Structural Changes Of Human Serum Albumin Research Articles

An ultrasensitive, selective pseudoesterase-activated fluorescent probe for monitoring acetylation modification, configuration transformation and biological levels of human serum albumin

Quantitative assays and the structural monitoring of human serum albumin (HSA) are essential for disease diagnosis, drug development, and physiological studies. Commonly used methods for quantifying HSA are based on the dye-HSA binding mechanism, which involves determining the absorption or fluorescence signal when the dye binds non-covalently to HSA. However, these methods generally suffer from poor selectivity and susceptibility to interference from co-existing species. Herein, an ultrasensitive and selective fluorescent probe, 2-(2-morpholinoethyl)-1,3-dioxo-2,3-dihydro-1 H-benzo[de]isoquinolin-6-yl cyclopropanecarboxylate (MDDH), was designed based on the pseudoesterase activity of HSA and used to monitor the acetylation modification, configuration transformation, and biological levels of HSA. Upon catalysis by HSA, the cyclopropanecarbonyl ester bound to MDDH undergoes hydrolysis, yielding a strong fluorescent signal. In contrast to conventional HSA-binding dyes, MDDH demonstrates superior sensitivity (with a 302-fold fluorescence enhancement), high selectivity for distinguishing between HSA and bovine serum albumin, and strong anti-interference against various coexisting species. By sensing HSA pseudoesterase activity, MDDH allows for the sensitive monitoring of structural changes in HSA, including aspirin-induced acetylation, guanidine hydrochloride-induced defolding and trypsin-induced cleavage. Moreover, MDDH exhibits strong stability, low cytotoxicity and good cell membrane permeability. It is suitable for the quantitative determination of HSA in the serum and urine, as well as for the fluorescence imaging of HSA in cells. Our work provides an efficient tool for monitoring the acetylation modifications, configuration changes, and biological content of HSA.

Exploring high hydrostatic pressure effects on anthocyanin binding to serum albumin and food-derived transferrins

This study investigated the effects of high hydrostatic pressure (HHP) on the binding interactions of cyanindin-3-O-glucoside (C3G) to bovine serum albumin, human serum albumin (HSA), bovine lactoferrin, and ovotransferrin. Fluorescence quenching revealed that HHP reduced C3G-binding affinity to HSA, while having a largely unaffected role for the other proteins. Notably, pretreating HSA at 500 MPa significantly increased its dissociation constant with C3G from 24.7 to 34.3 μM. Spectroscopic techniques suggested that HSA underwent relatively pronounced tertiary structural alterations after HHP treatments. The C3G-HSA binding mechanisms under pressure were further analyzed through molecular dynamics simulation. The localized structural changes in HSA under pressure might weaken its interaction with C3G, particularly polar interactions such as hydrogen bonds and electrostatic forces, consequently leading to a decreased binding affinity. Overall, the importance of pressure-induced structural alterations in proteins influencing their binding with anthocyanins was highlighted, contributing to optimizing HHP processing for anthocyanin-based products.

Structure of Human Serum Albumin at a Foam Surface.

Proteins can be adsorbed on the air-water interface (AWI), and the structural changes in proteins at the AWI are closely related to the foaming properties of foods and beverages. However, how these structural changes in proteins at the AWI occur is not well understood. We developed a method for the structural assessment of proteins in the foam state using hydrogen/deuterium exchange mass spectrometry. Adsorption sites and structural changes in human serum albumin (HSA) were identified in situ at the peptide-level resolution. The N-terminus and the loop (E492-T506), which contains hydrophobic amino acids, were identified as adsorption sites. Both the structural flexibility and hydrophobicity were considered to be critical factors for the adsorption of HSA at the AWI. Structural changes in HSA were observed after more than one minute of foaming and were spread widely throughout the structure. These structural changes at the foam AWI were reversible.

