Sucrose dependence on the human serum albumin-dehydroepiandrosterone binding: Thermodynamic and fractal approach

In this paper, the effect of sucrose concentration (x) on the dehydroepiandrosterone (DHEA)-human serum albumin (HSA) binding was investigated by a biochromatographic approach. A mathematical development based on fractal geometry is proposed to provide a more realistic picture of the DHEA-HSA binding. The fractal dimension D of the cavity surface and the thermodynamic data of the binding mechanism were calculated at different sucrose concentrations in the bulk solvent. Results showed that under a critical sucrose concentration value x_c (domain I), the enhancement of the DHEA-HSA binding intensity was principally due to the increase of hydrophobic interaction between DHEA and HSA cavity. Above x_c (domain II), the salting-out agent levelled the HSA cavity surface irregularity and, consequently, the DHEA affinity for the HSA decreased. Moreover, for the domain II, the HSA-DHEA binding and the thermodynamic data are discussed using fractal concept of surface fluctuations.     

Complex formation of Zn-, Ni-, and Pd-derivatives of purpurin-18 with serum albumin

We analyzed the spectral characteristics of the complexes of Zn²⁺, Ni²⁺, and Pd²⁺ derivatives of purpurin-18 with human serum albumin (HSA) in aqueous buffer at pH 7.0. Pd²⁺ in the coordination sphere of purpurin-18 decreased the affinity to HSA compared to the respective complexes of zinc and nickel derivatives. Since the formation of complexes with HSA is an important parameter of photodynamic activity of tetrapyrrolic compounds, the differential affinity of metal derivatives of purpurin-18 to this protein should be considered for the optimization of photosensitizers.     

Recognition of dicarboxylates in aqueous acetonitrile by a dinuclear zinc(II) complex of 2,2’-binaphthalene-based receptor

Molecular recognition of dicarboxylates by a dinuclear zinc(II) complex of 2,2?-binaphthalene bearing di(2-pyridyl)aminomethyl groups at 8- and 8?-positions ([Zn₂L]⁴⁺) by UV-vis and fluorescence spectral titrations in an aqueous solvent was investigated. In a series of α,ω-dicarboxylate, receptor [Zn₂L]⁴⁺ showed the highest binding affinity for succinate and UV-vis and fluorescence changes were also largest upon the addition of this guest. In a series of phthalate derivatives, phthalate was strongly bound by [Zn₂L]⁴⁺. The characteristic UV-vis and fluorescence changes strongly depend on the dihedral angle of two naphthyl groups of the complex [Zn₂L]⁴⁺· dicarboxylate estimated by spectral titrations and DFT calculations.     

Study of the binding equilibrium between Zn(II) and HSA by capillary electrophoresis-inductively coupled plasma optical emission spectrometry

A new method has been developed for following the interaction between zinc ion and human serum albumin (HSA) by capillary electrophoresis-inductively coupled plasma optical emission spectrometry. Under optimized experimental conditions, the detection limit (3σ) for free Zn²⁺ ion was found to be 1.34 μM by running 11 replicates of the reagent blank. The RSD was less than 3% and the recovery was more than 98.13%. The linear range of zinc ion concentration was between 5.1 μM and 0.3 M. The measured Zn(II)-HSA combination values of n₁ and K₁ for primary binding of Zn²⁺ to HSA were 1.09 and 2.29 × 10⁵ L mol⁻¹, respectively. The measured values of n₂ and K₂ for the non-specific binding of Zn²⁺ to HSA were 8.96 and 6.65 × 10³ L mol⁻¹, respectively. This new method allows rapid analysis of a small amount of sample, simple operation, while avoiding long periods of dialysis and eliminating interference from other metal ions. This method provides a reliable and convenient new way for studying interactions between metal ions and biomolecules.     

