Human Serum Albumin Ligands Research Articles

Effect of L-thyroxine, a Human Serum Albumin Natural Ligand, on the Kinetics of β-amyloid Peptide Fibril Formation

Ligands of human serum albumin (HSA) are capable of modulating its interaction with β-amyloid peptide (Aβ), which is a key factor in the pathogenesis of Alzheimer’s disease (AD). L-thyroxine (L-Tr), a natural HSA ligand, is associated with the pathogenesis of AD according to epidemiological and animal model studies. In this work, we studied the kinetics of Aβ fibril formation in the presence of L-Tr and HSA using a fluorescent test with thioflavin T. L-Tr had no significant effect on the inhibitory effect of HSA on fibril growth. At the same time, L-Tr itself had an inhibitory effect similar to that of HSA. Our data can partially explain the relationship between AD and thyroid pathologies.

Protein binding and anticancer activity of two newly synthesized Schiff base platinum (II) complexes: A theoretical and experimental study

Platinum-based complexes are effective chemotherapeutics which are applied in chemotherapy regimens to treat cancer. The present study is designed to investigate the interaction of new Schiff base-platinum complexes named Pt-246 and Pt-247 with human serum albumin (HSA) via spectroscopy techniques, molecular docking studies, and molecular dynamics (MD) simulations. Also, the toxicity effects of platinum complexes were studied on two cancer cell lines, HCT116 and MCF7 by MTT assay. The thermodynamic characteristics of the interaction between Pt-complexes and HSA were examined through fluorescence spectroscopy. According to the fluorescence analysis, the quenching mechanisms of Pt-246 and Pt-247 with HSA can be attributed to the dynamic model. Circular dichroism (CD) spectroscopy results indicated that the ordered secondary structure of HSA can be altered by the Pt-complexes. According to the docking study, the main interactions of Pt-246 and Pt-247 complexes with HSA were van der Waals, as well as hydrophobic and hydrogen bonding interactions. MD simulations revealed that the main interactions between HSA protein and ligands can be attributed to van der Waals forces. The Coulomb energy in HSA:Pt-247 was almost twice as large as in HSA:Pt-246, indicating that Pt-247 formed a far more stable complex. The IC50 values of Pt-246-treated MCF7 and HCT116 cell lines were determined 58.52 ± 1.19 (P-value 0.0001) and 42.68 ± 1.6 (P-value 0.054), respectively. On the other hand, the IC50 values of Pt-247-treated MCF7 and HCT116 cell lines were calculated 52.98 ± 3.2 (P-value 0.038) and 31.7 ± 1 (P-value 0.001), respectively. The current study concluded that Schiff base ligands can be employed in designing potential Pt-based complexes as therapeutic products with anti-cancer activities.

The role of human serum albumin in prevention and treatment of Alzheimer’s disease

Alzheimer’s disease (AD) has been and remains the main cause of dementia in aging patients. This neurodegenerative disease belongs to the progressive and socially significant ones. There are several hypotheses for the development of AD: the tau hypothesis, the amyloid cause, the cholinergic cause, the cause of oxidative stress and inflammation. The lack of a generally accepted understanding of the etiology and pathogenesis of AD hinders the development of new effective mechanisms for its treatment and prevention. In 2021, for the first time, a drug for pathogenetic therapy of AD (aducanumab) was approved, which helps to reduce the content of amyloid-β peptide (Aβ) in the brain of patients. Another promising approach to the treatment of AD, aimed at removing Aβ from the patient’s central nervous system, is the impact on human serum albumin (HSA), which carries 90% of Aβ in the blood serum and 40–90% of Aβ in the cerebrospinal fluid. In clinical practice, plasmapheresis has already been tested and shown to be effective with the replacement of one’s own HSA with a purified therapeutic albumin preparation. Another variant of this approach is to enhance the interaction of HSA with Aβ through the action of exogenous and endogenous HSA ligands, such as serotonin, ibuprofen and some unsaturated fatty acids. In vivo studies confirm the association of this group of ligands with the pathogenesis of AD. These substances are well-studied natural metabolites or drugs, which greatly simplifies the development of new methods of therapy and prevention of AD with their use. In general, a new direction of scientific research devoted to the study of HSA as a carrier and depot of Aβ in the blood and cerebrospinal fluid will expand our understanding of Aβ metabolism and its role in the pathogenesis of AD.

