Secondary Structure Of Human Serum Albumin Research Articles

Probing of the Interaction Between Human Serum Albumin and A New Synthesized Pd(II) Complex Using Spectroscopic Methods

Human serum albumin (HSA) is an abundant, multifunctional and nonglycosylated negatively charged plasma protein. HSA ascribed ligand-binding and transport properties, antioxidant functions and enzymatic activities. In the present study, the interaction and side effects of a new designed anti-cancer compound (1,10-phenanthroline butyl dithiocarbamato palladium(II) nitrate) on HSA have been investigated by different spectroscopic (UV-visible, fluorescence and circular dichroism (CD)) techniques at two temperatures of 25 and 37°C. By the analysis of fluorescence spectra, it was observed that this complex has an ability to quench the intrinsic fluorescence of HSA through a static quenching procedure. The number of binding sites and the association binding constants of Pd(II) complex were calculated at 25 and 37°C. Also, the negative ∆H° and positive ∆S˚ values for the interaction calculated by the vant’s Hoff equation showed that the electrostatic interaction has a major role in the binding process. The quantitative analysis of CD spectra represented that Pd(II) complex induces significantly alterations in the secondary structure of HSA via decreasing in the content of α helical structure of the protein. Our results suggest that the new synthesized Pd(II) complex can bind to the blood carrier protein of HSA and change the tertiary and secondary structure of the protein, which may be considered as side effects of this new synthesized drug.

Spectroscopic study of interaction between osthole and human serum albumin: Identification of possible binding site of the compound

The studies on the interaction between human serum albumin (HSA) and drugs have been an interesting research field in life science, chemistry and clinical medicine. Osthole possesses a variety of pharmacological activities including anti-tumor, anti-inflammation, anti-seizure, anti-hyperlipidemic and anti-osteoporosis effects. The interaction of osthole with HSA and its binding site in HSA by spectroscopic methods is the subject of this work. By monitoring the intrinsic fluorescence of the single Trp214 residue and performing site markers displacement measurements, the specific binding of osthole in the vicinity of Sudlow's site I of HSA has been clarified. The changes in the secondary structure of HSA after its complexation with ligand were studied with CD spectroscopy, which indicate that osthole induced only a slight decrease in the helix structural content of the protein. In addition, the mean distance between osthole and HSA fluorophores is estimated to be 4.96nm using Föster's equation on the basis of the fluorescence energy transfer. Furthermore, the synchronous fluorescence spectra show that the microenvironment of the tryptophan residues does not have obvious changes. Osthole can quench the intrinsic fluorescence of HSA by dynamic quenching, and analysis of the thermodynamic parameters of binding showed that hydrophobic interactions play an important role in the stabilizing of the complex. Increase of protein surface hydrophobicity (PSH) was also observed upon the osthole binding.

Exploring the mechanism of interaction between sulindac and human serum albumin: Spectroscopic and molecular modeling methods

In the present study, a combination of fluorescence, molecular modeling and circular dichroism (CD) approaches had been employed to investigate the interaction between sulindac and human serum albumin (HSA). Results of mechanism discussion demonstrated that the fluorescence quenching of HSA by sulindac was a static quenching procedure. Binding parameters calculated from the modified Stern–Volmer equation showed that sulindac bound to HSA with the binding affinities in the order of 105Lmol−1. The thermodynamic parameters (ΔH=−18.58kJmol−1; ΔS=37.26Jmol−1K−1) obtained by the van′t Hoff equation revealed that hydrophobic forces played a leading role in the formation of sulindac–HSA complex, but hydrogen bonds could not be omitted. Site marker competitive experiments revealed a displacement of warfarin by sulindac, which indicated that the binding site of sulindac to HSA located in the sub-domain IIA (Sudlow′s site I). The molecular docking study confirmed the specific binding mode and binding site obtained by fluorescence and site marker competitive experiments. CD and three-dimensional fluorescence spectroscopy were used to investigate the changes of HSA secondary structure and microenvironment in the presence of sulindac. Alterations of HSA conformation were observed with the reduction of α-helix from 60.1% (free HSA) to 57.3%, manifesting a slight unfolding of the polypeptides of protein.