Metal binding discrimination of the calmodulin Q41C/K75C mutant on Ca2+ and La3+

Calmodulin (CaM) is a multifunctional Ca²⁺-binding protein regulating the activity of many enzymes in response to fluctuation of the intracellular Ca²⁺ level. It has been shown that a CaM Q41C/K75C mutant (CaMSS) with a disulfide bond in the N-terminal domain exhibits greatly reduced affinity to Ca²⁺. In the present study, the experimental results revealed a unique metal binding pattern in CaMSS towards La³⁺ and Ca²⁺ separately: the mutant protein binds Ca²⁺ at site I, III and IV; however, it binds La³⁺ at site I, II and IV. A putative mechanism was proposed which is the conformation of site II (or site III) of CaMSS could be altered and thus loses its metal ion affinity in response to metal binding in the opposite terminal domain possibly through the long range domain interaction. The present work may offer new perspectives for understanding the mechanisms of specific metal ion affinity in CaM and for CaM-based protein design.     

Synthesis,characterization, HSA interaction,and antibacterial activity of a new water-soluble Pt(II) complex containing the drug cephalexin

Abstract

A new water-soluble platinum(II) complex, [Pt(CEX)Cl(DMSO)]Cl (CEX is cephalexin), was synthesized and characterized by physicochemical, spectroscopic, and computational methods. Multispectroscopic techniques were used to investigate the interaction of Pt(II) complex with human serum albumin (HSA) under the physiological conditions. The results of fluorescence titration indicated that the binding of the Pt(II) complex to HSA induced fluorescence quenching through static quenching mechanism with binding constant of 1.24?×?10⁴?M^?1 at 298?K. The thermodynamic parameters at different temperatures indicated that van der Waals forces, hydrogen bonds, and electrostatic forces play major roles in the stability of Pt(II) complex–HSA association. The displacement experiments using the site probes warfarin and ibuprofen substantiated that Pt(II) complex could bind to both site I and II of HSA. Furthermore, UV–Vis and fluorescence spectra were used to investigate the conformational changes of HSA molecule with the addition of Pt(II) complex. The binding constant of Pt(II) complex is more than two orders of magnitude higher than the corresponding value of cephalexin. These results indicate that the binding affinity of Pt(II) complex is stronger than the free drug. In addition, the antibacterial study showed that the MIC of platinum complex of cephalexin for variety of organisms was lower than free cephalexin.     

白杨素与不同构型人血清白蛋白的作用机制

采用多种光谱学手段研究了白杨素(CHR)和不同构型人血清白蛋白(HSA)相互作用的分子机制.研究表明,白杨素能使蛋白质荧光发射峰发生静态淬灭,同时,白杨素的紫外吸收谱带也发生了明显的位移,说明与蛋白质的结合可使白杨素分子中的酚羟基发生解离.蛋白质还可以引起白杨素荧光发射峰强度的明显增强.利用荧光淬灭和荧光增强两种模式计算得到的白杨素和人血清白蛋白在生理条件下(pH 7.4)的结合常数(KA)分别为(9.97±0.24)×104和(9.75±0.11)×104L mol-1,其结合比例为1:1.随着pH值的降低,蛋白质与白杨素的结合常数逐渐减小,这与蛋白质的构型变化有关.根据不同异构体血清蛋白质的结构特征,判定白杨素在蛋白质分子上的结合位置位于IIA亚域的Site I活性位点.结合分子模拟,讨论了白杨素与蛋白质分子的结合机制.     

Spectroscopic and Molecular Docking Approaches for Investigation of Interaction of Phellopterin with Human Serum Albumin