Steady-state and 3D fluorescence study reveals the binding of a dicoumarol analogue in subdomain IIA of human serum albumin with structural variation

Drugs exhibit pharmacological properties through their binding interactions with the target receptors. Coumarins and dicoumarols are known to exhibit diverse pharmacological potential. 3,3′-Carbonylbis (7-diethylamino coumarin) (CDC) is a bis-coumarin derivative with antioxidant activity. The interaction of CDC to human serum albumin (HSA) has been reported here. The fluorescence quenching of HSA by CDC at 303, 308, and 313 K revealed a dynamic quenching with a quenching constant of 104 M−1 order. The binding constant from spectroscopic methods was found to be 103 M−1 order. Thermodynamic parameters (∆S0, ∆H0, and ∆G0) were calculated from temperature studies using the Van't Hoff equation. A negative ∆G0 value showed the spontaneity of the interaction. A positive ∆S0 value and negative ∆H0 value indicated the major role of an electrostatic force. Further the binding was found to be an entropy-driven process. Site-specific marker displacement studies with 1-Anilino-8-naphthalene sulfonate (ANS), warfarin, and ibuprofen revealed CDC binding at site-1 in subdomain IIA. HSA conformational changes were verified using synchronous and 3D fluorescence studies. Secondary structural changes in HSA were visible from circular dichroism results. Molecular docking results corroborated the experimental finding showing CDC binding at site-1 in HSA.

Interactions of isoorientin and its Semi-synthetic analogs with human serum albumin

Isoorientin is a C-glycosyl flavone with a wide range of health beneficial effects and inhibits glycogen synthase kinase 3β (GSK-3β) potentially against Alzheimer’s disease. Its semi-synthetic derivatives have greater potency than isoorientin. The present study was aimed to determine the mechanism of interactions of isoorientin and its derivatives with human serum albumin (HSA) using multi-spectroscopic, microscale thermophoresis (MST) and computational studies. Spectra of steady-state fluorescence, UV–Vis, and time-resolved fluorescence indicated that isoorientin and its derivatives quenched the intrinsic fluorescence of HSA through a static quenching process. Isoorientin and its derivatives had a moderate affinity with HSA (Ka 7.7–14.9 × 104 M−1). The binding process was accompanied by an exothermic phenomenon, ΔG° of HSA-isoorientin and its derivatives systems were calculated as from −29.51 kJ mol−1 to −27.87 kJ mol−1. Displacement experiments with site-specific markers revealed that isoorientin and its derivatives bind to HSA at site II (subdomain IIIA) only. A reduction in the α-helical content of HSA-isoorientin and its derivatives complex was observed, because the conformational changes was structurally perturbed by the hydrophilic groups of the compounds. Further molecular modeling studies confirmed that the binding of isoorientin and its derivatives to the site II via hydrophobic interaction. The MST results confirmed the interactions between HSA and the compounds of interest. The esterase-like assay studies indicated that isoorientin and its derivatives shared the same binding site in HSA, and their induced structural changes of HSA may have been caused by partial unfolding of HSA. This work helps to understand transport, distribution, bioactivity, and design of flavonoid-based GSK-3β inhibitors.

Nanocorona Formation between Foodborne Nanoparticles Extracted from Roast Squid and Human Serum Albumin.

Foodborne nanoparticles (FNPs) produced by roasting have attracted the attention of people, owing to their safety risk to body health. Herein, we reported the formation, physicochemical properties, elemental composition, biodistribution, and binding with human serum albumin (HSA) of FNPs extracted from roast squid. The results showed that the FNP size gradually decreased from 4.1 to 2.3 nm as the roasting temperature changed from 190 to 250 °C. The main component elements of FNPs are carbon, oxygen, and nitrogen, and the carbon and nitrogen contents of FNPs increased with the roasting temperature rising. The surface of FNPs contained hydroxyl, amino, and carboxyl functional groups. The FNPs can emit fluorescence in ultraviolet light and show excitation-dependent emission behavior. Furthermore, it was found that the FNPs derived from roast squid could be accumulated in the stomach, intestine, and brain of BALB/c mice after oral feeding. Static fluorescence quenching of HSA was found by the Stern-Volmer equation and ultraviolet-visible spectrum analysis after interaction with the FNPs. After the addition of FNPs, the α-helix content of HSA decreased and the morphological height of HSA increased, which indicated that the FNPs could cause structural changes in HSA. The atomic force microscopy characterization showed the formation of nanocorona between FNPs and HSA.

Domain-Specific Stabilization of Structural and Dynamic Responses of Human Serum Albumin by Sucrose.

