Understanding the mode of binding mechanism of doripenem to human serum albumin: Spectroscopic and molecular docking approaches

The infections caused by multidrug resistant bacteria are widely treated with carabapenem antibiotics as a drug of choice, and human serum albumin (HSA) plays a vital role in binding with drugs and affecting its rate of delivery and efficacy. So, we have initiated this study to characterize the mechanism of doripenem binding and to locate its site of binding on HSA by using spectroscopic and docking approaches. The binding of doripenem leads to alteration of the environment surrounding Trp‐214 residue of HSA as observed by UV spectroscopic study. Fluorescence spectroscopic study revealed considerable interaction and complex formation of doripenem and HSA as indicated by K_sv and K_q values of the order of 10⁴ M^?1 and 10¹² M^?1 s^?1, respectively. Furthermore, doripenem quenches the fluorescence of HSA spontaneously on a single binding site with binding constant of the order of 10³ M^?1, through an exothermic process. Van der Waals forces and hydrogen bonding are the major forces operating to stabilize HSA‐doripenem complex. Circular dichroism spectroscopic study showed changes in the structure of HSA upon doripenem binding. Drug displacement and molecular docking studies revealed that the binding site of doripenem on HSA is located on subdomain IB and III A. This study concludes that, due to significant interaction of doripenem on either subdomain IB or IIIA of HSA, the availability of doripenem on the target site may be compromised. Hence, there is a possibility of unavailability of threshold amount of drug to be reached to the target; consequently, resistance may develop in the bacterial population.     

Insight into the interaction of antitubercular and anticancer compound clofazimine with human serum albumin: spectroscopy and molecular modelling

The binding of clofazimine to human serum albumin (HSA) was investigated by applying optical spectroscopy and molecular docking methods. Fluorescence quenching data revealed that clofazimine binds to protein with binding constant in the order of 10⁴ M^?1, and with the increase in temperature, Stern–Volmer quenching constants gradually decreased indicating quenching mode to be static. The UV–visible spectra showed increase in absorbance upon interaction of HSA with clofazimine which further reveals formation of the drug–albumin complex. Thermodynamic parameters obtained from fluorescence data indicate that the process is exothermic and spontaneous. Forster distance (R_o) obtained from fluorescence resonance energy transfer is found to be 2.05 nm. Clofazimine impelled rise in α-helical structure in HSA as observed from far-UV CD spectra while there are minor alterations in tertiary structure of the protein. Clofazimine interacts strongly with HSA inducing secondary structure in the protein and slight alterations in protein topology as suggested by dynamic light scattering results. Moreover, docking results indicate that clofazimine binds to hydrophobic pocket near to the drug site II in HSA.     

Studies on the interaction between vincamine and human serum albumin: a spectroscopic approach

The interaction between vincamine (VCM) and human serum albumin (HSA) has been studied using a fluorescence quenching technique in combination with UV/vis absorption spectroscopy, Fourier transform infrared (FT–IR) spectroscopy, circular dichroism (CD) spectroscopy and molecular modeling under conditions similar to human physiological conditions. VCM effectively quenched the intrinsic fluorescence of HSA via static quenching. The binding constants were calculated from the fluorescence data. Thermodynamic analysis by Van't Hoff equation revealed enthalpy change (ΔH) and entropy change (ΔS) were ?4.57 kJ/mol and 76.26 J/mol/K, respectively, which indicated that the binding process was spontaneous and the hydrophobic interaction was the predominant force. The distance r between the donor (HSA) and acceptor (VCM) was obtained according to the Förster's theory of non‐radiative energy transfer and found to be 4.41 nm. Metal ions, viz., Na⁺, K⁺, Li⁺, Ni²⁺, Ca²⁺, Zn²⁺ and Al³⁺ were found to influence binding of the drug to protein. The 3D fluorescence, FT–IR and CD spectral results revealed changes in the secondary structure of the protein upon interaction with VCM. Furthermore, molecular modeling indicated that VCM could bind to the subdomain IIA (site I) of HSA. Copyright © 2013 John Wiley & Sons, Ltd.     