The interaction of 2-mercaptobenzimidazole with human serum albumin as determined by spectroscopy, atomic force microscopy and molecular modeling

The interaction of 2-mercaptobenzimidazole (MBI) with human serum albumin (HSA) was studied in vitro by equilibrium dialysis under normal physiological conditions. This study used fluorescence, ultraviolet–visible spectroscopy (UV–vis), Fourier transform infrared (FT-IR), circular dichroism (CD) and Raman spectroscopy, atomic force microscopy (AFM) and molecular modeling techniques. Association constants, the number of binding sites and basic thermodynamic parameters were used to investigate the quenching mechanism. Based on the fluorescence resonance energy transfer, the distance between the HSA and MBI was 2.495nm. The ΔG0, ΔH0, and ΔS0 values across temperature indicated that the hydrophobic interaction was the predominant binding Force. The UV, FT-IR, CD and Raman spectra confirmed that the HSA secondary structure was altered in the presence of MBI. In addition, the molecular modeling showed that the MBI–HSA complex was stabilized by hydrophobic forces, which resulted from amino acid residues. The AFM results revealed that the individual HSA molecule dimensions were larger after interaction with MBI. Overall, this study suggested a method for characterizing the weak intermolecular interaction. In addition, this method is potentially useful for elucidating the toxigenicity of MBI when it is combined with the biomolecular function effect, transmembrane transport, toxicological testing and other experiments.

Multi-spectroscopic studies on the interaction of human serum albumin with astilbin: Binding characteristics and structural analysis

Five spectroscopic techniques were used to investigate the interaction of astilbin (ASN) with human serum albumin (HSA). UV–vis absorption measurements prove that ASN–HSA complex can be formed. The analysis of fluorescence spectra reveal that in the presence of ASN, quenching mechanism of HSA is considered as static quenching. The quenching rate constant kq, KSV and the binding constant K were estimated. According to the van't Hoff equation, the thermodynamic parameters enthalpy change (ΔΗ) and entropy change (ΔS) were calculated to be −12.94kJmol−1 and 35.92Jmol−1K−1, respectively. These indicate that the hydrophobic interaction is the major forces between ASN and HSA, but the hydrogen bond interaction cannot be excluded. The changes in the secondary structure of HSA which was induced by ASN were determined by circular dichroism (CD), Fourier transform infrared spectroscopy (FT-IR) and Raman spectroscopy.

Binding of methacycline to human serum albumin at subdomain IIA using multispectroscopic and molecular modeling methods

This study was designed to examine the interaction of methacyline (METC) with human serum albumin (HSA) by multispectroscopy and a molecular modeling method under simulative physiological conditions. The quenching mechanism was suggested to be static quenching based on fluorescence and ultraviolet-visible (UV-Vis) spectroscopy. According to the Vant' Hoff equation, the values of enthalpy (∆H) and entropy change (∆S) were calculated to be -95.29 kJ/mol and -218.13 J/mol/K, indicating that the main driving force of the interaction between HSA and METC were hydrogen bonds and van der Waals's forces. By performing displacement measurements, the specific binding of METC in the vicinity of Sudlow's site I of HSA was clarified. An apparent distance of 3.05 nm between Trp214 and METC was obtained via the fluorescence resonance energy transfer (FRET) method. Furthermore, the binding details between METC and HSA were further confirmed by molecular docking studies, which revealed that METC was bound at subdomain IIA through multiple interactions, such as hydrophobic effect, polar forces, hydrogen bonding, etc. The results of three-dimensional fluorescence and Fourier transform infrared (FTIR) spectroscopy showed that METC caused conformational and some microenvironmental changes in HSA and reduced the α-helix significantly in the range of 52.3-40.4% in HSA secondary structure. Moreover, the coexistence of metal ions such as Ca(2+), Al(3+), Fe(3+), Zn(2+), Cu(2+), Cr(3+) and Cd(2+) can decrease the binding constants of METC-HSA.

