Spectroscopic investigation of Bovine Liver Catalase interactions with a novel phen-imidazole derivative of platinum

Successful clinical experience of using cisplatin and its derivatives in cancer therapy has encouraged scientists to synthesize new metal complexes with the aim of interacting with special targets such as proteins In this regard, biological effects of [Pt(FIP)(Phen)](NO₃)₂ compound which contains a novel phen-imidazole ligand, FIP, was investigated on bovine liver catalase (BLC) structure and function. Various spectroscopic methods such as UV–visible, fluorescence, and circular dichroism (CD) were applied at two temperatures 25 and 37°C for kinetics and structural studies. As a consequence, the enzymatic activity decreased slightly with increasing the platinum compound’s concentration up to 30 μM and then remained constant at near 80% after this concentration. On the other hand, the fluorescence quenching measurements revealed that despite slight changes in activity, catalase experiences notable alterations in three-dimensional environment around the chromophores of the enzyme structure with increasing platinum complex concentration. Moreover, quenching data showed that BLC has two binding sites for Pt complex and hydrogen bonding interactions play a major role in the binding process. Furthermore, CD spectroscopy data showed that Pt(II) complex induces significant decrease in α-helix content of the secondary structure of BLC, but notable increase in random coil proportion accompanying a slight decrease in β-sheet content. All in all, hydrogen bonding interactions which are mainly involved in the binding process of the novel phen-imidazole compound to BLC significantly alter the protein structure but slightly change its function. This might be a promising outcome for chemotherapists and medicinal chemists to investigate in vivo properties of this novel metal complex with significant binding tendency to a macromolecule in the low concentrations without decreasing its intrinsic function.     

Spectroscopic and molecular simulation studies on the interaction of di‐(2‐ethylhexyl) phthalate and human serum albumin

Di‐(2‐ethylhexyl) phthalate (DEHP) is widely used as a plasticizer in industrial production, but may have a potential health risk. In this study, the binding characteristics of DEHP with human serum albumin (HSA) in aqueous solution at pH 7.4 were determined using UV/vis absorption, fluorescence, Fourier transform infrared (FTIR) spectroscopy and circular dichroism (CD), along with a molecular simulation technique. Analysis of the fluorescence titration data at different temperatures suggested that the fluorescence quenching mechanism of HSA by DEHP was static. The calculated thermodynamic parameters indicated that hydrophobic forces played a predominant role in formation of the DEHP–HSA complex, but hydrogen bonds could not be omitted. Site marker competitive experiments and denaturation studies showed that the binding of DEHP to HSA primarily took place in subdomain IIA of HSA, and molecular docking results further corroborated the binding sites. The synchronous fluorescence, UV/vis absorption, FTIR and CD spectra revealed that the addition of DEHP induced changes in the secondary structure of HSA. Protein surface hydrophobicity (PSH) tests indicated that DEHP binding to HSA caused an increase in the PSH. Moreover, the effects of some metal ions on the binding constant of DEHP − HSA interaction were also investigated. Copyright © 2014 John Wiley & Sons, Ltd.     

Study on the interaction of chromate with bovine serum albumin by spectroscopic method

The interaction between two chromates [sodium chromate (Na₂CrO₄) and potassium chromate K₂CrO₄)] and bovine serum albumin (BSA) in physiological buffer (pH 7.4) was investigated by the fluorescence quenching technique. The results of fluorescence titration revealed that two chromates could strongly quench the intrinsic fluorescence of BSA through a static quenching procedure. The apparent binding constants K and number of binding sites n of chromate with BSA were obtained by the fluorescence quenching method. The thermodynamic parameters enthalpy change (ΔH), entropy change (ΔS) were negative, indicating that the interaction of two chromates with BSA was driven mainly by van der Waals forces and hydrogen bonds. The process of binding was a spontaneous process in which Gibbs free energy change was negative. The distance r between donor (BSA) and acceptor (chromate) was calculated based on Forster’s non-radiative energy transfer theory. The results of UV–Vis absorption, synchronous fluorescence, three-dimensional fluorescence and circular dichroism (CD) spectra showed that two chromates induced conformational changes of BSA.     

Study on the mechanism of the interaction between acteoside and pepsin using spectroscopic techniques

The interaction of acteoside with pepsin has been investigated using fluorescence spectra, UV/vis absorption spectra, three‐dimensional (3D) fluorescence spectra and synchronous fluorescence spectra, along with a molecular docking method. The fluorescence experiments indicate that acteoside can quench the intrinsic fluorescence of pepsin through combined quenching at a low concentration of acteoside, and static quenching at high concentrations. Thermodynamic analysis suggests that hydrogen bonds and van der Waal's forces are the main forces between pepsin and acteoside. According to the theory of Förster's non‐radiation energy transfer, the binding distance between pepsin and acteoside was calculated to be 2.018 nm, which implies that energy transfer occurs between acteoside and pepsin. In addition, experimental results from UV/vis absorption spectra, 3D fluorescence spectra and synchronous fluorescence spectra imply that pepsin undergoes a conformation change when it interacts with acteoside. Copyright © 2015 John Wiley & Sons, Ltd.     