Binding of fluphenazine with human serum albumin in the presence of rutin and quercetin: An evaluation of food‐drug interaction by spectroscopic techniques

The interactions between human serum albumin (HSA) and fluphenazine (FPZ) in the presence or absence of rutin or quercetin were studied by fluorescence, absorption and circular dichroism (CD) spectroscopy and molecular modeling. The results showed that the fluorescence quenching mechanism was static quenching by the formation of an HSA–FPZ complex. Entropy change (ΔS ⁰) and enthalpy change (ΔH ⁰) values were 68.42 J/(mol? K) and ?4.637 kJ/mol, respectively, which indicated that hydrophobic interactions and hydrogen bonds played major roles in the acting forces. The interaction process was spontaneous because the Gibbs free energy change (ΔG ⁰) values were negative. The results of competitive experiments demonstrated that FPZ was mainly located within HSA site I (sub‐domain IIA). Molecular docking results were in agreement with the experimental conclusions of the thermodynamic parameters and competition experiments. Competitive binding to HSA between flavonoids and FPZ decreased the association constants and increased the binding distances of FPZ binding to HSA. The results of absorption, synchronous fluorescence, three‐dimensional fluorescence, and CD spectra showed that the binding of FPZ to HSA caused conformational changes in HSA and simultaneous effects of FPZ and flavonoids induced further HSA conformational changes.     

Probing the interaction of anticancer drug temsirolimus with human serum albumin: molecular docking and spectroscopic insight

The binding interaction between temsirolimus, an important antirenal cancer drug, and HSA, an important carrier protein was scrutinized making use of UV and fluorescence spectroscopy. Hyper chromaticity observed in UV spectroscopy in the presence of temsirolimus as compared to free HSA suggests the formation of complex between HSA and temsirolimus. Fluorescence quenching experiments clearly showed quenching in the fluorescence of HSA in the presence of temsirolimus confirming the complex formation and also confirmed that static mode of interaction is operative for this binding process. Binding constant values obtained through UV and fluorescence spectroscopy reveal strong interaction; temsirolimus binds to HSA at 298 K with a binding constant of 2.9 × 10⁴ M^?1implying the strength of interaction. The negative Gibbs free energy obtained through Isothermal titration calorimetry as well as quenching experiments suggests that binding process is spontaneous. Molecular docking further provides an insight of various residues that are involved in this binding process; showing the binding energy to be -12.9 kcal/mol. CD spectroscopy was retorted to analyze changes in secondary structure of HSA; increased intensity in presence of temsirolimus showing changes in secondary structure of HSA induced by temsirolimus. This study is of importance as it provides an insight into the binding mechanism of an important antirenal cancer drug with an important carrier protein. Once temsirolimus binds to HSA, it changes conformation of HSA which in turn can alter the functionality of this important carrier protein and this altered functionality of HSA can be highlighted in variety of diseases.     

Insights into In Vitro Binding of Parecoxib to Human Serum Albumin by Spectroscopic Methods

Herein, we report the effect of parecoxib on the structure and function of human serum albumin (HSA) by using fluorescence, circular dichroism (CD), Fourier transforms infrared (FTIR), three‐dimensional (3D) fluorescence spectroscopy, and molecular docking techniques. The Stern–Volmer quenching constants K_SV and the corresponding thermodynamic parameters ΔH, ΔG, and ΔS have been estimated by the fluorescence quenching method. The results indicated that parecoxib binds spontaneously with HSA through van der Waals forces and hydrogen bonds with binding constant of 3.45 × 10⁴ M^?1 at 298 K. It can be seen from far‐UV CD spectra that the α‐helical network of HSA is disrupted and its content decreases from 60.5% to 49.6% at drug:protein = 10:1. Protein tertiary structural alterations induced by parecoxib were also confirmed by FTIR and 3D fluorescence spectroscopy. The molecular docking study indicated that parecoxib is embedded into the hydrophobic pocket of HSA.     

