)] binding to human serum albumin (HSA) and of ferric HSA–heme [HSA-heme-Fe(III)]-mediated peroxynitrite isomerization by isoniazid and rifampicin is reported. Moreover, the allosteric inhibition of isoniazid and rifampicin binding to HSA by heme-Fe(III) has been investigated. Data were obtained at pH 7.2 and 20.0 °C. The affinity of isoniazid and rifampicin for HSA [K ₀ = (3.9 ± 0.4) × 10⁻⁴ and (1.3 ± 0.1) × 10⁻⁵ M, respectively] decreases by about 1 order of magnitude upon heme-Fe(III) binding to HSA [K _h = (4.3 ± 0.4) × 10⁻³ and (1.2 ± 0.1) × 10⁻⁴ M, respectively]. As expected, the heme-Fe(III) affinity for HSA [H ₀ = (1.9 ± 0.2) × 10⁻⁸ M] decreases by about 1 order of magnitude in the presence of saturating amounts of isoniazid and rifampicin [H _d = (2.1 ± 0.2) × 10⁻⁷ M]. In the absence and presence of CO₂, the values of the second-order rate constant (l _on) for peroxynitrite isomerization by HSA-heme-Fe(III) are 4.1 × 10⁵ and 4.3 × 10⁵ M⁻¹ s⁻¹, respectively. Moreover, isoniazid and rifampicin inhibit dose-dependently peroxynitrite isomerization by HSA-heme-Fe(III) in the absence and presence of CO₂. Accordingly, isoniazid and rifampicin impair in a dose-dependent fashion the HSA-heme-Fe(III)-based protection of free l-tyrosine against peroxynitrite-mediated nitration. This behavior has been ascribed to the pivotal role of Tyr150, a residue that either provides a polar environment in Sudlow’s site I (i.e., the binding pocket of isoniazid and rifampicin) or protrudes into the heme-Fe(III) cleft, depending on ligand binding to Sudlow’s site I or to the FA1 pocket, respectively. These results highlight the role of drugs in modulating heme-Fe(III) binding to HSA and HSA-heme-Fe(III) reactivity.     

Multispectroscopic insight,morphological analysis and molecular docking studies of CuII-based chemotherapeutic drug entity with human serum albumin (HSA) and bovine serum albumin (BSA)

The interaction studies of Cu^II nalidixic acid–DACH chemotherapeutic drug entity, [C₃₆H₅₀N₈O₆Cu] with serum albumin proteins, viz., human serum albumin (HSA) and bovine serum albumin (BSA) employing UV–vis, fluorescence, CD, FTIR and molecular docking techniques have been carried out. Complex [C₃₆H₅₀N₈O₆Cu] demonstrated strong binding affinity towards serum albumin proteins via hydrophobic contacts with binding constants, K?=?3.18?×?10⁵ and 7.44?×?10⁴ M^–1 for HSA and BSA, respectively implicating a higher binding affinity for HSA. The thermodynamic parameters ΔG, ΔH and ΔS at different temperatures were also calculated and the interaction of complex [C₃₆H₅₀N₈O₆Cu] with HSA and BSA was found to be enthalpy and entropy favoured, nevertheless, complex [C₃₆H₅₀N₈O₆Cu] demonstrated higher binding affinity towards HSA than BSA evidenced from its higher binding constant values. Time resolved fluorescence spectroscopy (TRFS) was carried out to validate the static quenching mechanism of HSA/BSA fluorescence. The collaborative results of spectroscopic studies indicated that the microenvironment and the conformation of HSA and BSA (α–helix) were significantly perturbed upon interaction with complex [C₃₆H₅₀N₈O₆Cu]. Hirshfeld surfaces analysis and fingerprint plots revealed various intermolecular interactions viz., N–H····O, O–H····O and C–H····O linkages in a 2–dimensional framework that provide crucial information about the supramolecular architectures in the complex. Molecular docking studies were carried out to ascertain the preferential binding mode and affinity of complex [C₃₆H₅₀N₈O₆Cu] at the target site of HSA and BSA. Furthermore, only for Transmission electroscopy microscopy micrographs of HSA and BSA in presence of complex [C₃₆H₅₀N₈O₆Cu] revealed major protein morphological transitions and aggregation which validates efficient delivery of complex by serum proteins to the target site.

Communicated by Ramaswamy H. Sarma     

Fluorescence spectroscopy and docking study in two flavonoids,isolated tectoridin and its aglycone tectorigenin,interacting with human serum albumin: a comparison study

Two flavonoids, tectoridin (TD) isolated from rhizomes of Iris tectorum and hydrolyzed aglycone tectorigenin (TG) were prepared and characterized to compare their different interaction ability with human serum albumin (HSA). Based on the results, the affinity of TG–HSA was stronger than that of TD–HAS, and TG combined more closely with HSA than did TD. HSA fluorescence was quenched by TD/TG. The interactions between TD/TG and HSA involved static quenching. The thermodynamic parameters indicated that both binding processes were spontaneous; hydrogen binding and van der Waals force were the main forces between TD and HSA, whereas a hydrophobic interaction was the main binding force between TG and HSA. Synchronous and 3D fluorescence spectra showed that the binding of TD/TG to HSA induced conformational changes. Moreover, a docking study confirmed the experimental results. Copyright © 2015 John Wiley & Sons, Ltd.     