The mechanism of interaction between human serum albumin (HSA) and natural product phellopterin (PL) from Angelica dahurica was investigated by spectroscopic techniques with molecular docking under simulated physiological conditions. The experimental results showed that the fluorescence of HSA was regularly quenched by PL, and the quenching constants (K_SV) decreased with increasing temperature, which indicated that the quenching mechanism was a static quenching procedure. The binding constants (K_A) were larger than 10^?5 M^?1 and the number of binding sites (n) was approximate to 1 at different temperatures, which indicated that the binding affinity was hige and there was just one main binding site in HSA for PL. According to thermodynamic parameters from Van't Hoff equation, the binding process of PL with HSA was spontaneous and exothermic process due to ΔG < 0, and the electrostatic force played major role in the binding between PL and HSA according to ΔH < 0 and ΔS > 0. The binding distance (r) was calculated to be about 3.35 nm, which implied that the energy transfer from HSA to PL occurred with high possibility according to the theory of Förster's non-radiation energy transfer. The microenvironment and conformation of HSA changed with the addition of PL based on the results of synchronous and three-dimensional fluorescence methods. The molecular docking analysis revealed the binding locus of PL to HSA in subdomain IIIA (Sudlow's site II).     

Metal binding and interdomain thermodynamics of mammalian metallothionein-3: enthalpically favoured Cu+ supplants entropically favoured Zn2+ to form Cu4+ clusters under physiological conditions

Metallothioneins (MTs) are a ubiquitous class of small metal-binding proteins involved in metal homeostasis and detoxification. While known for their high affinity for d¹⁰ metal ions, there is a surprising dearth of thermodynamic data on metals binding to MTs. In this study, Zn²⁺ and Cu⁺ binding to mammalian metallothionein-3 (MT-3) were quantified at pH 7.4 by isothermal titration calorimetry (ITC). Zn²⁺ binding was measured by chelation titrations of Zn₇MT-3, while Cu⁺ binding was measured by Zn²⁺ displacement from Zn₇MT-3 with competition from glutathione (GSH). Titrations in multiple buffers enabled a detailed analysis that yielded condition-independent values for the association constant (K) and the change in enthalpy (ΔH) and entropy (ΔS) for these metal ions binding to MT-3. Zn²⁺ was also chelated from the individual α and β domains of MT-3 to quantify the thermodynamics of inter-domain interactions in metal binding. Comparative titrations of Zn₇MT-2 with Cu⁺ revealed that both MT isoforms have similar Cu⁺ affinities and binding thermodynamics, indicating that ΔH and ΔS are determined primarily by the conserved Cys residues. Inductively coupled plasma mass spectrometry (ICP-MS) analysis and low temperature luminescence measurements of Cu-replete samples showed that both proteins form two Cu₄⁺–thiolate clusters when Cu⁺ displaces Zn²⁺ under physiological conditions. Comparison of the Zn²⁺ and Cu⁺ binding thermodynamics reveal that enthalpically-favoured Cu⁺, which forms Cu₄⁺–thiolate clusters, displaces the entropically-favoured Zn²⁺. These results provide a detailed thermodynamic analysis of d¹⁰ metal binding to these thiolate-rich proteins and quantitative support for, as well as molecular insight into, the role that MT-3 plays in the neuronal chemistry of copper.

Metallothioneins (MTs) are a ubiquitous class of small metal-binding proteins involved in metal homeostasis and detoxification.     

Mechanism of interaction of hypoglycemic agents glimepiride and glipizide with human serum albumin

The mechanism of interaction of hypoglycemic drugs, glimepiride and glipizide with human serum albumin (HSA) has been studied using fluorescence spectroscopy. The results are discussed in terms of the binding parameters, thermodynamics of the binding process, nature of forces involved in the interaction, identification of drug binding site on serum albumin and the fluorescence quenching mechanism involved. The association constants were of the order of 10⁵ and glipizide was found to have much higher affinity for HSA than glimepiride at all temperatures. Thermodynamic parameters for the binding suggested that hydrophobic interactions are primarily involved in the binding of these drugs to HSA. However, glimepiride and glipizide appear to cause temperature-dependent conformational changes in the albumin molecule and, therefore, the nature of interaction varied with temperature. Glimepiride and glipizide bind to both site I and site II on HSA, but the primary interaction occurs at site II. The binding region in site II is different for the two drugs. Stern-Volmer analysis of quenching data indicated that tryptophan residues of HSA are not fully accessible to the drugs and a predominantly dynamic quenching mechanism is involved in the binding. Results can provide useful insight into prediction of competitive displacement of these drugs by other co-administered drugs and excipients, resulting in serious fluctuations of the blood glucose levels in diabetic patients.      