Combination mode of antimalarial drug mefloquine and human serum albumin: Insights from spectroscopic and docking approaches

The interaction between mefloquine (MEF), the antimalarial drug, and human serum albumin (HSA), the main carrier protein in blood circulation, was explored using fluorescence, absorption, and circular dichroism spectroscopic techniques. Quenching of HSA fluorescence with MEF was characterized as static quenching and thus confirmed the complex formation between MEF and HSA. Association constant values for MEF-HSA interaction were found to fall within the range of 3.79-5.73 × 10⁴ M^˗1 at various temperatures (288, 298, and 308 K), which revealed moderate binding affinity. Hydrogen bonds and hydrophobic interactions were predicted to connect MEF and HSA together in the MEF-HSA complex, as deduced from the thermodynamic data (ΔS = +133.52 J mol⁻¹ K⁻¹ and ΔH = +13.09 kJ mol⁻¹) of the binding reaction and molecular docking analysis. Three-dimensional fluorescence spectral analysis pointed out alterations in the microenvironment around aromatic amino acid (tryptophan and tyrosine) residues of HSA consequent to the addition of MEF. Circular dichroic spectra of HSA in the wavelength ranges of 200-250 and 250-300 nm hinted smaller changes in the protein's secondary and tertiary structures, respectively, induced by MEF binding. Noncovalent conjugation of MEF to HSA bettered protein thermostability. Site marker competitive drug displacement results suggested HSA Sudlow's site I as the MEF binding site, which was also supported by molecular docking analysis.     

Investigation of the interaction of aurantio‐obtusin with human serum albumin by spectroscopic and molecular docking methods

The interaction between human serum albumin (HSA) and aurantio‐obtusin was investigated by spectroscopic techniques combined with molecular docking. The Stern–Volmer quenching constants (K_SV) decreased from 8.56 × 10⁵ M^?1 to 5.13 × 10⁵ M^?1 with a rise in temperatures from 289 to 310 K, indicating that aurantio‐obtusin produced a static quenching of the intrinsic fluorescence of HSA. Time‐resolved fluorescence studies proved again that the static quenching mechanism was involved in the interaction. The sign and magnitude of the enthalpy change as well as the entropy change suggested involvement of hydrogen bonding and hydrophobic interaction in aurantio‐obtusin–HSA complex formation. Aurantio‐obtusin binding to HSA produced significant alterations in secondary structures of HSA, as revealed from the time‐resolved fluorescence, Fourier transform infrared (FT‐IR) spectroscopy, three‐dimensional (3D) fluorescence and circular dichroism (CD) spectral results. Molecular docking study and site marker competitive experiment confirmed aurantio‐obtusin bound to HSA at site I (subdomain IIA).     

Probing the binding of two 19‐nortestosterone derivatives to human serum albumin: insights into the interactions of steroid hormone drugs with functional biomacromolecule

Norethindrone acetate (NETA) is a fatty acid ester of norethindrone (NET) that can convert to its more active parent compound NET when orally administered. To study the interactions of NETA and NET with human serum albumin (HSA), we applied fluorescence spectroscopy, circular dichroism (CD), and molecular docking. The effects of metal ions on the HSA–NETA/NET system were also explored. Fluorescence data showed that the quenching mechanism of HSA by NETA and NET was consistent with a static model and that the binding constant of NETA was higher than that of NET. Thermodynamic parameters indicated that hydrogen bonds and van der Waals forces were the main forces maintaining the stability of the HSA–NETA/NET complex. Molecular modeling studies revealed that NETA and NET were bound within subdomain IIA of HSA, in accordance with the site probe results. Synchronous fluorescence spectroscopy, CD, and three‐dimensional fluorescence spectroscopy further confirmed that the binding of NETA/NET to HSA changed the secondary structure of the protein. All other metal ions, except for Ca²⁺, decreased the K value of the HSA–NETA/NET system with enhancement of the maximum effectiveness of NETA/NET. Three commercially available steroid hormone drugs influenced the binding ability of NETA on HSA to different extents. This study provides novel insights into the interactions between HSA and NETA/NET, as well as a solid foundation for future research on drug pharmacokinetics and pharmacodynamics. Copyright © 2016 John Wiley & Sons, Ltd.     