Investigation on the Binding Behavior of Ellagic Acid to Human Serum Albumin in Aqueous Solution

Ellagic acid (EA), one of the polyphenols in fruits and nuts, has pharmacological activity. To explore binding behavior of EA to protein, human serum albumin (HSA) was chosen and investigated by fluorescence spectroscopy, Fourier transform infrared (FT-IR) spectroscopy and molecular modeling in aqueous solution. Fluorescence titration results indicated that EA effectively quenched the intrinsic fluorescence of HSA by static quenching and the binding process was spontaneous. According to the Scatchard equation, there was only one class of binding sites can bind to HSA, the binding constants at three different temperatures (298, 310 and 318 K) were 8.47 × 104, 7.39 × 104 and 6.00 × 104, respectively. It was found by FT-IR spectra that EA altered HSA secondary structure. Thermodynamic analysis showed that hydrophobic interaction and hydrogen bonds played an important role in stabilizing EA–HSA complex. A molecular docking study suggested that the HSA residues for EA binding located in sub-domain IIA.

The Investigation of the Binding of 6-Mercaptopurine to Site I on Human Serum Albumin

6-Mercaptopurine (6-MP) is one of a large series of purine analogues which has been found active against human leukemias. The equilibrium dialysis, circular dichroism (CD) and molecular docking were employed to study the binding of 6-MP to human serum albumin (HSA). The binding of 6-MP to HSA in the equilibrium dialysis experiment was detected by measuring the displacement of 6-MP by specific markers for site I on HSA, warfarin (RWF), phenylbutazone (PhB) and n-butyl p-aminobenzoate (ABE). It was shown, according to CD data, that binding of 6-MP to HSA leads to alteration of HSA secondary structure. Based on the findings from displacement experiment and molecular docking simulation it was found that 6-MP was located within binding cavity of subdomain IIA and the space occupied by site markers overlapped with that of 6-MP. Displacement of 6-MP by the RWF or PhB was not up the level expected for a competitive mechanism, therefore displacement of 6-MP was rather by non-cooperative than that the direct competition. Instead, in case of the interaction between ABE and 6-MP, when the little enhancement of the binding of ABE by 6-MP was found, the interaction could be via a positively cooperative mechanism.

Binding of triclosan to human serum albumin: insight into the molecular toxicity of emerging contaminant

The interaction between triclosan (TCS) and human serum albumin (HSA) was investigated in order to obtain the binding mechanism, binding constant, the type of binding force, the binding distance between the donor and acceptor, and the effect of TCS on the conformation change of HSA. A HSA solution was added to the quartz cell and then titrated by successive addition of TCS. The fluorescence quenching spectra and synchronous spectra were recorded with the excitation and emission slits of the passage of band set at 10 and 20nm. Three-dimensional fluorescence spectra of HSA were recorded before and after the addition of TCS. The capillary electrophoresis was conducted with the pressure injection mode at 0.5psi for 5s, separation under 25kV, and detection at 214nm. Fluorescence data indicated the fluorescence quenching of HSA by TCS was static quenching, and the quenching constants (K ( a )) were 1.14 × 10(5), 8.75 × 10(4), 6.67 × 10(4), and 5.00 × 10(4) at 293, 298, 303, and 309K, respectively. The thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS) for the interaction were calculated to be -37.9kJmol(-1) and 32.6J mol(-1)K(-1). The binding distance between TCS and tryptophan residues of HSA was obtained to be 1.81nm according to Fǒrster nonradioactive energy transfer theory. The UV-Vis absorption spectroscopy, the synchronous fluorescence spectroscopy, three-dimensional fluorescence spectroscopy, and circular dichroism spectroscopy revealed the alterations of HSA secondary structure in the presence of TCS. Finally, the interaction between TCS and HSA was further confirmed by capillary electrophoresis. TCS was bound to HSA to form the TCS-HSA complex, with the binding distance of 1.81nm. Hydrophobic interaction and hydrogen bond were dominated in the binding. TCS could change the secondary conformation of HSA. This work provides an insight into noncovalent interaction between emerging pollutants and protein, helping to elucidate the toxic mechanism of such pollutants.

