Stability Of Human Serum Albumin Research Articles

Interaction and Binding Kinetics of Different Substituted Pyrazines with HSA: Based on Multispectral, Physiological Activity, and Molecular Dynamics Simulations

Pyrazine is a kind of compound containing pyrazine ring structure, which has attracted the attention of researchers because of its special aroma and healthcare function. It is currently used in spices, pharmaceuticals, food additives, and other industries. Human serum albumin (HSA) is a transport and storage protein in the human circulatory system. Previous research reported that pyrazine can interact with albumin, but the interaction between pyrazine and HSA has not been reported. Consequently, this study is based on the multispectral method and molecular dynamics (MD) simulation, exploring the interaction between different pyrazines and HSA. The results show that the four pyrazines mainly quench the endogenous fluorescence of HSA through the static quenching mechanism. Molecular docking indicates that hydrophobic forces play a major role in the binding of pyrazines with HSA. At the same time, synchronous fluorescence spectroscopy, circular dichroism (CD), three-dimensional fluorescence spectroscopy, and resonance light scattering (RLS) showed that each pyrazine compound can change the conformation of HSA. MD simulation results show that the combination of four pyrazines and HSA can enhance the stability of HSA. Among them, 2,3,5,6-tetramethylpyrazine (TTMP) and 2,3,5-trimethylpyrazine (TMTP) binding is the most stable and the most unstable combination with HSA, respectively. In addition, the four pyrazines could affect the physiological activity of HSA. This study is of great significance to explore the mechanism of action of pyrazine compounds in vivo and can provide useful information for the application of pyrazine compounds in food.

Human Serum Albumin in the Presence of Small Platinum Nanoparticles

Noble metal materials, especially platinum nanoparticles (Pt NPs), have immense potential in nanomedicine as therapeutic agents on account of their high electron density and their high surface area. Intravenous injection is proposed as the best mode to deliver the product to patients. However, our understanding of the reaction of nanoparticles with blood components, especially proteins, is far behind the explosive development of these agents. Using synchrotron radiation circular dichroism (SRCD), we investigated the structural and stability changes of human serum albumin (HSA) upon interaction with PEG-OH coated Pt NPs at nanomolar concentrations, conditions potentially encountered for intravenous injection. There is no strong complexation found between HSA and Pt NPs. However, for the highest molar ratio of NP:HSA of 1:1, an increase of 18 °C in the thermal unfolding of HSA was observed, which is attributed to increased thermal stability of HSA generated by preferential hydration. This work proposes a new and fast method to probe the potential toxicity of nanoparticles intended for clinical use with intravenous injection.

Understanding the intricacy of protein in hydrated deep eutectic solvent: Solvation dynamics, conformational fluctuation dynamics, and stability

Deep eutectic solvents (DESs) are potential biocatalytic media due to their easy preparation, fine-tuneability, biocompatibility, and most importantly, due to their ability to keep protein stable and active. However, there are many unanswered questions and gaps in our knowledge about how proteins behave in these alternate media. Herein, we investigated solvation dynamics, conformational fluctuation dynamics, and stability of human serum albumin (HSA) in 0.5 Acetamide/0.3 Urea/0.2 Sorbitol (0.5Ac/0.3Ur/0.2Sor) DES of varying concentrations to understand the intricacy of protein behaviour in DES. Our result revealed a gradual decrease in the side-chain flexibility and thermal stability of HSA beyond 30 % DES. On the other hand, the associated water dynamics around domain-I of HSA decelerate only marginally with increasing DES content, although viscosity rises considerably. We propose that even though macroscopic solvent properties are altered, a protein feels only an aqueous type of environment in the presence of DES. This is probably the first experimental study to delineate the role of the associated water structure of the enzyme for maintaining its stability inside DES. Although considerable effort is necessary to generalize such claims, it might serve as the basis for understanding why proteins remain stable and active in DES.