High-performance affinity chromatography and the analysis of drug interactions with modified proteins: binding of gliclazide with glycated human serum albumin

This study used high-performance affinity chromatography (HPAC) to examine the binding of gliclazide (i.e., a sulfonylurea drug used to treat diabetes) with the protein human serum albumin (HSA) at various stages of modification due to glycation. Frontal analysis conducted with small HPAC columns was first used to estimate the number of binding sites and association equilibrium constants (K _a) for gliclazide with normal HSA and glycated HSA. Both normal and glycated HSA interacted with gliclazide according to a two-site model, with a class of high-affinity sites (average K _a, 7.1–10 × 10⁴ M⁻¹) and a group of lower-affinity sites (average K _a, 5.7–8.9 × 10³ M⁻¹) at pH 7.4 and 37 °C. Competition experiments indicated that Sudlow sites I and II of HSA were both involved in these interactions, with the K _a values for gliclazide at these sites being 1.9 × 10⁴ and 6.0 × 10⁴ M⁻¹, respectively, for normal HSA. Two samples of glycated HSA had similar affinities to normal HSA for gliclazide at Sudlow site I, but one sample had a 1.9-fold increase in affinity at this site. All three glycated HSA samples differed from normal HSA in their affinity for gliclazide at Sudlow site II. This work illustrated how HPAC can be used to examine both the overall binding of a drug with normal or modified proteins and the site-specific changes that can occur in these interactions as a result of protein modification.     

The Crystal Structure of a Homodimeric Pseudomonas Glyoxalase I Enzyme Reveals Asymmetric Metallation Commensurate with Half‐of‐Sites Activity

The Zn inactive class of glyoxalase I (Glo1) metalloenzymes are typically homodimeric with two metal‐dependent active sites. While the two active sites share identical amino acid composition, this class of enzyme is optimally active with only one metal per homodimer. We have determined the X‐ray crystal structure of GloA2, a Zn inactive Glo1 enzyme from Pseudomonas aeruginosa. The presented structures exhibit an unprecedented metal‐binding arrangement consistent with half‐of‐sites activity: one active site contains a single activating Ni²⁺ ion, whereas the other contains two inactivating Zn²⁺ ions. Enzymological experiments prompted by the binuclear Zn²⁺ site identified a novel catalytic property of GloA2. The enzyme can function as a Zn²⁺/Co²⁺‐dependent hydrolase, in addition to its previously determined glyoxalase I activity. The presented findings demonstrate that GloA2 can accommodate two distinct metal‐binding arrangements simultaneously, each of which catalyzes a different reaction.     

A novel lophine-based fluorescence probe and its binding to human serum albumin

The binding of a lophine-based fluorescence probe, 4-[4-(4-dimethylaminophenyl)-5-phenyl-1H-imidazol-2-yl]benzoic acid methyl ester (DAPIM) with human serum albumin (HSA) was investigated by fluorescence spectroscopy under physiological conditions. While DAPIM shows extreme low fluorescence in aqueous solution, DAPIM binding with HSA emits strong fluorescence at 510 nm. The binding constant and binding number determined by Scatchard plot was 3.65 × 10⁶ M⁻¹ and 1.07, respectively. Competitive binding between DAPIM and other ligands such as warfarin, valproic acid, diazepam and oleic acid, were also studied fluorometrically. The results indicated that the primary binding site of DAPIM to HSA is site II at subdomain IIIA. DAPIM can be a useful fluorescence probe for the characterization of drug-binding sites. In addition to the interaction study, because the fluorescence intensity of DAPIM increased in proportion to HSA concentration, its potential in HSA assay for serum sample was also evaluated.     