Synthesis, characterization and interaction studies of copper based drug with Human Serum Albumin (HSA): Spectroscopic and molecular docking investigations

A new water soluble copper(II) complex, [Cu(glygly)(ssz)(H2O)]⋅6H2O, 1 derived from dipeptide (glycyl glycine anion) and sulfasalazine was synthesized and characterized by elemental analysis (CHN), molar conductance measurements and spectroscopic methods (IR, UV–vis, ESI-MS). The complex 1 is non-ionic in nature and possess octahedral geometry around Cu(II) metal ion. The interaction of complex 1 with Human Serum Albumin (HSA) was investigated under physiological condition in Tris–HCl buffer solution at pH 7.4 by means of various spectroscopic methods (fluorescence, CD and FTIR) and molecular docking technique. The results of fluorescence titration revealed that the complex 1 strongly quench the intrinsic fluorescence of HSA through a static quenching procedure. Binding constants (Kb) and the number of binding sites (n≈1) were calculated using modified Stern–Volmer equations. The thermodynamic parameters ΔG at different temperatures were calculated subsequently the value of ΔH and ΔS was also calculated which revealed that the hydrophobic and hydrogen bonding interactions play a major role in HSA–complex 1 association. The distance r between donor (HSA) and acceptor (complex 1) was obtained according to fluorescence resonance energy transfer and the alterations of HSA secondary structure induced by complex 1 were confirmed by FT-IR and CD measurements.

Surface modification of PAMAM dendrimer improves its biocompatibility

Modification of dendrimer surface groups is one of the methods available to obtain compounds characterized by reduced toxicity. This article reports results of preliminary biocompatibility studies of a modified polyamidoamine dendrimer of the fourth generation. Reaction with dimethyl itaconate resulted in transformation of surface amine groups into pyrrolidone derivatives. Interaction of the modified dendrimer with human serum albumin (HSA) was analyzed. The influence of the dendrimer on mouse neuroblastoma cell line viability and its hemolytic properties were also investigated. The binding constant between analyzed dendrimer and HSA was found to be equal to 1.2 × 105 ± 0.2 × 105 M–1. Small changes in HSA secondary structure were observed. The analyzed dendrimer revealed minor toxic activity, as diminishment in cell viability was observed only for dendrimer concentrations higher than 2 mg/mL. Moreover, under the applied experimental conditions, no hemolytic activity was observed. Those observations point to the potential of the analyzed compound for further studies toward its applicability in nanomedicine. From the Clinical EditorThis basic science paper explores the biocompatibility of fourth generation surface modified PAMAM dendrimers for future preclinical applications.

Probing the Interaction of Human Serum Albumin With Bilirubin in the Presence of Aspirin by Multi-Spectroscopic, Molecular Modeling and Zeta Potential Techniques: Insight on Binary and Ternary Systems