Biomolecular interaction of a platelet aggregation inhibitor, 3,4-methylenedioxy-β-nitrostyrene with human serum albumin: multi-spectral and computational characterization

Abstract

Molecular interaction of the 3,4-methylenedioxy-β-nitrostyrene (MNS), an inhibitor of platelet aggregation with the main transport protein, albumin from human serum (HSA) was explored using absorption, fluorescence and circular dichroism (CD) spectroscopy in combination with in silico analyses. The MNS–HSA complexation was corroborated from the fluorescence and absorption spectral results. Implication of static quenching mechanism for MNS–HSA system was predicted from the Stern–Volmer constant, K_SV-temperature relationship as well as the bimolecular quenching rate constant, k_q values. Stabilization of the complex was affirmed by the value of the binding constant (K_a = 0.56-1.48?×?10⁴ M^?1). Thermodynamic data revealed that the MNS–HSA association was spontaneously driven mainly through hydrophobic interactions along with van der Waal’s interaction and H-bonds. These results were well supported by in silico interpretations. Far-UV and near-UV CD spectral results manifested small variations in the protein’s secondary and tertiary structures, respectively, while three-dimensional fluorescence spectra displayed microenvironmental fluctuations around protein’s fluorophores, upon MNS binding. Significant improvement in the protein’s thermostability was evident from the temperature-stability results of MNS-bound HSA. Binding locus of MNS, as identified by competitive drug displacement findings as well as in silico analysis, was found to be located in subdomain IIA (Sudlow’s site I) of the protein.

Communicated by Ramaswamy H. Sarma     

Multi-spectroscopic,thermodynamic and molecular docking studies to investigate the interaction of eplerenone with human serum albumin

The binding of small molecular drugs with human serum albumin (HSA) has a crucial influence on their pharmacokinetics. The binding interaction between the antihypertensive eplerenone (EPL) and HSA was investigated using multi-spectroscopic techniques for the first time. These techniques include ultraviolet-visible (UV-vis) spectroscopy, Fourier-transform infrared (FTIR), native fluorescence spectroscopy, synchronous fluorescence spectroscopy and molecular docking approach. The fluorescence spectroscopic study showed that EPL quenched HSA inherent fluorescence. The mechanism for quenching of HSA by EPL has been determined to be static in nature and confirmed by UV absorption and fluorescence spectroscopy. The modified Stern–Volmer equation was used to estimate the binding constant (K_b) as well as the number of bindings (n). The results indicated that the binding occurs at a single site (K_b = 2.238 × 10³ L mol⁻¹at 298 K). The enthalpy and entropy changes (∆H and ∆S) were 58.061 and 0.258 K J mol⁻¹, respectively, illustrating that the principal intermolecular interactions stabilizing the EPL–HSA system are hydrophobic forces. Synchronous fluorescence spectroscopy revealed that EPL binding to HSA occurred around the tyrosine (Tyr) residue and this agreed with the molecular docking study. The Förster resonance energy transfer (FRET) analysis confirmed the static quenching mechanism. The esterase enzyme activity of HSA was also evaluated showing its decrease in the presence of EPL. Furthermore, docking analysis and site-specific markers experiment revealed that EPL binds with HSA at subdomain IB (site III).     

Interaction of a tyrosine kinase inhibitor,vandetanib with human serum albumin as studied by fluorescence quenching and molecular docking

Interaction of a tyrosine kinase inhibitor, vandetanib (VDB), with the major transport protein in the human blood circulation, human serum albumin (HSA), was investigated using fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and molecular docking analysis. The binding constant of the VDB–HSA system, as determined by fluorescence quenching titration method was found in the range, 8.92–6.89?×?10^3?M^?1 at three different temperatures, suggesting moderate binding affinity. Furthermore, decrease in the binding constant with increasing temperature revealed involvement of static quenching mechanism, thus affirming the formation of the VDB–HSA complex. Thermodynamic analysis of the binding reaction between VDB and HSA yielded positive ΔS (52.76 J?mol^?1 K^?1) and negative ΔH (?6.57?kJ?mol^?1) values, which suggested involvement of hydrophobic interactions and hydrogen bonding in stabilizing the VDB–HSA complex. Far-UV and near-UV CD spectral results suggested alterations in both secondary and tertiary structures of HSA upon VDB-binding. Three-dimensional fluorescence spectral results also showed significant microenvironmental changes around the Trp residue of HSA consequent to the complex formation. Use of site-specific marker ligands, such as phenylbutazone (site I marker) and diazepam (site II marker) in competitive ligand displacement experiments indicated location of the VDB binding site on HSA as Sudlow’s site I (subdomain IIA), which was further established by molecular docking results. Presence of some common metal ions, such as Ca²⁺, Zn²⁺, Cu²⁺, Ba²⁺, Mg²⁺, and Mn²⁺ in the reaction mixture produced smaller but significant alterations in the binding affinity of VDB to HSA.     