Human Serum Albumin (HSA) is the most abundant protein present in human blood plasma. It is a large multi-domain protein with 585 amino acid residues. Due to its importance in human body, studies on the interaction of HSA with different external agent is of vital interest. The denaturation and renaturation of HSA in presence of external agents are of particular interest as they affect the biological activity of the protein. The objective of this work is to study the domain-specific and overall structural and dynamical changes occurring to HSA in the presence of a denaturing agent, urea and a renaturing agent, sucrose. In order to carry out the domain-specific studies, HSA has been tagged using N-(7- dimethylamino-4-methylcoumarin-3-yl) iodoacetamide (DACIA) at Cys-34 of domain-I and pnitrophenyl coumarin ester (NPCE) at Tyr-411 site in domain-III, separately. Steady-state absorption, emission and solvation dynamic measurements have been carried out in order to monitor the domain-specific alteration of HSA caused by the external agents. The overall structural change of HSA have been monitored using circular dichroism spectroscopy. The α-helicity of HSA was found to decrease from 65% to 11% in presence of urea and was found to further increase to 25% when sucrose is added, manifesting the denaturing and renaturing effects of urea and sucrose, respectively. The steady state studies show that domain-III is more labile towards denaturation as compared to domain-I. The presence of an intermediate state is observed during the denaturation process. The stabilization of this intermediate state in presence of sucrose is attributed as the reason for the stabilization of HSA by sucrose. From solvation dynamics studies, it could be seen that the solvation time of DACIA inside domain-I of HSA decreases and increases regularly with increasing concentrations of urea and sucrose, respectively, while in the case of NPCE-tagged domain-III, the effect of sucrose on solvation time is evident only at high concentrations of urea. The denaturing and renaturing effects of urea and sucrose could be clearly seen from the steady state studies and circular dichroism spectroscopy measurements. A regular change in solvation time could only be observed in the case of domain-I and not in domain-III. The results indicate that the renaturing effect of sucrose on domain-III is not very evident when protein is in its native state, but is evident in when the protein is denatured.

Influence of Antipsychotic Drug Risperidone on Human Serum Albumin Affinity to Organic Anions

Risperidone is an antipsychotic drug. In blood, this drug binds mainly to human serum albumin (HSA) and is also transported by HSA. To study certain details of the interaction between risperidone and HSA, a fluorescent dye CAPIDAN was used as a reporter. This dye specifically fluoresces from HSA in serum and is highly sensitive to structural changes in HSA including pathology-induced changes. Interaction of CAPIDAN with HSA has been studied using time-resolved fluorescence techniques. The addition of phenylbutazone, a marker for the HSA drug-binding site I, leads to displacement of CAPIDAN from this site due to direct competition between phenylbutazone and the dye. The addition of risperidone induces a response of CAPIDAN fluorescence that is highly similar to its response to phenylbutazone. This response depends strongly on ionic strength and is very similar in both cases, phenylbutazone and risperidone. This similarity suggests that risperidone binds to HSA in the region of site I. In this site, the risperidone molecule probably covers the positive charge of Arginine 218 or Arginine 222 preventing their interaction with the CAPIDAN negatively charged carboxyl group. This effect was observed both in isolated HSA and in serum, suggesting similarity of the interaction. Thus, risperidone is able to prevent binding of organic anions (i.e. CAPIDAN as a drug-like molecule) to HSA.

Redox responsive curcumin-loaded human serum albumin nanoparticles: Preparation, characterization and in vitro evaluation

The purpose of this study was to fabricate redox-responsive human serum albumin (HSA) nanoparticles (NPs) through self-assembly of HSA molecules for incorporation of curcumin (CCM) as a hydrophobic drug molecule. The structural changes of HSA in self-assembly process of nanoparticle formation were investigated using fluorescence, UV–vis, circular dichroism spectroscopy and X-ray diffraction. Spectroscopy data show changes in secondary and tertiary structures of HSA in the process of nanoparticle formation, which can be indicative of the interaction of the hydrophobic drug with HSA molecules. Unlike free CCM, nanoparticulate curcumin is readily dispersed in aqueous medium. Furthermore, self-assembled HSA-CCM NPs release CCM in an environment imitating the intracellular environment with the trigger of acidic pH and redox potential. The in vitro release studies showed that at pH7.4 only 26% CCM was released from CCM-loaded HSA NPs in 48h. However, the release of CCM was significantly accelerated in the presence of 10mM glutathione (GSH) at pH7.4 or pH5.5, in which 57% and 70% of CCM was released, respectively. The incorporation of CCM in HSA nanoparticles enhanced the cellular uptake of CCM in comparison with free CCM results in higher anticancer efficacy against MCF-7 cells.