Identification of pyrazosulfuron-ethyl binding affinity and binding site subdomain IIA in human serum albumin by spectroscopic methods

Pyrazosulfuron-ethyl (PY) is a sulfonylurea herbicide developed by DuPont which has been widely used for weed control in cereals. The determination of PY binding affinity and binding site in human serum albumin (HSA) by spectroscopic methods is the subject of this work. From the fluorescence emission, circular dichroism and three-dimensional fluorescence results, the interaction of PY with HSA caused secondary structure changes in the protein. Fluorescence data demonstrated that the quenching of HSA fluorescence by PY was the result of the formation of HSA–PY complex at 1:1 molar ratio, a static mechanism was confirmed to lead to the fluorescence quenching. Hydrophobic probe 8-anilino-1-naphthalenesulfonic acid (ANS) displacement results show that hydrophobic patches are the major sites for PY binding on HSA. The thermodynamic parameters ΔH° and ΔS° were calculated to be ?36.32 kJ mol^?1 and ?35.91 J mol^?1 K^?1, which illustrated van der Waals forces and hydrogen bonds interactions were the dominant intermolecular force in stabilizing the complex. Also, site marker competitive experiments showed that the binding of PY to HSA took place primarily in subdomain IIA (Sudlow's site I). What presented in this paper binding research enriches our knowledge of the interaction between sulfonylurea herbicides and the physiologically important protein HSA.     

Albumin-mediated alteration of plasma zinc speciation by fatty acids modulates blood clotting in type-2 diabetes

Zn²⁺ is an essential regulator of coagulation and is released from activated platelets. In plasma, the free Zn²⁺ concentration is fine-tuned through buffering by human serum albumin (HSA). Importantly, the ability of HSA to bind/buffer Zn²⁺ is compromised by co-transported non-esterified fatty acids (NEFAs). Given the role of Zn²⁺ in blood clot formation, we hypothesise that Zn²⁺ displacement from HSA by NEFAs in certain conditions (such as type 2 diabetes mellitus, T2DM) impacts on the cellular and protein arms of coagulation. To test this hypothesis, we assessed the extent to which increasing concentrations of a range of medium- and long-chain NEFAs reduced Zn²⁺-binding ability of HSA. Amongst the NEFAs tested, palmitate (16 : 0) and stearate (18 : 0) were the most effective at suppressing zinc-binding, whilst the mono-unsaturated palmitoleate (16 : 1c9) was markedly less effective. Assessment of platelet aggregation and fibrin clotting parameters in purified systems and in pooled plasma suggested that the HSA-mediated impact of the model NEFA myristate on zinc speciation intensified the effects of Zn²⁺ alone. The effects of elevated Zn²⁺ alone on fibrin clot density and fibre thickness in a purified protein system were mirrored in samples from T2DM patients, who have derranged NEFA metabolism. Crucially, T2DM individuals had increased total plasma NEFAs compared to controls, with the concentrations of key saturated (myristate, palmitate, stearate) and mono-unsaturated (oleate, cis-vaccenate) NEFAs positively correlating with clot density. Collectively, these data strongly support the concept that elevated NEFA levels contribute to altered coagulation in T2DM through dysregulation of plasma zinc speciation.

Zn²⁺ is an essential regulator of coagulation. In plasma, Zn²⁺ availability is fine-tuned by human serum albumin (HSA). Here we show that elevated fatty acid levels contribute to altered coagulation in type-2 diabetes through Zn²⁺ mishandling by HSA.     

Synthesis,structural determination and HSA interaction studies of a new water-soluble Cu(II) complex derived from 1,10-phenanthroline and ranitidine drug