Virtual Screening of Human Serum Albumin Mutants to Optimize the Search for its Forms that Increase Affinity to Amyloid-Β Peptide

A promising approach to the treatment of Alzheimer's disease (AD) is the removal of amyloid-β peptide (Aβ) from the patient's central nervous system by acting on human serum albumin (HSA). HSA carries 90% of Aβ in blood serum and 40-90% of Aβ in the cerebrospinal fluid (CNS). In this work, virtual screening of all possible mutant forms of HSA based on the data of the I-Mutant service made it possible to predict changes in HSA stability and identify the most “sensitive” regions of its polypeptide chain to substitutions. The data obtained will be used to optimize the search for HSA forms with increased affinity to Aβ, as well as to study the mechanisms underlying the modulating effects of HSA ligands on its interaction with Aβ, which can become the basis for the development of new approaches to therapy and prevention of AD.

Systematic search for peptide and protein ligands of human serum albumin capable of affecting its interaction with amyloid β peptide

Background. Human serum albumin (HSA) is a natural buffer of amyloid-β peptide (Aβ), a key factor in the development of Alzheimer’s disease (AD). A promising approach to the AD prevention is to reduce the concentration of free Aβ by targeted stimulation of the interaction between HSA and Aβ. This approach can be implemented by increasing the affinity of HSA to Aβ through the action of HSA ligands, which was previously demonstrated for some low molecular weight ligands. The aim of the study was to search for peptide and protein ligands of human serum albumin capable of affecting its interaction with Aβ. Materials and methods. To perform a systematic search for peptides/proteins, HSA ligands that are capable of affecting Aβ-HSA interaction, we analyzed the DrugBank, BioGRID, and IntAct databases. As criteria for selecting candidates, along with physicochemical characteristics (molecular weight, solubility, blood-brain barrier passage, molar concentration), we used the requirements of extracellular proteins localization and strict association with AD, according to the DisGeNET and Open Targets Platform databases as well as Alzforum online resource. The algorithms for searching and analyzing the obtained data were implemented using the high-level programming language Python. Results. A candidate panel of 11 peptides and 34 proteins was formed. The most promising candidates include 4 peptides (liraglutide, exenatide, semaglutide, insulin detemir) and 4 proteins (S100A8, transferrin, C1 esterase inhibitor, cystatin C). Conclusions. Selected peptide and protein candidates are subject to experimental verification regarding their effect on the HSA-Aβ interaction and can become the basis for the development of first-in-class drugs for the prevention of Alzheimer’s disease.

Evaluation of Single-Nucleotide Polymorphisms in Human Serum Albumin Associated with Alzheimer's Disease

Human serum albumin (HSA) is a natural buffer for amyloid в peptide (Аβ), which is a key factor in the development of Alzheimer's disease (AD). An increase in HSA affinity to Аβ can be achieved via HSA saturation with low-molecular-weight ligands, such as serotonin or specific fatty acids. The conducted analysis of the genomic data of exomes (WES) associated with AD (ADSP database) revealed the presence of a single-nucleotide polymorphism of the HSA gene at the binding sites of ibuprofen, arachidonic and oleic acids. Research into the properties of the revealed genetic variants of HSA should be carried out to determine those variants that are susceptible to the modulatory action of HSA ligands, thus increasing its affinity to Aβ.

Concomitant binding of two fluorescent probes at site-I of human serum albumin: The protein acting as a scaffold enabling fluorescence resonance energy transfer.

Human serum albumin (HSA) is the primary drug carrier in the blood plasma. Here, I aimed to show that two ligands can be accommodated simultaneously in the binding site-I of HSA. To do so, I studied the interaction inside the protein among site-I ligands of HSA via fluorescence resonance energy transfer (FRET), synchronous fluorescence, red edge excitation shift (REES), and induced circular dichroism (ICD). Warfarin (WAR), coumarin-153 (C153), 6-(p-toluidino)-2-naphthalenesulfonic acid sodium salt (TNS), dansylglycine (DGY), and 4-(dicyanomethylene)-2-methyl-6-(4-dimethylaminostyryl)-4H-pyran (DCM) were enrolled in the investigation. I found that WAR can transfer energy to C153 only in the presence of the protein. In addition, the presence of WAR at site-I altered the protein microenvironment felt by C153. The alteration was detected by measuring the synchronous fluorescence, REES, and ICD in C153. The findings were validated by measuring the energy transfer from TNS to DCM and the alteration in synchronous fluorescence and REES. FRET was not observed using WAR as donor and DGY as acceptor. The result is consistent, as DGY is a site-II ligand at a higher WAR distance. In all studied cases, the effects were only observed in the presence of HSA. In conclusion, the protein acted as a scaffold approximating the ligands. These findings prove that more than one ligand can simultaneously be complex at site-I of HSA.