Investigating Interaction Between Biochanin A and Human Serum Albumin by Multi-spectroscopic and Molecular Simulation Methods

Biochanin A (BCA), the most abundant isoflavone in chickpeas, presents a wide range of biological activities, such as hypolipidaemic, anti-oxidative, anti-proliferative, and estrogen-like effects. We investigated the interaction between BCA and human serum albumin (HSA) via several techniques. UV–Vis absorption spectroscopy verified the conformational variation of HSA after BCA addition, and fluorescence spectroscopy revealed the relevant binding parameters. Circular dichroism spectroscopy was used to estimate the secondary structural changes of HSA with and without BCA. Molecular docking and dynamics simulations were then applied to study the characteristics of HSA with BCA. Energy decomposition analysis was used to prove that Trp214 in subdomain IIA of HSA is the most likely binding site of BCA. Van der Waals forces and hydrophobic interactions may play important roles during the binding process. All of our results showed that BCA presents significant binding affinity to HSA, thus confirming that the role of HSA has as an efficient transporter of biomolecules.

Comprehensive insight into the binding of sunitinib, a multi-targeted anticancer drug to human serum albumin

Binding studies between a multi-targeted anticancer drug, sunitinib (SU) and human serum albumin (HSA) were made using fluorescence, UV-vis absorption, circular dichroism (CD) and molecular docking analysis. Both fluorescence quenching data and UV-vis absorption results suggested formation of SU–HSA complex. Moderate binding affinity between SU and HSA was evident from the value of the binding constant (3.04×104M−1), obtained at 298K. Involvement of hydrophobic interactions and hydrogen bonds as the leading intermolecular forces in the formation of SU–HSA complex was predicted from the thermodynamic data of the binding reaction. These results were in good agreement with the molecular docking analysis. Microenvironmental perturbations around Tyr and Trp residues as well as secondary and tertiary structural changes in HSA upon SU binding were evident from the three-dimensional fluorescence and circular dichroism results. SU binding to HSA also improved the thermal stability of the protein. Competitive displacement results and molecular docking analysis revealed the binding locus of SU to HSA in subdomain IIA (Sudlow's site I). The influence of a few common ions on the binding constant of SU–HSA complex was also noticed.

Determination on the binding of thiadiazole derivative to human serum albumin: a spectroscopy and computational approach

4-[3-acetyl-5-(acetylamino)-2,3-dihydro-1,3,4-thiadiazole-2-yl]phenyl benzoate from the family of thiadiazole derivative has been newly synthesized. It has good anticancer activity as well as antibacterial and less toxic in nature, its binding characteristics are therefore of huge interest for understanding pharmacokinetic mechanism of the drug. The binding of thiadiazole derivative to human serum albumin (HSA) has been investigated by studying its quenching mechanism, binding kinetics and the molecular distance, r between the donor (HSA) and acceptor (thiadiazole derivative) was estimated according to Forster’s theory of non-radiative energy transfer. The Gibbs free energy (ΔG), enthalpy (ΔH) and entropy (ΔS) changes of temperature-dependent Kb was calculated, which explains that the reaction is spontaneous and exothermic. The microenvironment of HSA have also been studied using synchronous fluorescence spectroscopy, and the feature of thiadiazole derivative-induced structural changes of HSA have been carried using Fourier transform infrared spectroscopy and the Molecular modelling simulations explore the hydrophobic and hydrogen bonding interactions.

Investigation on the protein-binding properties of icotinib by spectroscopic and molecular modeling method

Icotinib is a highly-selective epidermal growth factor receptor tyrosine kinase inhibitor with preclinical and clinical activity in non-small cell lung cancer, which has been developed as a new targeted anti-tumor drug in China. In this work, the interaction of icotinib and human serum albumin (HSA) were studied by three-dimensional fluorescence spectra, ultraviolet spectra, circular dichroism (CD) spectra, molecular probe and molecular modeling methods. The results showed that icotinib binds to Sudlow's site I in subdomain IIA of HSA molecule, resulting in icotinib-HSA complexes formed at ground state. The number of binding sites, equilibrium constants, and thermodynamic parameters of the reaction were calculated at different temperatures. The negative enthalpy change (ΔHθ) and entropy change (ΔSθ) indicated that the structure of new complexes was stabilized by hydrogen bonds and van der Waals power. The distance between donor and acceptor was calculated according to Förster's non-radiation resonance energy transfer theory. The structural changes of HSA caused by icotinib binding were detected by synchronous spectra and circular dichroism (CD) spectra. Molecular modeling method was employed to unfold full details of the interaction at molecular level, most of which could be supported by experimental results. The study analyzed the probability that serum albumins act as carriers for this new anticarcinogen and provided fundamental information on the process of delivering icotinib to its target tissues, which might be helpful in understanding the mechanism of icotinib in cancer therapy.