Comparative investigation on interaction mechanism and native conformation of human serum albumin with organometallic iridium(III) complexes via spectroscopic and electrochemical approaches

Organometallic iridium(III) complexes, as high-efficient phototherapic drugs for cancer therapy and tumor treatment, have attracted widespread interest in diverse areas. To further clarify the biological functions of iridium(III) complexes, the interaction mechanism and native conformation of human serum albumin (HSA) affected by three organometallic iridium(III) complexes containing curcuminoid ligands were systematically and comparably investigated via various spectroscopic and electrochemical approaches. The results revealed that three iridium(III) complexes reacted with HSA tightly at its Sudlow's site I under the binding forces of van der Waals interaction, protonation, and hydrogen bond, leading to the hypochromic effect in the absorption spectrum of HSA and the static quenching in the intrinsic fluorescence of HSA. Three iridium(III) complexes disturbed the native conformation of HSA by reducing its α-helical content, finally reducing the melting temperature and the thermal stability of HSA. In comparison, the iridium(III) complex containing curcuminoid ligand with bigger spatial steric-hindrance effect and stronger chemical polarity was prone to binding with HSA more tightly and subsequently affecting its native conformation and biological function more prominently. This research finding reveals the interaction mechanisms of organometallic iridium(III) complexes with HSA and evaluates the function of curcuminoid ligands during their binding interactions, which pays the way for the targeted design and the potential application of novel iridium(III) complexes in biological fields.

Biophysical insight into the anti-fibrillation potential of Glyburide for its possible implication in therapeutic intervention of amyloid associated diseases

Protein aggregation is an underlying cause of many neurodegenerative diseases. Also, the overlapping pathological disturbances between neurodegenerative diseases and type-2 diabetes mellitus have urged the scientific community to explore potential of already available anti-diabetic medications in impeding amyloid formation too. Recent study brief out promising potential of an anti-diabetic drug Glyburide(GLY) as an inhibitor of amyloid fibrillation utilizing several biophysical techniques, computational methods and imaging tools. The mechanism of interaction was elucidated and the structural alterations in human serum albumin(HSA) as well as the microenvironment changes of its fluorophores(tryptophan, tyrosine) upon interacting with GLY were studied by spectroscopic techniques like Circular dichroism and synchronous fluorescence. Binding studies detailing about the GLY-HSA complex distance and the energy transfer efficiency was obtained by Fluorescence resonance energy transfer. For aggregation inhibition studies, the existence and size of aggregates formed in HSA and their inhibition by GLY was determined by Turbidity assay, Dynamic light scattering and Rayleigh light scattering along with dye binding assays. The ThT kinetics measurements analysis suggested that GLY deaccelerates fibrillation by decrement of apparent rate(Kapp) constant. The inhibitory effect of GLY might be attributed to native structure stabilization of HSA by obstruction into β-sheet conversion as confirmed by CD spectroscopy results. Amyloid inhibition and suppression of amyloid-induced hemolysis by GLY was further delineated by TEM and SEM analysis respectively. All these findings for the first time report the new facet of the anti-amyloidogenic potential of GLY, making it a promising candidate to treat neurodegenerative diseases too in the near future.

Experimental and Computational Characterisation of the Molecular Interactions between 1‐Butyl‐1‐methyl‐pyrrolidin‐1‐ium bis(trifluoromethanesulphonyl)imide and Human Serum Albumin

AbstractRecently, ionic liquids (ILs) have emerged as a therapeutic agent for industrial‐based pharmacology. In this regard, the current study highlights the key aspects of IL, (1‐butyl‐1‐methyl‐pyrrolidine‐1‐ium bis(trifluoromethanesulphonyl)imide), [Pyr C4 NTf3−] such as pharmacodynamics and pharmacokinetics. We performed a detailed experimental and computational analysis of [Pyr C4 NTf3−] with human serum albumin (HSA) using various spectroscopic techniques viz. UV‐vis, steady‐state fluorescence, synchronous fluorescence, time‐resolved fluorescence, steady‐state anisotropy and tensiometry study along with molecular docking. Absorption and emission spectral studies revealed the complex formation between [Pyr C4 NTf3−] and HSA where the binding constant (Kb) was found to be 5.5×103 Lmol−1. Thermodynamic parameters, Gibb's free energy change (ΔG), molar enthalpy change (ΔH), and molar entropy change (ΔS) revealed that the complex formation process is spontaneous, and enthalpy driven where involvement of hydrogen bond and van der Waal forces play a major role. Further, CD spectroscopy and ANS experiment showed the stability of HSA in presence of [Pyr C4 NTf3−]. A site marker study suggested that the binding of [Pyr C4 NTf3−] occurred at site II in HSA which is well supported by a molecular docking study. Also, MD simulation was done which revealed the stability of the complex formed between HSA and [Pyr C4 NTf3−]. The study might help in utilizing [Pyr C4 NTf3−], IL as a therapeutic agent in the pharmaceutical industries.