Adsorption of Ribose Nucleotides on Manganese Oxides with Varied Mn/O Ratio: Implications for Chemical Evolution

Manganese exists in different oxidation states under different environmental conditions with respect to redox potential. Various forms of manganese oxides, namely, Manganosite (MnO), Bixbyite (Mn₂O₃), Hausmannite (Mn₃O₄) and Pyrolusite (MnO₂) were synthesized and their possible role in chemical evolution studied. Adsorption studies of ribose nucleotides (5′-AMP, 5′-GMP, 5′-CMP and 5′-UMP) on these manganese oxides at neutral pH, revealed a higher binding affinity to manganosite (MnO) compared to the other manganese oxides. That manganese oxides having a lower Mn-O ratio show higher binding affinity for the ribonucleotides indirectly implies that such oxides may have provided a surface onto which biomonomers could have been concentrated through selective adsorption. Purine nucleotides were adsorbed to a greater extent compared to the pyrimidine nucleotides. Adsorption data followed Langmuir adsorption isotherms, and X _m and K _L values were calculated. The nature of the interaction and mechanism was elucidated by infrared spectral studies conducted on the metal-oxide and ribonucleotide-metal-oxide adducts.     

Features of the binding of the fluorescent probe K-35 to albumin

A study was made of the binding of a fluorescent probe K-35 (N-(carboxyphenyl)imide of 4-(dimethylamino)naphthalic acid), used as an indicator of albumin structural changes in pathology, to human serum albumin (HSA). Based on the data on the fluorescence decay of the probe, four types of site of K-35 binding to HSA have been recognized, which differ in fluorescence decay time (τ) and binding constant (K). Probe molecules bound to the first type of site have a decay time of 8–10 ns; this value corresponds to a high fluorescence quantum yield of about 0.7. These sites have a maximal binding constant, K ₁ = 5 · 10⁴ M⁻¹. The τ₂ of the second type of site is close to 3.6 ns and K ₂ = 1 · 10⁴ M⁻¹, which is much lower than K ₁; however, the number of these sites is several times greater. The number of sites of the third type and the binding constant are close to those of the second type, but the decay time τ₃ is 1 ns, which is significantly lower than τ₂. The binding of K-35 to sites of the second and the third types is characterized by a positive cooperativity. Their properties are similar but not completely identical. The total number of sites of these three types is about two per one HSA molecule. There are also one-two sites of the fourth type where bound K-35 molecules have a very short decay time τ₄ ≪ 1, i.e., are virtually nonfluorescent, and K ₄ = 1 · 10⁴ M⁻¹. The major contribution to the steady-state fluorescence is made by probe molecules bound to sites of the first and second types. As a rule, the concentration of albumin binding sites in blood is significantly higher than the concentration of metabolites and xenobiotics transferred by albumin. Therefore, the metabolite—or the probe in these experiments—is distributed among different sites in accordance with their K _i n _i values (n _i is the number of sites of the i-th type per albumin molecule). The low occupancy of the sites results in an approximately equal number of K-35 molecules bound to different sites of types 1, 2, and 3. The competition of K-35 with phenylbutazone, a marker of the albumin drug-binding site I, allows one to suggest that the K-35 site of the first type is localized exactly in the drug site I region, while the sites of the second and third types are close to it.     

Investigation on the Interaction of Norgestrel with Human Serum Albumin Using Spectroscopy and Molecular‐Docking Method

The interaction of norgestrel with human serum albumin (HSA) was investigated by spectroscopy and molecular‐docking methods. Results of spectroscopy methods suggested that the quenching mechanism of norgestrel on HSA was static quenching and that the quenching process was spontaneous. Negative values of thermodynamic parameters (ΔG, ΔH, and ΔS) indicated that hydrogen bonding and van der Waals forces dominated the binding between norgestrel and HSA. Three‐dimensional fluorescence spectrum and circular dichroism spectrum showed that the HSA structure was slightly changed by norgestrel. Norgestrel mainly bound with Sudlow site I based on a probe study, as confirmed by molecular‐docking results. Competition among similar structures indicated that ethisterone and norethisterone affected the binding of norgestrel with HSA. CH₃ in R₁ had little effect on norgestrel binding with HSA. The surface hydrophobicity properties of HSA, investigated using 8‐anilino‐1‐naphthalenesulfonic acid, was changed with norgestrel addition.     