Here, we report on the effect of aspirin (ASA), on the binding parameters with regard to bilirubin (BR) to human serum albumin (HSA). Two different classes of binding sites were detected. Binding to the first and second classes of the binding sites was dominated by hydrophobic forces in the case of HSA-BR, whereas in the case of the ternary system, binding to the first and second classes of the binding sites was achieved by electrostatic interaction. The binding constant (Ka) and number of binding site (n) obtained were 1.6 × 106 M−1 and 0.98, respectively, for the primary binding site in the case of HSA-BR, and 3.7 × 106 M−1 and 0.84, respectively, in the presence of ASA (ternary complex) at δex = 280 nm. The progressive quenching of the protein fluorescence as the BR concentration increased indicated an arrangement of the domain IIA in HSA. Changes in the environment of the aromatic residues were also observed by synchronous fluorescence spectroscopy (SFS). Changes of the secondary structure of HSA involving a decrease of α-helical and β-sheet contents and increased amounts of turns and unordered conformations were mainly found at high concentrations of BR. For the first time, the relationship between the structural parameters of HSA-BR by RLS for determining the critical induced aggregation concentration (CCIAC) of BR in the absence and presence of ASA was investigated, and there was a more significant enhancement in the case of the ternary mixture as opposed to the binary one. Changes in the zeta potential of HSA and the HSA-ASA complex in the presence of BR demonstrated a hydrophobic adsorption of this anionic ligand onto the surface of HSA in the binary system as well as both electrostatic and hydrophobic adsorption in the case of the ternary complex. By performing docking experiments, it was found that the acting forces between BR and HSA were mainly hydrophobic > hydrogen bonding > electrostatic interactions, and consequently BR had a long storage time in blood plasma, especially in the presence of ASA. This was due to the electrostatic interaction force between the BR and HSA being stronger in (HSA-ASA) BR than in the HSA-BR complex. In addition, it was demonstrated that, in the presence of ASA, the first binding site of BR on HSA was altered, but the parameters of binding did not become significantly modified, and thus the affinity of BR barely changed with and without ASA.

Comparative studies on the interactions of honokiol and magnolol with human serum albumin

In this study, the binding modes of honokiol (HK) and magnolol (MG) with human serum albumin (HSA) have been established under imitated physiological condition, which was very important to understand the pharmacokinetics and toxicity of HK or MG. The experimental results proved that the fluorescence of HSA was quenched by HK or MG through a static quenching procedure. The binding constants of HK-HSA and MG-HSA complexes were 5.304 and 263.755×10(4) L mol(-1) at 298 K, respectively. The binding process was a spontaneous molecular interaction procedure, in which the hydrophobic interaction played a major role in the formation of the HK-HSA complex, whereas, the binding interaction between MG and HSA might involve the hydrophobic interaction strongly and electrostatic interaction. In addition, the effect of HK/MG on the secondary structure of HSA was analyzed using CD, UV-vis absorption, Fourier transform infrared (FT-IR), synchronous fluorescence and three-dimensional fluorescence spectra. According to Förster no-radiation energy transfer theory, the binding distance of HSA to HK or MG was calculated to be 1.842 or 1.238 nm. Besides, the effects of common ions on the binding constants of HSA-HK/MG systems were also discussed.

Probing the Interaction of Human Serum Albumin with Ciprofloxacin in the Presence of Silver Nanoparticles of Three Sizes: Multispectroscopic and ζ Potential Investigation

The binding of ciprofloxacin to human serum albumin (HSA) in the presence and absence of silver nanoparticles of three sizes was investigated for the first time. For this purpose fluorescence spectroscopy, circular dichroism, UV-vis spectroscopy, and ζ potential techniques were employed under physiological conditions. The titration results indicated that ciprofloxacin quenched the fluorescence intensity of HSA through a static mechanism, but in the presence of Ag nanoparticles of sizes I and II there were two different kinds of interaction behavior. Results of circular dichroism indicated that the secondary structure of HSA was modified with increasing ciprofloxacin concentration. A comparison between resonance light scattering of binary and ternary systems allowed us to estimate the effect of silver nanoparticles on the initial formation and aggregation of ciprofloxacin with HSA. The ζ-potential results suggested induced conformational changes on HSA, thus confirming the experimental and theoretical results.