A new water-soluble Cu(II) complex containing ranitidine drug and 1,10-phenanthroline was synthesized and characterized by elemental analysis, molar conductivity, spectroscopic and computational methods. In vitro human serum albumin (HSA)-interaction studies of Cu(II) complex were performed by employing fluorescence spectroscopy in combination with UV–vis absorption and circular dichroism (CD) spectroscopies. The results of fluorescence titration showed that Cu(II) complex strongly quenched the intrinsic fluorescence of HSA through a static quenching mechanism with an intrinsic binding constant (6.05 × 10⁴ M^?1) at 286 K. The thermodynamic parameters ΔG, ΔH, and ΔS at different temperatures were calculated and suggested that the hydrophobic and hydrogen bonding interactions play major roles in Cu(II) complex-HSA association. The displacement experiments using warfarin and ibuprofen as site I and II probes proved that the Cu(II) complex could bind to site I (subdomain IIA) of HSA. Finally, CD spectra indicated that the interaction of the Cu(II) complex with HSA leads to an increase in the α-helical content. The main result of this study was the finding that the binding affinity of the Cu(II) complex to HSA is three orders of magnitude stronger than that of ranitidine drug.     

Fluorometric and molecular modeling deciphering the non-covalent interaction between cyromazine and human serum albumin

Cyromazine (CMZ) had been believed to be one of the safest pesticides and widely used for many years until its carcinogenesis was revealed recently. In this work, the interaction between cyromazine and human serum albumin (HSA) was systematically investigated by multiple spectroscopic methods and molecular docking techniques using warfarin and flufenamic acid as probes. The results demonstrated the fluorescence of HSA had been quenched by CMZ through static mechanism, with new non-covalent complexes formed at ground state. Fluorescence probe experiments indicated CMZ bound to Sudlow’s site I in subdomain IIA of HSA, having no competition with site marker in site II. The number of binding sites, equilibrium constants and thermodynamic parameters were calculated by monitoring the binding equilibriums at different temperatures. The positive enthalpy change (ΔH ^θ) and entropy change (ΔS ^θ) implied the binding was mainly conducted by hydrophobic interactions. The binding was an endothermic, spontaneous (ΔG ^θ?<?0) and entropy-driven process which made the energy distribution of the system more evenly. The force of interaction and the conformation of binding pocket were displayed by molecular simulation and discussed at molecular level. Circular dichroism (CD) spectra indicated distorted α-helix content of HSA decreased while other fine secondary structure increased when CMZ was added.     

Metal Ions Enhance the Affinities of Flavonoids for Human Serum Albumin in Vitro

The affinities of apigenin, chrysin, daidzein and quercetin for human serum albumin (HSA) were studied in the presence and absence of Pb²⁺, Ni²⁺, Zn²⁺, Mg²⁺ and Mn²⁺. The fluorescence intensities of HSA decrease remarkably with increasing concentration of these four flavonoids. Adding apigenin and chrysin resulted in blue-shifts of HSA from ?? _em=336 to 332 nm and 330 nm, respectively. However, quercetin showed an obvious red-shift of HSA from ?? _em=336 to 347 nm whereas daidzein hardly affected the ?? _em of HSA. The ?? _em shifts induced by flavonoids in the presence of mental ions were much bigger than those in the absence of these ions. Pb²⁺, Ni²⁺, Zn²⁺, Mg²⁺ and Mn²⁺ increased the quenching constants of these four flavonoids for HSA by 19.2?% to 43?%, 47.7?% to 117?%, 23.3?% to 64.4?%, 9.29?% to 42.2?% and 18?% to 55.6?%, respectively. The affinities of apigenin, chrysin, daidzein and quercetin for HSA increased about 9.49?%, 3.63?%, 5.73?% and 2.32?%, respectively, in the presence of Pb²⁺. Ni²⁺ improved the affinities of apigenin, chrysin, daidzein and quercetin for HSA by about 4.79?%, 0.85?%, 11.91?% and 10.55?%, respectively. Zn²⁺ enhanced the affinities of apigenin, chrysin, daidzein and quercetin for HSA by about 1.03?%, 1.34?%, 1.96?% and 13.14?%, respectively. Mg²⁺ increased the affinities of apigenin, chrysin, daidzein and quercetin for HSA by about 2.03?%, 0.7?%, 1.39?% and 2.07?%, respectively. Mn²⁺ increased the affinities of apigenin, chrysin, daidzein and quercetin for HSA by about 2.46?%, 6.71?%, 12.3?% and 4.10?%, respectively.