Interaction of human serum albumin with novel imidazole derivatives studied by spectroscopy and molecular docking

This study was a detailed characterization of the interaction of a series of imidazole derivatives with a model transport protein, human serum albumin (HSA). Fluorescence and time‐resolved fluorescence results showed the existence of a static quenching mode for the HSA–imidazole derivative interaction. The binding constant at 296 K was in the order of 10⁴ M^–1, showing high affinity between the imidazole derivatives and HSA. A site marker competition study combined with molecular docking revealed that the imidazole derivatives bound to subdomain IIA of HSA (Sudlow's site I). Furthermore, the results of synchronous, 3D, Fourier transform infrared, circular dichroism and UV–vis spectroscopy demonstrated that the secondary structure of HSA was altered in the presence of the imidazole derivatives. The specific binding distance, r, between the donor and acceptor was obtained according to fluorescence resonance energy transfer. Copyright © 2015 John Wiley & Sons, Ltd.     

Synthesis,characterization, DNA and HSA binding studies of isomeric Pd (II) antitumor complexes using spectrophotometry techniques

Two new Palladium(II) isomeric complexes, [Pd (Gly)(Leu)](I) and [Pd (Gly)(Ile)](II), where Gly is glycine, and Leu and Ile are isomeric amino acids (leucine and isoleucine), have been synthesized and characterized by elemental analysis, molar conductivity measurements, FT-IR, ¹H NMR, and UV–Vis. The complexes have been tested for their In vitro cytotoxicity against cancer cell line K₅₆₂ and their binding properties to calf thymus DNA (CT-DNA) and human serum albumin (HSA) have also been investigated by multispectroscopic techniques. Interactions of these complexes with CT-DNA were monitored using gel electrophoresis. The energy transfer from HSA to these complexes and the binding distance between HSA and the complexes (r) were calculated. The results obtained from these studies indicated that at very low concentrations, both complexes effectively interact with CT-DNA and HSA. Fluorescence studies revealed that the complexes strongly quench DNA bound ethidium bromide as well as the intrinsic fluorescence of HSA through the static quenching procedures. Binding constant (K_b), apparent biomolecular quenching constant (k_q), and number of binding sites (n) for CT-DNA and HSA were calculated using Stern–Volmer equation. The calculated thermodynamic parameters indicated that the hydrogen binding and vander Waals forces might play a major role in the interaction of these complexes with HSA and DNA. Thus, we propose that the complexes exhibit the groove binding with CT-DNA and interact with the main binding pocket of HSA. The complexes follow the binding affinity order of I > II with DNA- and II > I with HSA-binding.     

Reversible two-step unfolding of heme–human serum albumin: a 1H-NMR relaxometric and circular dichroism study

Human serum albumin (HSA) participates in heme scavenging, the bound heme turning out to be a reactivity center and a powerful spectroscopic probe. Here, the reversible unfolding of heme–HSA has been investigated by ¹H-NMR relaxometry, circular dichroism, and absorption spectroscopy. In the presence of 6 equiv of myristate (thus fully saturating all available fatty acid binding sites in serum heme–albumin), 1.0 M guanidinium chloride induces some unfolding of heme–HSA, leading to the formation of a folding intermediate; this species is characterized by increased relaxivity and enhanced dichroism signal in the Soret region, suggesting a more compact heme pocket conformation. Heme binds to the folding intermediate with K _d = (1.2 ± 0.1) × 10⁻⁶ M. In the absence of myristate, the conformation of the folding intermediate state is destabilized and heme binding is weakened [K _d = (3.4 ± 0.1) × 10⁻⁵ M]. Further addition of guanidinium chloride (up to 5 M) brings about the usual denaturation process. In conclusion, myristate protects HSA from unfolding, stabilizing a folding intermediate state in equilibrium with the native and the fully unfolded protein, envisaging a two-step unfolding pathway for heme–HSA in the presence of myristate.     

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.     

Resonance energy transfer,pH‐induced folded states and the molecular interaction of human serum albumin and icariin

Icariin is a flavonol glycoside with a wide range of pharmacological and biological activities. The pharmacological and biological functions of flavonoid compounds mainly originate from their binding to proteins. The mode of interaction of icariin with human serum albumin (HSA) has been characterized by fluorescence spectroscopy and far‐ and near‐UV circular dichroism (CD) spectroscopy under different pH conditions. Fluorescence quenching studies showed that the binding affinity of icariin with HSA in the buffer solution at different pH values is: K_a (pH 4.5) > K_a (pH 3.5) > K_a (pH 9.0) > K_a (pH 7.0). Red‐edge excitation shift (REES) studies revealed that pH had an obvious effect on the mobility of the tryptophan microenvironment and the addition of icariin made the REES effect more distinct. The static quenching mechanism and number of binding sites (n ≈ 1) were obtained from fluorescence data at three temperatures (298, 304 and 310 K). Both ?H⁰ < 0 and ??⁰ < 0 suggested that hydrogen bonding and van der Waal's interaction were major driving forces in the binding mechanism, and this was also confirmed by the molecular simulation results. The distance r between the donor (HSA) and the acceptor (icariin) was calculated based on Förster non‐radiation energy transfer theory. We found that pH had little impact on the energy transfer between HSA and icariin. Far‐ and near‐UV CD spectroscopy studies further indicated the influence of pH on the complexation process and the alteration in the protein conformation upon binding. Copyright © 2015 John Wiley & Sons, Ltd.