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.

Human Serum Albumin Misfolding in Aging and Disease.

Age-dependent conformational stability of human serum albumin was determined by the method of fluorescent bilayer liposome assay. After pre-heating at 80 °C, albumin in the sera of 74-year-old healthy subjects exhibited hydrophobic effects on liposomes and made liposomal membrane phospholipids more susceptible to hydrolysis by the lipolytic enzyme phospholipase A2. In contrast, albumin in the sera of 24-year-old individuals was stable at 80 °C and displayed no increased hydrophobic effects on liposomes. The results suggest that albumin in the sera of 74-year-old subjects is more easily converted to a misfolded form in which its protein structure is altered when compared to albumin in the sera of 24-year-old individuals. Misfolded albumin can lose its ability to carry out its normal homeostatic functions and may promote alterations in membrane integrity under inflammatory conditions. However, our investigation has limitations that include the lack of testing sera from large numbers of individuals across a broad range of age to validate our preliminary observations of age-dependent differences in albumin stability and its interactions with liposomes.

A multi-spectroscopic and computational simulations study to delineate the interaction between antimalarial drug hydroxychloroquine and human serum albumin

Hydroxychloroquine (HCQ), a quinoline based medicine is commonly used to treat malaria and autoimmune diseases such as rheumatoid arthritis. Since, human serum albumin (HSA) serves as excipient for vaccines or therapeutic protein drugs, it is important to understand the effect of HCQ on the structural stability of HSA. In this study, the binding mechanism of HCQ and their effect on stability of HSA have been studied using various spectroscopic techniques and molecular dynamic simulation. The UV-VIS results confirmed the strong binding of HCQ with HSA. The calculated thermodynamics parameters confirmed that binding is spontaneous in nature and van der Waals forces and hydrogen bonding are involved in the binding system which is also confirmed by molecular docking results. The steady-state fluorescence confirms the static quenching mechanism in the interaction system, which was further validated by time-resolved fluorescence. The synchronous fluorescence confirmed the more abrupt binding of HCQ with tryptophan residue of HSA compared to Tyr residue of HSA. Isothermal titration calorimetry (ITC) was done to validate the thermodynamics parameters of HSA-HCQ complex in one experiment, supporting the values obtained from the spectroscopic techniques. The circular dichroism (CD) demonstrated that the HCQ affected the secondary structure of HSA protein by reducing their α-helical content. The docking and molecular dynamic simulation results further helped in understanding the effect of HCQ on conformational changes of HSA. Overall, present work defined the physicochemical properties and interaction mechanism of HCQ with HSA that have extensively been elucidated by both in vitro and in silico approaches. Communicated by Ramaswamy H. Sarma

Evaluation of pH change effects on the HSA folding and its drug binding characteristics, a computational biology investigation.

The binding of therapeutics to human serum albumin (HSA), which is an abundant protein in plasma poses a major challenge in drug discovery. Although HSA has several binding pockets, the binding site I on D2 and binding site II on D3 are the main binding pockets of HSA. To date, a few experiments have been conducted to examine the effects of the potential of hydrogen (pH) changes on HSA attributes. In the present investigation, the effect of acidic (pH7.1) and basic states (pH7.7) on HSA structure and its drug binding potency were examined in comparison with the physiological state (pH 7.4). For this purpose, molecular dynamics (MD), free energy landscape (FEL), principal component analysis (PCA), probability distribution function (PDF), tunnel-cavity investigation, secondary structure analysis, docking study, and free energy investigation were employed to investigate the effect of pH changes on the structural characteristics of HSA at the atomic level. The results obtained from this study revealed the significant effect of pH alterations on the secondary and tertiary structure of HSA. In addition, HSA stability and its drug binding ability can be severely affected following pH changes. Given that pH change frequently occurs in various diseases such as cancer, diabetes, and kidney failure, therefore, pharmaceutical companies should allocate specific consideration to this subject throughout their drug design experiments.