Chlorine Atoms of an Aripiprazole Molecule Control the Geometry and Motion of Aripiprazole and Deschloro-aripiprazole in Subdomain IIIA of Human Serum Albumin.

Aripiprazole (ARP), an antipsychotic drug, binds more strongly to human serum albumin (HSA) than the other ARP derivatives. In addition, the signs for the extrinsic Cotton effects for HSA complexed with ARP or deschloro-ARP are reversed. In this study, we report on a structural–chemical approach using circular dichroism (CD) spectroscopic analysis, X-ray crystallographic analysis, and molecular dynamics simulations. The objective was to examine the relationship between the induced CD spectra and the structural features of the HSA complexes with ARP or deschloro-ARP. The intensity of the induced CD spectra of the HSA complexes with ARP or deschloro-ARP was reduced with increasing temperature. We determined the crystal structure of the HSA complexed with deschloro-ARP in this study and compared it to HSA complexed with ARP that we reported previously. The comparison of these structures revealed that both ARP and deschloro-ARP were bound at the site II pocket in HSA and that the orientation of the molecules was nearly identical. Molecular dynamics simulations indicated that the molecular motions of ARP and deschloro-ARP within the site II pocket were different from one another and the proportion of stacking interaction formations of Tyr411 with the dihydroquinoline rings of ARP and deschloro-ARP was also different. These findings indicate that the induced CD spectra are related to the molecular motions and dynamic interactions of ARP and deschloro-ARP in HSA and may help to understand the molecular recognition and motion that occurs within the binding site for the other HSA ligands more clearly.

Crystallographic analysis of interaction between cisplatin and human serum albumin: Effect of fatty acid

Metallodrugs are important for anticancer treatments. They bind mainly to human serum albumin (HSA) in blood circulation, greatly modulating their pharmacokinetics and anticancer efficacy. Fatty acid (FA) is one of the most important endogenous ligands of HSA with tight binding to HSA and affecting its conformation. However, the effect of fatty acids on metallodrugs interaction with HSA is unknown. Here we identify the binding sites of a widely used metallodrug, cisplatin, in HSA in the presence or absence of a representative fatty acid, myristate, by X-ray crystallography. Our crystal structures indicate that the sidechain of residue Met548 becomes more exposed to solvent in the presence of fatty acid, and is the main Pt binding site together with Met329 in HSA:Myr:cisplatin ternary structure. An undoubted new Pt binding site is detected at His338 in the presence of fatty acid, and additional two sites are also identified at His146 and His440 + K436. In addition, we revealed the mechanism of cisplatin-induced HSA aggregation, which is due to the crosslinking between Met298 and His510 of two HSA molecules.

Effect of Fatty Acids and Uremic Toxins on the Binding of Nateglinide, an Insulin Secretagogue, to Site II on Human Serum Albumin.

Nateglinide (NAT) is used to treat diabetes, stimulating pancreatic islet β-cells with residual insulin secretory capacity to increase insulin secretion. NAT has been reported to bind to human serum albumin (HSA), but the detail is still unclear. In the current study, we investigated the location and the affinity for the binding of NAT to HSA. Quantitative analysis data from the ultrafiltration experiment indicated that NAT binds strongly to a primary site on HSA with a high affinity. The presence of diazepam (DZP) or ibuprofen (IB), the specific site II ligands of HSA, decreased the binding constants of NAT respectively, without the significant changes in the number of binding sites. Whereas warfarin (WF), a site I specific ligand, did not affect the binding of NAT. Fluorescent replacement experiment showed that NAT replaced dansylsarcosine (DNSS), a site II probe of HSA, but not WF. An increasing level of myristate and uremic toxins, indoxyl sulphate (IS), indoxyl acetate (IA) and p-cresyl sulphate (PCS), during renal disease significantly increased the concentration of unbound NAT. These findings suggest that NAT specifically binds to site II of HSA and the binding capacity and pharmacokinetics of NAT change in renal diseases.

Ibuprofen Favors Binding of Amyloid-β Peptide to Its Depot, Serum Albumin.