Insights into the binding of thiosemicarbazone derivatives with human serum albumin: spectroscopy and molecular modelling studies

4-[(1Z)-1-(2-carbamothioylhydrazinylidene)ethyl]phenyl acetate [Ace semi],4-[(1Z)-1-(2-carbamothioylhydrazinylidene)ethyl]phenyl propanoate [Pro semi] from the family of thiosemicarbazones derivative has been newly synthesized. It has good anticancer activity as well as antibacterial and it is also less toxic in nature, its binding characteristics are therefore of huge interest for understanding pharmacokinetic mechanism of the drug. The binding of thiosemicarbazone derivative to human serum albumin (HSA) has been investigated by studying its quenching mechanism, binding kinetics and the molecular distance (r) between donor (HSA) and acceptor (thiosemicarbazone derivative) was estimated according to Forster’s theory of non-radiative energy transfer using fluorescence spectroscopy. The binding dynamics has been elaborated using synchronous fluorescence spectroscopy, and the feature of thiosemicarbazone derivative induced structural changes of HSA has been studied by circular dichorism, Fourier transform infrared spectroscopy. Molecular modelling simulations explore the hydrophobic interaction and hydrogen bonding which stabilizes the interaction.

Molecular interactions of benzophenone UV filters with human serum albumin revealed by spectroscopic techniques and molecular modeling

Benzophenone (BP)-type UV filters have been widely used in many personal care products to protect human from UV exposure. Their dermal applications can cause direct human health risk following accumulation in bloodstream. Few studies have addressed whether BP-type UV filters could bind and alter the structure and function of human serum albumin (HSA), the major carrier protein in plasma. Four benzophenones, BP-1, BP-2, BP-3 and BP-8 were selected to investigate their potentially toxic interactions with HSA and the intrinsic binding mechanism using combined spectroscopies and molecular docking techniques. Four benzophenones significantly quench the intrinsic fluorescence of HSA via static mode. The competitive binding fluorescence assay and molecular docking both revealed that the benzophenones bind at site II of HSA. Their binding constants range from 1.91×104M−1 to 12.96×104M−1 at 296K. BP-8 interacts with HSA mainly through hydrogen bonding interactions and van der Waals interactions, while hydrophobic interactions and electrostatic interactions are dominant for interactions between BP-1, BP-2, BP-3 and HSA. Molecular docking revealed that the changes in structural moiety and hydrophobicity of four benzophenones account for their different binding affinities. As further revealed by circular dichroism and time-resolved fluorescence decay, these benzophenones cause global and local structural changes of HSA, which illustrates their potential toxicity to cause structural damage of HSA. Two degradation products of BP-3 have higher binding affinities to HSA, suggesting higher potencies in causing adverse effects on human health.

Molecular dynamics study of conformational changes in human serum albumin by binding of fatty acids

Human serum albumin (HSA) binds with fatty acids under normal physiologic conditions. To date, there is little published information on the tertiary structure of HSA-fatty acid complex in aqueous solution. In the present study, we used molecular dynamics (MD) simulations to elucidate possible structural changes of HSA brought about by the binding of fatty acids. Both unliganded HSA and HSA-fatty acid complex models for MD calculations were constructed based on the X-ray crystal structures. Five myristates (MYRs) were bound in the HSA-fatty acid complex model. In the present MD study, the motion of domains I and III caused by the binding of MYR molecules increased the radius of gyration of HSA. Root-mean-square fluctuations from the MD simulations revealed that the atomic fluctuations of the specific amino acids at drug-binding site I that can regulate the drug-binding affinity were increased by the binding of MYR molecules. Primary internal motions, characterized by the first three principal components, were observed mainly at domains I and III in the principal component analysis for trajectory data. The directional motion projected on the first principal component of unliganded HSA was conserved in HSA-MYR complex as the third principal directional motion with higher frequency. However, the third principal directional motion in unliganded HSA turned into the first principal directional motion with lower frequency in the HSA-MYR complex. Thus, the present MD study provides insights into the possible conformational changes of HSA caused by the binding of fatty acids.