Conformation and Thermodynamic Properties of the Binding of Vitamin C to Human Serum Albumin

The binding of vitamin C, L-ascorbic acid (AsA), with human serum albumin (HSA) was investigated by various spectroscopic techniques under simulated physiological conditions. The fluorescence quenching constants (K SV) at four different temperatures (292, 298, 304, and 310 K) were obtained. The thermodynamic parameters ΔH ∘ and ΔS ∘ were calculated to be 6.02 kJ⋅mol−1 and 84.55 J⋅mol−1⋅K−1 using the van’t Hoff equation. Additional experiments to determine the stoichiometry (n) were carried out using isothermal titration calorimetry (ITC) and cyclic voltammetry (CV). The distance, r, between AsA and the tryptophan residues of HSA was calculated to be 3.7 nm according to Forster’s non-radiation energy transfer theory. The effect of AsA on the conformation of HSA was studied by means of three dimensional fluorescence spectra and CD spectra. The results indicate that the presence of AsA resulted in a slight change of the HSA secondary structure. The effect of common ions on the binding of AsA to HSA was also examined.

Binding of Phthalate Plasticizers to Human Serum Albumin in Vitro: A Multispectroscopic Approach and Molecular Modeling

As endocrine-disrupting chemicals, a few frequently used phthalate plasticizers were banned or restricted for use as additives in food in some countries. The interaction mechanisms between three phthalate plasticizers with human serum albumin (HSA) were studied by fluorescence (quenching, synchronous, and three-dimensional), UV-vis absorption, circular dichroism (CD), and Fourier transform infrared (FT-IR) spectroscopy, in combination with molecular modeling under simulative physiological conditions, respectively. The results obtained from fluorescence quenching data revealed that the plasticizers-HSA interaction altered the conformational strcture of HSA. Meanwhile, the alterations of HSA secondary structure in the presence of phthalate plasticizers were investigated. The binding distances for the plasticizers-HSA system were provided by the efficiency of fluorescence resonance energy transfer. Furthermore, the thermodynamic analysis implied that hydrophobic forces were the main interaction for the plasticizers-HSA system, which agreed well with the results from the molecular modeling study.

Interaction and photo-induced cleavage studies of a copper based chemotherapeutic drug with human serum albumin: spectroscopic and molecular docking study

The interaction of new dinuclear copper(ii) complex 1; [Cu(2)(glygly)(2)(ppz)(H(2)O)(4)]·2H(2)O, derived from dipeptide (glycyl glycine) and piperazine as a metallopeptide drug with human serum albumin (HSA) was examined by means of fluorescence spectroscopy which revealed that complex 1 has a strong ability to quench the intrinsic fluorescence of HSA through a static quenching procedure. The alterations of HSA secondary structure in the presence of complex 1 were confirmed by UV-visible, FT-IR, CD and 3D fluorescence spectroscopy. The binding constants (K), and binding site number (n), corresponding thermodynamic parameters ΔG, ΔH and ΔS at different temperatures were calculated. The molecular docking technique was utilized to ascertain the mechanism and mode of action towards the molecular target HSA indicating that complex 1 was located at the entrance of site I by electrostatic and hydrophobic forces, consistent with the corresponding experimental results. Complex 1 shows efficient photo-induced HSA cleavage activity, indicating the involvement of hydroxyl radicals as the reactive species. Furthermore, the cytotoxicity of 1 was examined on a panel of human tumor cell lines of different histological origins showing significant GI(50) values specifically towards MIAPACA2, A498 and A549 tumor cell lines. These results complement previous biological studies of new specific target metallopeptides, providing additional information about possibilities of their transport and disposition in blood plasma.

Spectroscopic Investigation on the Interaction of Pyrimidine Derivative, 2-Amino-6-hydroxy-4-(3,4-dimethoxyphenyl)-pyrimidine-5-carbonitrile with Human Serum Albumin: Mechanistic and Conformational Study