An efficient non-chromatographic method to separate human serum albumin from cryo-poor plasma

ABSTRACT Cryo-poor plasma (CPP) which remains after separating cryoprecipitate from the fresh frozen plasma (FFP) unit from human plasma has rare clinical application and can be stored for a maximum of 5 years, depending on storage temperature. In spite of its rare clinical applications that make it as the waste product in blood banking, it has higher concentrations (31.7 g/lit (±0.7)) of albumin than FFP (28.9 g/lit (±0.5)). In this study to develop a rapid, efficient, and inexpensive method to separate human serum albumin (HSA) from CPP, we have benefited from the advantage of the higher thermal stability of HSA. We have investigated the combination of heat treatment of CPP before applying polyethylene glycol as a preferential precipitating agent. In the first step, the effect of various temperatures on proteins’ precipitation profile has been investigated. The resulted effective temperatures (50 and 55 °C) are selected to treat CPP before mixing it with several concentrations and molecular weights of PEG. The pattern of precipitated proteins has been analyzed by the sodium dodecyl sulfate-PAGE and size exclusion chromatography methods. According to achieved data, PEG 8 kDa at concentrations of 20–25% (w/v), with previous heat treatment at 50 °C, showed the significant selective separation of HSA.

Structural insights into the binding behavior of flavonoids naringenin with Human Serum Albumin

The natural polyphenolic compounds present in fruits, vegetables, nuts, and tea are flavonoids. naringenin is one of the most critical flavonoids derivative in certain eatable fruits, such as citrus. Human Serum albumin (HSA) is a protein that is widely used as a bearer for drug binding studies. The spectroscopic and computational methods were employed in this analysis to peruse the efficacy of naringenin on the HSA structure. Analysis of fluorescence demonstrated the static quenching mechanism. Results from the different experiments have shown that naringenin binds to HSA. At three temperatures, the binding constant, the number of binding sites, and temperature parameters (somewhere between ΔH°, somewhere between ΔS°, and somewhere between ΔG°) were calculated. The experiences of Van der Waals and bonding hydrogen were considered to be the main forces. The results of molecular dynamics simulation and thermal denaturation show that naringenin increases the stability of HSA and decreases its flexibility. Due to the decline in the gyration radius (Rg) in attendance of naringenin, protein is more folded. The amount of RMSD decreases in the presence of naringenin, indicating which HSA becomes more stable by binding to naringenin. The mean RMSF of free HSA in the presence of naringenin demonstrated a decrease in protein flexibility during the simulation. ASA values show that most of the Trp and Tyr residues moved outward from the protein (more polar medium).

Development of Oxadiazole-Sulfonamide-Based Compounds as Potential Antibacterial Agents.

In this work, substituted 1,2,4-oxadiazoles (OX1–OX27) were screened against five bacterial strains, identified to be OX7 and OX11 as growth inhibitors with minimum inhibitory concentration (MIC) values of 31.25 and 15.75 μg/mL, respectively. The growth inhibitory property of OX7 and OX11 was further validated by disk diffusion, growth curve, and time kill curve assays. Both disrupted biofilm formation with 92–100% reduction examined by the XTT assay were further visualized by scanning electron microscopy analysis. These compounds in combination with ciprofloxacin also exhibit synergy against Escherichia coli cells. With insignificant cytotoxic behavior on HEK293 cells, human red blood cells, and Galleria mellonella larvae, OX11 was tested against 28 multidrug resistant environmental isolates of bacteria and showed inhibition of Kluyvera georgiana and Citrobacter werkmanii strains with 32 and 16 μg/mL MIC values, respectively. The synergistic behavior of OX11 with ampicillin showed many fold reductions in MIC values against K. georgiana and Klebsiella pneumoniae multidrug resistant strains. Further, transmission electron microscopy analysis of OX11-treated E. coli cells showed a significantly damaged cell wall, which resulted in the loss of integrity and cytosolic oozing. OX11 showed significant changes in the secondary structure of human serum albumin (HSA) in the presence of OX11, enhancing HSA stability. Overall, the study provided a suitable core for further synthetic alterations and development as an antibacterial agent.