Interaction of meropenem with ‘N’ and ‘B’ isoforms of human serum albumin: a spectroscopic and molecular docking study

Carbapenems are used to control the outbreak of β-lactamases expressing bacteria. The effectiveness of drugs is influenced by its interaction with human serum albumin (HSA). Strong binding of carbapenems to HSA may lead to decreased bioavailability of the drug. The non-optimal drug dosage will provide a positive selection pressure on bacteria to develop resistance. Here, we investigated the interaction between meropenem and HSA at physiological pH 7.5 (N-isoform HSA) and non-physiological pH 9.2 (B-isoform HSA). Results showed that meropenem quenches the fluorescence of both ‘N’ and ‘B’ isoforms of HSA (ΔG < 0 and binding constant ~10⁴ M^?1). Electrostatic interactions and van der Waal interactions along with H-bonds stabilized the complex of meropenem with ‘N’ and ‘B’ isoforms of HSA, respectively. Molecular docking results revealed that meropenem binds to HSA near Sudlow’s site II (subdomain IIIA) close to Trp-214 with a contribution of a few residues of subdomain IIA. CD spectroscopy showed a change in the conformation of both the isoforms of HSA upon meropenem binding. The catalytic efficiency of HSA (only N-isoform) on p-nitrophenyl acetate was increased primarily due to a decrease in K_m and an increase in k_cat values. This study provides an insight into the molecular basis of interaction between meropenem and HSA.     

Exploring the interaction between tyrphostin 9 and human serum albumin using biophysical and computational methods

Abstract

Tyrphostin 9 (Tyr 9) is a potent platelet-derived growth factor receptor (PDGFR) inhibitor, which induces apoptosis in various cancer cell types. The binding of Tyr 9 to the major transport protein, human serum albumin (HSA) was investigated using several spectroscopic techniques and molecular docking method. Fluorescence quenching titration results showed progressive decrease in the protein fluorescence with increasing drug concentrations. A decreasing trend of the Stern-Volmer constant, K _sv with increasing temperature characterized the drug-induced quenching as static quenching, thus pointed towards the formation of Tyr 9–HSA complex. The binding constant of Tyr 9–HSA interaction was found to lie within the range 3.48–1.69?×?10⁵ M^?1 at three different temperatures, i.e. 15 °C, 25 °C and 35?°C, respectively and suggested intermediate binding affinity between Tyr 9 and HSA. The drug–HSA complex seems to be stabilized by hydrophobic forces, van der Waals forces and hydrogen bonds, as suggested from the thermodynamic data as well as molecular docking results. The far-UV and the near-UV CD spectral results showed slight alteration in the secondary and tertiary structures, respectively, of the protein upon Tyr 9 binding. Interaction of Tyr 9 with HSA also produced microenvironmental perturbations around protein fluorophores, as evident from the three-dimensional fluorescence spectral results but increased protein’s thermal stability. Both competitive drug binding results and molecular docking analysis suggested Sudlow’s Site I of HSA as the preferred Tyr 9 binding site.