In the present study, fluorescence spectroscopy in combination with UV–vis absorption spectroscopy and synchronous fluorescence spectroscopy (SFS) was employed to investigate the binding affinity of pyrimidine derivative, 2-amino-6-hydroxy-4-(3,4-dimethoxyphenyl)-pyrimidine-5-carbonitrile (AHDMPPC) to human serum albumin (HSA) under the physiological conditions. In the mechanism discussion, it was proved that the fluorescence quenching of HSA by AHDMPPC is a result of the formation of AHDMPPC–HSA complex. The quenching mechanism and number of binding sites (n ≈ 1) were obtained by fluorescence titration data. Binding parameters calculated from Stern–Volmer method showed that the AHDMPPC bind to HSA with the binding affinities of the order 104 L mol–1. The thermodynamic parameters studies revealed that the binding was characterized by negative enthalpy and positive entropy changes −13.06 kJ/mol and 51.34 J/mol K–1 (from the Van’t Hoff equation) and suggest that the binding reaction was exothermic and hydrophobic interaction is the predominant intermolecular forces stabilizing the complex. The specific binding distance (r = 2.25 nm) between donor HSA and acceptor AHDMPPC was obtained according to fluorescence resonance energy transfer (FRET). Furthermore, the synchronous spectral result, three–dimensional fluorescence spectra and circular dichroism (CD) indicates that the secondary structure of HSA was changed in the presence of AHDMPPC.

Binding of fullerol to human serum albumin: Spectroscopic and electrochemical approach

The potential impact of human exposure to carbonaceous nanomaterials in the environment becomes a concerning issue. Here we report on the interaction of fullerol with human serum albumin (HSA) using spectroscopic and electrochemical methods. The water-soluble fullerene derivative (fullerol) was synthesized and characterized by IR, 1H NMR, TG-DSC, XRD, HR-TEM, etc. The spectroscopic methods show that the fluorescence quenching of HSA by fullerol is the result of the formation of an HSA-fullerol complex. Binding parameters such as ΔG, ΔH and ΔS were calculated, and the quenching constant Ka at different temperatures was determined using the modified Stern–Volmer equation. The electrochemical experiments further confirmed the conclusions. In addition, the influences of coexisting heavy metal ions have also been studied in the present system. The circular dichroism spectra (CD), 3D fluorescence spectra and FT-IR spectra results suggest that the secondary structure of HSA was changed by fullerol. Based on the site marker competitive experiments, we can predict the possible binding position of fullerol on the HSA was located at the site of sub domain II A. Furthermore, the distance r between donor (HSA) and acceptor (fullerol) was obtained according to the famous fluorescence resonance energy transfer (FRET) mechanism.

Separate and simultaneous binding effects through a non-cooperative behavior between cyclophosphamide hydrochloride and fluoxymesterone upon interaction with human serum albumin: Multi-spectroscopic and molecular modeling approaches

This study was designed to examine the interaction of two anti-breast cancer drugs, i.e., fluoxymesterone (FLU) and cyclophosphamide (CYC), with human serum albumin (HSA) using different kinds of spectroscopic, zeta potential and molecular modeling techniques under imitated physiological conditions. The RLS technique was utilized to investigate the effect of the two anticancer drugs on changes of the protein conformation, both separately and simultaneously. Our study suggested that the enhancement in RLS intensity was attributed to the formation of a new complex between the two drugs and the protein. Both drugs demonstrated a powerful ability to quench the fluorescence of HSA, and the fluorescence quenching action was much stronger when the two drugs coexisted. The quenching mechanism was suggested to be static as confirmed by time-resolved fluorescence spectroscopy results. The effect of both drugs on the conformation of HSA was analyzed using synchronous fluorescence spectroscopy. Our results revealed that the fluorescence quenching of HSA originated from the Trp and Tyr residues, and demonstrated a conformational change of HSA with the addition of both drugs. The binding distances between HSA and the drugs were estimated by the Förster theory, and it was revealed that nonradiative energy transfer from HSA to both drugs occurred with a high probability. According to CD measurements, the influence of both drugs on the secondary structure of HSA in aqueous solutions was also investigated and illustrated that the α-helix content of HSA decreased with increasing drug concentration in both systems. Moreover, the zeta-potential experiments revealed that both drugs induced conformational changes on HSA. Docking studies were also performed and demonstrated that a reduction of the binding affinity between the drugs and HSA occurred in the presence of both drugs.