Investigation on conformational variation and fibrillation of human serum albumin affected by molybdenum disulfide quantum dots

In this work, binding interaction between molybdenum disulfide quantum dots (MoS2 QDs) and human serum albumin (HSA) was researched deeply to dissect the conformational variation and fibrillation of HSA affected by MoS2 QDs. The results revealed that MoS2 QDs bound strongly with HSA with molar ratio of 1:1 under the joint actions of hydrogen bond and van der Waals force, leading to the static fluorescence quenching of HSA. MoS2 QDs caused the secondary structure transition of HSA from α-helix stepwise to β-turn, β-sheet, and random coil gradually. MoS2 QDs reduced both the molar enthalpy change and the melting temperature of HSA, reducing the thermal stability of HSA significantly. It is worth noting that MoS2 QDs inhibited the fibrillation process of HSA according to the reduced hydrophobic environment and the disturbance of disulfide bonds in HSA network structure. These results reveal the precise binding mechanism of MoS2 QDs with HSA at molecular level, providing indispensable information for the potential application of MoS2 QDs in biological fields.

A pH-dependent protein stability switch coupled to the perturbed pKa of a single ionizable residue

The contribution of electrostatic interactions in protein stability has not been fully understood. Burial of an ionizable amino acid inside the hydrophobic protein core can affect its ionization equilibrium and shift its pKa differentially in the native (N) and unfolded (U) states of a protein and this coupling between the folding/unfolding cycle and the ionization equilibria of the ionizable residue can substantially influence the protein stability. Here, we studied the coupling of the folding/unfolding cycle with the ionization of a buried ionizable residue in a multi-domain protein, Human Serum Albumin (HSA) using fluorescence spectroscopy. A pH-dependent change in the stability of HSA was observed in the near native pH range (pH 6.0–9.0). The protonation-deprotonation equilibrium of a single thiol residue that is buried in the protein structure was identified to give rise to the pH-dependent protein stability. We quantified the pKa of the thiol residue in the N and the U states. The mean pKa of the thiol in the N state was upshifted by 0.5 units to 8.7 due to the burial of the thiol in the protein structure. Surprisingly, the mean pKa of the thiol in the U state was observed to be downshifted by 1.3 units to 6.9. These results indicate that some charged residues are spatially proximal to the thiol group in the U state. Our results suggest that, in addition to the N state, electrostatic interactions in the U state are important determinants of protein stability.

The influence of sodium phosphate buffer on the stability of various proteins: Insights into protein-buffer interactions

Present study delineates the effects of sodium phosphate buffer (PBS) solutions on the stability of various proteins including lysozyme (Lys), bromelain (BM), hemoglobin (Hb), and human serum albumin (HSA). Different spectroscopic methods such as UV–visible, steady state fluorescence, temperature dependent fluorescence spectroscopy, Fourier transforms infrared spectroscopy (FTIR), circular dichroism (CD) spectroscopy and dynamic light scattering (DLS) show that PBS buffer protects protein structures. Efficacy of PBS stabilization from external additive factors follows order as lysozyme > BM > Hb. The thermal stability of HSA in PBS buffer solution was not detected, since no distinct and clear fluorescence transition was observed. However, results from other biophysical techniques reveal the native structure of HSA stabilized in presence of PBS. Moreover, increasing concentration of PBS also provides structural stabilization of HSA. This confirms the potentiality of PBS buffer as protecting agents for the proteins/enzymes. However, in case of Hb structure perturbed with the addition of higher concentrations of PBS. Our study confirms that efficacy of protein stabilization depends on type and concentration of buffer used. The present study is very useful for providing the alternative media to replace traditional volatile organic compounds or expensive modern solvents for biochemical applications.

Effect of ketoprofen-based ionic liquids on secondary structure and thermal stability of human serum albumin

This is the first study on the interactions of ionic liquids (ILs) composed of cations amino acid esters and anion ketoprofen (KETO) with human serum albumin (HSA). The effect of the ILs on the thermal unfolding of the HSA was negligible in comparison with that of the KETO. For the IL series, the denaturating transitions tend to shift to lower temperatures by 1–3 °C. The secondary structure analyses have shown that KETO, [L-LeuOEt][KETO] and [L-ValOBu][KETO] cause folding, and HSA molecule becomes more coiled. In the presence of [L-ValOPr][KETO] and [L-ValOiPr][KETO], HSA structure although partially unfolded seemed very close to that of the native protein. In contrast, [L-ValOEt][KETO] induces the same conformational changes of HSA as ketoprofen. For the latter, we performed binding assays and we found that the affinity of the HSA to bind the IL is comparable to that for the ketoprofen. The estimated binding constants were 3.1 × 104 and 1.4 × 104 L mol−1 for ketoprofen and [L-ValOEt][KETO], respectively. This study on KETO-ILs could be a basis to the development of a new drug formulation.