Communicated by Ramaswamy H. Sarma     

Study of the Enantioselective Interaction of Diclofop and Human Serum Albumin by Spectroscopic and Molecular Modeling Approaches In Vitro

In this contribution, the enantioselective interactions between diclofop (DC) and human serum albumin (HSA) were explored by steady‐state and 3D fluorescence, ultraviolet‐visible spectroscopy (UV‐vis), and molecular modeling. The binding constants between R‐DC and HSA were 0.9213 × 10⁵, 0.9118 × 10⁵, and 0.9009 × 10⁵ L · mol^‐1 at 293, 303, 313 K, respectively. Moreover, the binding constants of S‐DC for HSA were 1.4766 × 10⁵, 1.2899 × 10⁵, and 1.0882 × 10⁵ L · mol^‐1 at 293, 303, and 313 K individually. Such consequences markedly implied the binding between DC enantiomers and HSA were enantioselective with higher affinity for S‐DC. Steady‐state fluorescence study evidenced the formation of DC‐HSA complex and there was a single class of binding site on HSA. The thermodynamic parameters (ΔH, ΔS, ΔG) of the reaction clearly indicated that hydrophobic effects and H‐bonds contribute to the formation of DC‐HSA complex, which was in excellent agreement with molecular simulations. In addition, both site‐competitive replacement and molecular modeling suggested that DC enantiomers were located within the binding pocket of Sudlow's site II. Furthermore, the alterations of HSA secondary structure in the presence of DC enantiomers were verified by UV‐vis absorption and 3D fluorescence spectroscopy. This study can provide important insight into the enantioselective interaction of physiological protein HSA with chiral aryloxyphenoxy propionate herbicides and gives support to the use of HSA for chiral pesticides ecotoxicology and environmental risk assessment. Chirality 25:719–725, 2013. © 2013 Wiley Periodicals, Inc.     

Fipronil recognition by the FA1 site of human serum albumin

Fipronil is a broad‐spectrum pesticide widely used in agriculture, horticulture, and forestry. Because fipronil can cause a variety of toxic effects in animals and humans, its use is authorized as a pesticide in veterinary medicinal products for pets, but not for the treatment of livestock animals whose products are intended for consumption. Recently, however, the presence of fipronil residues has been detected in the eggs and meat of layer hens from farms located in different European countries. Given the relevance of fipronil toxicity for human health, it is important to gain information concerning its fate in the human body, including its binding mode to human serum albumin (HSA), the most abundant protein in plasma. Here, the inhibition of heme‐Fe(III) binding to the fatty acid site 1 (FA1) of HSA by fipronil is reported. Docking simulations support functional data, indicating that the FA1 site is the preferential cleft for fipronil recognition by HSA. The affinity of fipronil for HSA (K_f = 1.9 × 10^?6 M, at pH 7.3, and 20.0°C) may be relevant in vivo. Indeed, HSA could play a pivotal role in fipronil transport and scavenging, thus reducing the pesticide‐free plasmatic levels, with consequent reduced systemic toxicity. In turn, fipronil binding to the FA1 site of HSA could impair the recognition of endogenous and exogenous molecules.     

Binding of Janus kinase inhibitor tofacitinib with human serum albumin: multi-technique approach

In this report, we have investigated the binding affinity of tofacitinib with human serum albumin (HSA) under simulated physiological conditions by using UV–visible spectroscopy, fluorescence quenching measurements, dynamic light scattering (DLS), differential scanning calorimetry (DSC) and molecular docking methods. The obtained results demonstrate that fluorescence intensity of HSA gets quenched by tofacitinib and quenching occurs in static manner. Binding parameters calculated from modified Stern–Volmer equation shows that the drug binds to HSA with a binding constant in the order of 10⁵. Synchronous fluorescence data deciphered the change in the microenvironment of tryptophan residue in HSA. UV spectroscopy and DLS measurements deciphered complex formation and reduction in hydrodynamic radii of the protein, respectively. Further DSC results show that tofacitinib increases the thermo stability of HSA. Hydrogen bonding and hydrophobic interaction are the main binding forces between HSA and tofacitinib as revealed by docking results.     