The deposition of amyloid-β peptide (Aβ) in the brain is a critical event in the progression of Alzheimer’s disease (AD). This Aβ deposition could be prevented by directed enhancement of Aβ binding to its natural depot, human serum albumin (HSA). Previously, we revealed that specific endogenous ligands of HSA improve its affinity to monomeric Aβ. We show here that an exogenous HSA ligand, ibuprofen (IBU), exerts the analogous effect. Plasmon resonance spectroscopy data evidence that a therapeutic IBU level increases HSA affinity to monomeric Aβ40/Aβ42 by a factor of 3–5. Using thioflavin T fluorescence assay and transmission electron microcopy, we show that IBU favors the suppression of Aβ40 fibrillation by HSA. Molecular docking data indicate partial overlap between the IBU/Aβ40-binding sites of HSA. The revealed enhancement of the HSA–Aβ interaction by IBU and the strengthened inhibition of Aβ fibrillation by HSA in the presence of IBU could contribute to the neuroprotective effects of the latter, previously observed in mouse and human studies of AD.

Serotonin Promotes Serum Albumin Interaction with the Monomeric Amyloid β Peptide.

Prevention of amyloid β peptide (Aβ) deposition via facilitation of Aβ binding to its natural depot, human serum albumin (HSA), is a promising approach to preclude Alzheimer’s disease (AD) onset and progression. Previously, we demonstrated the ability of natural HSA ligands, fatty acids, to improve the affinity of this protein to monomeric Aβ by a factor of 3 (BBRC, 510(2), 248–253). Using plasmon resonance spectroscopy, we show here that another HSA ligand related to AD pathogenesis, serotonin (SRO), increases the affinity of the Aβ monomer to HSA by a factor of 7/17 for Aβ40/Aβ42, respectively. Meanwhile, the structurally homologous SRO precursor, tryptophan (TRP), does not affect HSA’s affinity to monomeric Aβ, despite slowdown of the association and dissociation processes. Crosslinking with glutaraldehyde and dynamic light scattering experiments reveal that, compared with the TRP-induced effects, SRO binding causes more marked changes in the quaternary structure of HSA. Furthermore, molecular docking reveals distinct structural differences between SRO/TRP complexes with HSA. The disintegration of the serotonergic system during AD pathogenesis may contribute to Aβ release from HSA in the central nervous system due to impairment of the SRO-mediated Aβ trapping by HSA.

Spectroscopic and molecular docking investigation of the binding of a bioactive mercaptobenzimidazole-functionalized Schiff base to human serum albumin

This study was conducted to examine the binding of a newly synthesized Schiff base derived from 2-hydroxy-1-naphthaldehyde and 5-amino-2-mercaptobenzimidazole with human serum albumin (HSA) employing various biophysical techniques. The thermal-based fluorescence quenching data indicated that static quenching occurred between the ligand and HSA. The fluorescence results expose that ligand quenches the intrinsic fluorescence of HSA through a static quenching procedure. The thermodynamic parameters of the binding interaction, obtained using van't Hoff equation indicated the spontaneity of the reaction. The stability of the HSA–ligand complex resulted from hydrogen bonding and hydrophobic interactions, which afforded a substantial binding affinity between ligand and HSA. UV–Vis and circular dichroism data indicated that ligand binding induced conformational changes in HSA. The energy transfer efficiency determined according to Fӧorster's theory. Absorption, distribution, metabolism, and excretion (ADME) and Lipinski’s drug likeness of the ligand was predicted, revealing that it had auspicious physicochemical properties for oral bioavailability. Furthermore, molecular modeling was also employed to determine the location of the ligand in HSA binding sites, which revealed that the ligand interacted with polar and apolar residues of site I (subdomain IIA) of HSA, predominantly through hydrophobic and hydrogen bonding interactions.

Untargeted Defining Protein-Metabolites Interaction Based on Label-Free Kinetic Size Exclusion Chromatography-Mass Spectrometry.

The specific interactions between protein and metabolites (PMIs) are closely related to many cellular processes and play a vital role in signal transduction and regulating material and energy metabolism. However, most of the available analytical strategies for PMIs involve chemical modification of metabolites or immobilization of protein, which has restricted current PMIs study mainly to lipid-protein and hydrophobic metabolites. In this work, a label-free online kinetic size exclusion chromatography-mass spectrometry (KSEC-MS) method combined with untargeted metabolomics was developed to define PMIs in a complex system. The metabolite mixture and target protein were injected into the SEC column sequentially without preincubation, and the separation results of KSEC were monitored by global metabolite profiling with mass spectrometry. The potential ligands in the metabolite mixture can be discovered if their migration patterns were affected by the target protein and the variation was positively correlated with the concentration of target protein. To verify this approach, carbonic anhydrase was first selected as a test protein, and acetazolamide as its known inhibitor was successfully defined. Furthermore, human serum albumin (HSA) as the common transport carrier of metabolites was selected as a target protein to demonstrate the usefulness of this approach. Multiple endogenous ligands of HSA were simultaneously defined from the extracted metabolites of human serum; most of them are polar metabolites rather than nonpolar lipids. This approach can provide a novel way for mapping and identifying unknown PMIs in a complex system, especially for polar metabolites-protein interactions.