Unveiling the interaction mechanism of alogliptin benzoate with human serum albumin: Insights from spectroscopy, microcalorimetry, and molecular docking and molecular dynamics analyses.

The interaction between a DPP-4 inhibitor, alogliptin benzoate (AB), and human serum albumin (HSA) was systematically investigated via spectroscopy, microcalorimetry and molecular simulations. Steady-state fluorescence and time-resolved fluorescence spectrometry illustrated that the fluorescence quenching type of AB to HSA was static and caused by the formation of ground state AB-HSA complex. Isothermal titration calorimetry (ITC) combined with fluorescence spectra revealed that the affinity of AB to the subdomain IIA of HSA was moderate with a binding constant in the order of 104. Molecular docking analysis and thermodynamic parameters demonstrated that this combination was maintained by hydrogen bonding along with van der Waals force and hydrophobic force. Circular dichroism and three-dimensional (3D) fluorescence showed that AB increased the hydrophobicity of Trp residue and the α-helix content of HSA by 1.99%. Microdifferential scanning calorimetry revealed that the addition of AB enhanced the thermal stability of HSA. The action forces, binding stability, binding sites, and protein structure of the AB-HSA system were evaluated via molecular dynamics analysis in the simulated environment. On the basis of molecular docking, MD simulation constructed a more reliable 3D model of the AB-HSA complex in terms of spatial structure.

Conformational structure variation of human serum albumin after binding interaction with black phosphorus quantum dots

Herein, binding interaction between black phosphorus quantum dots (BPQDs) and human serum albumin (HSA) was systematically characterized for deep illustration of conformational structure variation of HSA affected by BPQDs. The results confirmed that the intrinsic fluorescence of HSA was statically quenched by BPQDs mainly through van der Waals interaction and hydrogen bond. BPQDs bound strongly with the site I of HSA to form ground state complex with molar ratio of 1 to 1. The secondary structure of HSA was changed obviously after its binding interaction with BPQDs, and the α-helix structure of HSA was transformed to the β-sheet structure. The melting temperature of HSA was decreased after its binding interaction with BPQDs, suggesting that BPQDs promoted the thermal denaturation process of HSA. BPQDs could also reduce the molar enthalpy change and the thermal stability of HSA. These results explored the exact conformational structure variation of HSA after its binding interaction with BPQDs, which provides vital information for possible biological influence of BPQDs on human beings.

A Comprehensive Study on Theoretical and Experimental Effects of Nicotinic Acid and Picolinic Acid on the Structure and Stability of Human Serum Albumin

The interaction of nicotinic acid (Nic) and picolinic acid (Pic), as two pyridine carboxylic acids, with human serum albumin (HSA) as a major transport protein in the blood was investigated using UV-Vis, fluorimetry, circular dichroism (CD), and molecular docking studies. The melting point (Tm) and ΔG0(298K) of HSA, as two thermodynamic parameters, were obtained from thermal denaturation of HSA with and without the presence of Nic and Pic. Tm values of 332.5, 336.4, and 333.9 K, and ΔG0(298K) of 97.4, 99.9, and 118.9 kJ/mol were recorded for HSA alone and following incubation with Nic and Pic, respectively. In chemical denaturation experiments utilizing guanidine hydrochloride (GuHCl), value of ΔG0H2O of 12.5, 16, and 15.3 kJ/mol, [Ligand]1/2 of 2.2, 2.4, and 2.3 M, and m of 5.6, 6.6, and 6.6 kJ/(mol × M) were recorded, respectively. The results of CD, UV-Vis spectroscopy, and molecular dynamics (MD) simulations showed that the binding of Nic and Pic to HSA induced conformational changes in HSA. Furthermore, the study of molecular docking indicated that the binding affinity of the Nic and Pic to site І (subdomain ІІA) is greater than that of site ІI (subdomain ІІIA) of HSA. These results provide valuable insights into the binding mechanisms of Nic and Pic to a plasma protein that is known to play an important role in the delivery of drugs to target organs.