A comparison investigation of DNP-binding effects to HSA and HTF by spectroscopic and molecular modeling techniques

This paper describes the interaction between 2,4-dinitrophenol (DNP) with the two drug carrier proteins – human serum albumin (HSA) and human holo transferrin (HTF). Hence, binding characteristics of DNP to HSA and HTF were analyzed by spectroscopic and molecular modeling techniques. Based on results obtained from fluorescence spectroscopy, DNP had a strong ability to quench the intrinsic fluorescence of HSA and HTF through a static quenching procedure. The binding constant and the number of binding sites were calculated as 2.3?×?10¹¹?M^?1 and .98 for HSA, and 1.7?×?10¹¹?M^?1 and 1.06 for HTF, respectively. In addition, synchronous fluorescence results showed that the microenvironment of Trp had a slight tendency of increasing its hydrophobicity, whereas the microenvironment of the Tyr residues of HSA did not change and that of HTF showed a significant trend (red shift of about 4?nm) of an increase in polarity. The distance between donor and acceptor was obtained by the Förster energy according to fluorescence resonance energy transfer, and was found to be 3.99 and 3.72?nm for HSA and HTF, respectively. The critical induced aggregation concentration (C_CIAC) of the drug on both proteins was determined and confirmed by an inflection point of the zeta potential behavior. Circular dichroism data revealed that the presence of DNP caused a decrease of the α-helical content of HSA and HTF, and induced a remarkable mild denaturation of both proteins. The molecular modeling data confirmed our experimental results. This study is deemed useful for determining drug dosage.     

Insights into the binding of paclitaxel to human serum albumin: multispectroscopic studies

The interaction of paclitaxel with human serum albumin (HSA) was studied using fluorescence, resonance light scattering, ultraviolet‐visible, circular dichroism and Fourier transform infrared spectroscopy at pH 7.4. Fluorescence data revealed that the fluorescence quenching of HSA by paclitaxel was a static quenching procedure. Time‐resolved fluorescence data also confirmed the quenching mode, which present a constant decay time of about 5 ns. The binding sites were approximately 1 and the binding constant suggested a weak association (324/M at 298 K), which is helpful for the release of the drug to targeted organs. The thermodynamic parameters, ΔG^○, ΔH° and ΔS° were calculated as – 1.06 × 10⁴ J/mol, 361 J/mol per K and 9.7 × 10⁴ J/mol respectively at 298 K, suggesting that binding was spontaneous and was driven mainly by hydrophobic interactions. The binding distance between HSA and paclitaxel was determined to be 2.23 nm based on the Förster theory. Analysis of circular dichroism, ultraviolet‐visible, three‐dimensional fluorescence, Fourier transform infrared and resonance light scattering spectra demonstrated that HSA conformation was slightly altered in the presence of paclitaxel and dimension of the individual HSA molecules were larger after interacting with paclitaxel. These results were confirmed by a molecular docking study. Copyright © 2013 John Wiley & Sons, Ltd.     

Titanium dioxide nanoparticles preferentially bind in subdomains IB,IIA of HSA and minor groove of DNA

Titanium dioxide nanoparticles (TiO₂-NPs) interaction with human serum albumin (HSA) and DNA was studied by UV–visible spectroscopy, spectrofluorescence, circular dichroism (CD), and transmission electron microscopy (TEM) to analyze the binding parameters and protein corona formation. TEM revealed protein corona formation on TiO₂-NPs surface due to adsorption of HSA. Intrinsic fluorescence quenching data suggested significant binding of TiO₂-NPs (avg. size 14.0 nm) with HSA. The Stern–Volmer constant (K_sv) was determined to be 7.6 × 10² M^?1 (r² = 0.98), whereas the binding constant (K_a) and number of binding sites (n) were assessed to be 5.82 × 10² M^?1 and 0.97, respectively. Synchronous fluorescence revealed an apparent decrease in fluorescence intensity with a red shift of 2 nm at Δλ = 15 nm and Δλ = 60 nm. UV–visible analysis also provided the binding constant values for TiO₂-NPs–HSA and TiO₂-NPs-DNA complexes as 2.8 × 10² M^?1 and 5.4 × 10³ M^?1. The CD data demonstrated loss in α-helicity of HSA and transformation into β-sheet, suggesting structural alterations by TiO₂-NPs. The docking analysis of TiO₂-NPs with HSA revealed its preferential binding with aromatic and non-aromatic amino acids in subdomain IIA and IB hydrophobic cavity of HSA. Also, the TiO₂-NPs docking revealed the selective binding with A-T bases in minor groove of DNA.