Monolith weak affinity chromatography for μg-protein-ligand interaction study

Affinity monolith columns of 375 nL (effective length 8.5 cm, internal diameter 75 μm) were developed for protein-ligand affinity investigations needing only 3 μg of human serum albumin (HSA). To promote specific interactions and avoid non-specific ones, different combinations of monolithic supports and bio-functionalization pathways were evaluated. Silica and glycidylmethacrylate based monoliths were in-situ synthesized and grafted with HSA. Two direct grafting methods epoxy-amine and Schiff Base plus the streptavidin-biotin method were compared. The columns were evaluated by frontal analysis with ligands of known affinity for HSA. It is shown that a classical capillary electrophoresis instrument equipped with an external pressure device can be used to do weak affinity chromatography at low pressure (less than 1.2 MPa) in a fully automated way and with very low reagent consumption. The grafting pathways were compared in terms of (i) total and active amounts of immobilized protein, (ii) non-specific interactions, (iii) protein denaturation. According to these criteria, the organic monoliths combined with the streptavidin-biotin approach provided the best results. This immobilization pathway led to the highest active protein content (40 pmol of HSA per 8.5-cm column) with less than 10% non-specific interactions and 84% protein activity. The target grafting step lasts only 10 min and is UV-monitored, the UV breakthrough curve giving the exact amount of bound protein. This novel approach was validated by Kd measurements of 3 known ligands of HSA. Streptavidin generic monolith columns could be stored at 4 °C for 3 months maintaining activity. μg of a biotin modified sensitive protein could be attached to a stable streptavidin monolith for immediate interaction studies avoiding stability problems. This development was subsequently extended to another protein of higher pharmaceutical interest: the N-terminal domain of HSP90. Affinity was measured for two known ligands and determined Kd values were in accordance with the literature, proving that our technique is applicable to other proteins.

Rapid Investigation and Screening of Bioactive Components in Simo Decoction via LC-Q-TOF-MS and UF-HPLC-MD Methods.

Simo decoction (SMD), as a traditional medicine, is widely used in the treatment of gastrointestinal dysmotility in China. In this study, a combined method of liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS) and ultrafiltration high-performance liquid chromatography molecular docking (UF-HPLC-MD) was efficiently employed to identify and screen bioactive ingredients in SMD. Ninety-four major constituents were identified or tentatively characterized by comparing their retention times and mass spectra with standards or literature data by using LC-Q-TOF-MS, and the ascription of those compounds were classified for the first time. Among them, 13 bioactive ingredients, including norisoboldine, eriocitrin, neoeriocitrin, narirutin, hesperidin, naringin, neohesperidin, hesperitin-7-O-glucoside, linderane, poncirin, costunolide, nobiletin, and tangeretin, were primarily identified as the human serum albumin (HSA) ligands at a range of docking scores from −29.7 to −40.6 kJ/mol by UF-HPLC-MD. The results indicate the systematic identification and screening of HSA ligands from Simo decoction guided by LC-Q-TOF-MS and UF-HPLC-MD represents a feasible and efficient method that could be extended for the identification and screening of other bioactive ingredients from natural medicines.

Metal-catalyzed oxidation of human serum albumin does not alter the interactive binding to the two principal drug binding sites

It is well known that various physiological factors such as pH, endogenous substances or post-translational modifications can affect the conformational state of human serum albumin (HSA). In a previous study, we reported that both pH- and long chain fatty acid-induced conformational changes can alter the interactive binding of ligands to the two principal binding sites of HSA, namely, site I and site II. In the present study, the effect of metal-catalyzed oxidation (MCO) caused by ascorbate/oxygen/trace metals on HSA structure and the interactive binding between dansyl-L-asparagine (DNSA; a site I ligand) and ibuprofen (a site II ligand) at pH 6.5 was investigated. MCO was accompanied by a time-dependent increase in carbonyl content in HSA, suggesting that the HSA was being oxidized. In addition, The MCO of HSA was accompanied by a change in net charge to a more negative charge and a decrease in thermal stability. SDS-PAGE patterns and α-helical contents of the oxidized HSAs were similar to those of native HSA, indicating that the HSA had not been extensively structurally modified by MCO. MCO also caused a selective decrease in ibuprofen binding. In spite of the changes in the HSA structure and ligand that bind to site II, no change in the interactive binding between DNSA and ibuprofen was observed. These data indicated that amino acid residues in site II are preferentially oxidized by MCO, whereas the spatial relationship between sites I and II (e.g. the distance between sites), the flexibility or space of each binding site are not altered. The present findings provide insights into the structural characteristics of oxidized HSA, and drug binding and drug-drug interactions on oxidized HSA.

The Interaction of a New Schiff Base Ligand with Human Serum Albumin: Molecular Docking and Molecular Dynamics Simulation Studies

ABSTRACTThe interaction of a new heterocyclic Schiff base bearing pyridine and pyrimidine cycles, with human serum albumin (HSA) using molecular docking and molecular dynamics simulation methods was examined. Molecular docking studies showed that the ligand was bonded to the IB domain of the protein. It was found that there was one hydrogen bond interaction between HSA and the ligand. The standard Gibbs free energy for binding of the ligand to HSA was calculated as −9.63 kcal.mol−1. The results of the molecular dynamics simulation showed that the root mean square deviation (RMSD) of the non-liganded HSA and the HSA–ligand complex reached equilibration after 1000 ps. The study of the radius of gyration revealed that there was a conformational change when the HSA–ligand complex was formed. Finally, analyzing the RMS fluctuations (RMSF) suggested that the structure of the ligand binding site remained approximately rigid during the simulation.

Drug-protein binding of Danhong injection and the potential influence of drug combination with aspirin: Insight by ultrafiltration LC–MS and molecular modeling

Danhong injection (DHI) is a widely used Chinese medicine injection (CMI) for the clinical treatment of cardiovascular and cerebrovascular diseases. In this study, a simple and efficient in vitro method based on ultrafiltration LC–MS and molecular modeling has been developed to study the human serum albumin (HSA) binding of the compounds in DHI. Seven major components including protocatechuic aldehyde, p-coumaric acid, salvianolic acid D, rosmarinic acid, salvianolic acid E, lithospermic acid and salvianolic acid B were identified as HSA ligands and their binding degrees in the proposed non-saturated model were 26.17, 37.69, 99.77, 91.78, 96.91, 99.42 and 98.10%, respectively. Considering the drug-HSA binding property of the compounds in DHI may change during drug combination therapy, competitive binding assay was carried out to evaluate the influence of aspirin on the DHI-HSA binding. Experimental results revealed that the salvianolic acids in DHI had stronger binding ability to HSA than sodium salicylate. To further verify the results above, molecular modeling and probe displacement assay were conducted to investigate the optimum binding site and binding affinity of the ligands on HSA. Our findings suggested that the established method could be a powerful tool to study the drug-HSA binding property of CMIs.

Influence of Taxifolin on the Human Serum Albumin–Propranolol Interaction: Multiple Spectroscopic and Chemometrics Investigations and Molecular Dynamics Simulation

The aim of the present study is to characterize the competition between propranolol (PROP) and taxifolin (TAX) in binding to human serum albumin (HSA) in a physiological buffer (pH 7.4) using multiple spectroscopic, chemometrics and molecular dynamics simulation measurements. Fluorescence analysis was used to determine the binding and quenching properties of HSA–ligand complexes in binary and ternary systems. These spectral data were further analyzed by the multivariate curve resolution-alternating least squares method. In addition, the concentration profiles and pure spectra of three species (HSA, ligand and HSA–ligand complex) and the apparent equilibrium constants K app were evaluated. Fluorescence spectroscopy showed that in the presence of TAX, the binding constant of HSA–PROP increased. The effect of ligands on the secondary structure of the protein has been analyzed by using Fourier transform infrared spectra. The conformational change of the protein was analyzed using synchronous fluorescence spectroscopy, three-dimensional fluorescence spectra and molecular dynamics (MD) simulation. The results of synchronous fluorescence and three-dimensional fluorescence spectra show that PROP alters the microenvironment around the tryptophan (Trp) and tyrosine (Tyr) residues in the presence of TAX. According to the MD simulation, these ligands can interact with the protein, affecting the secondary structure of HSA and modifying its tertiary structure. MD simulations and experimental data support each other.