How the surface functionalized nanoparticles affect conformation and activity of proteins: Exploring through protein-nanoparticle interactions

To understand the effect of counter ions (Na⁺) on the secondary conformation and functionality of the lysozyme, we have studied the interaction of lysozyme with counterion associated iron oxide nanoparticles (IONPs). The investigation was carried out at pH 7.4 and 9.0, with three different types of NPs, namely, bare IONPs, low molecular weight chitosan modified IONPs (LMWC-IONPs) and the counterion (Na⁺) associated sodium tripolyphosphate IONPs (STP-LMWC-IONPs) and confirmed by using various spectroscopy techniques. The difference in UV–vis absorbance (ΔA) between native and STP-LMWC-IONPs interacted hen egg white lysozyme (HEWL) was greater than that between native and NPs interacted HEWL at pH 9.0 compared with pH 7.4. Furthermore, STP-LMWC-IONPs exhibited quenching effect on lysozyme fluorescence spectrum at pH 9.0 due to binding of Na⁺ counterions to the protein, confirming denaturation of the latter. After HEWL interaction with STP-LMWC-IONPs (pH 9.0), CD spectra revealed a conformational change in the secondary structure of HEWL. Also, counterion induced lysozyme inactivation, due to interaction with nanoparticles at pH 9.0, was confirmed by enzymatic activity assay involving lysis of Micrococcus lysodeikticus. In conclusion, pH 9.0 was observed to be a more favorable condition, compared to pH 7.4, for the strongest electrostatic interaction between lysozyme and NPs. We postulate that the counterions in nanoparticle surface-coating can ameliorate protein misfolding or unfolding and also prevent their aggregation and, therefore, can be considered as a powerful and potential therapeutic strategy to treat incurable neurodegenerative disorders.     

Exploration of interaction of canthaxanthin with human serum albumin by spectroscopic and molecular simulation methods

The interaction between the food colorant canthaxanthin (CA) and human serum albumin (HSA) in aqueous solution was explored by using fluorescence spectroscopy, three‐dimensional fluorescence spectra, synchronous fluorescence spectra, UV–vis absorbance spectroscopy, circular dichroism (CD) spectra and molecular docking methods. The thermodynamic parameters calculated from fluorescence spectra data showed that CA could result in the HSA fluorescence quenching. From the K_SV change with the temperature dependence, it was concluded that HSA fluorescence quenching triggered by CA is the static quenching and the number of binding sites is one. Furthermore, the secondary structure of HSA was changed with the addition of CA based on the results of synchronous fluorescence, three‐dimensional fluorescence and CD spectra. Hydrogen bonds and van der Waals forces played key roles in the binding process of CA with HSA, which can be obtained from negative standard enthalpy (ΔH) and negative standard entropy (ΔS). Furthermore, the conclusions were certified by molecular docking studies and the binding mode was further analyzed with Discovery Studio. These conclusions can highlight the potential of the interaction mechanism of food additives and HSA.     

Biophysical studies on the interactions of jatrorrhizine with bovine serum albumin by spectroscopic and molecular modeling methods

The interaction between jatrorrhizine (JAT) and bovine serum albumin (BSA) has been studied. The studies were carried out in a buffer medium at pH 7.4 using fluorescence spectroscopy, UV–vis spectroscopy, and molecular modeling methods. The results of fluorescence quenching and UV–vis absorption spectra experiments indicated the formation of the complex of BSA–JAT. Binding parameters were determined using the Stern–Volmer equation and Scatchard equation. The results of thermodynamic parameters ΔG, ΔH and ΔS at different temperatures indicate that the electrostatic interactions and hydrogen bonds play a major role for JAT–BSA association. Site marker competitive displacement experiments and molecular modeling calculation demonstrating that JAT is mainly located within the hydrophobic pocket of the subdomain IIIA of BSA. Furthermore, The distance between donor (BSA) and acceptor (JAT) was estimated according to fluorescence resonance energy transfer.     

Spectroscopic analysis on the interaction of ferulic acid and tetramethylpyrazine with trypsin

The interaction of trypsin with tetramethylpyrazine (TMP) and ferulic acid (FA) was studied using fluorescence, synchronous fluorescence, UV–vis absorption, circular dichroism (CD) and three‐dimensional (3D) fluorescence spectra techniques. Using fluorescence quenching calculations, the bimolecular quenching constant (k_q), apparent quenching constant (K_SV), effective binding constant (K_a) and binding site number (n) were obtained. The distance r between donor and acceptor was found to be 2.049 and 1.281 nm for TMP–trypsin and FA–trypsin complexes. TMP and FA can quench the fluorescence intensity of trypsin by a static quenching procedure. Thermodynamic parameters calculated on the basis of different temperatures revealed that the binding of trypsin to TMP/FA mainly depended on van der Waals' forces and hydrogen bonds. The effect of TMP and FA on the conformation of trypsin was analyzed using synchronous fluorescence, CD, 3D fluorescence spectra and molecular docking studies. Copyright © 2013 John Wiley & Sons, Ltd.     

Probing the behavior of bovine serum albumin upon binding to atenolol: insights from spectroscopic and molecular docking approaches

Molecular interaction of atenolol, a selective β₁ receptor antagonist with the major carrier protein, bovine serum albumin (BSA), was investigated under imitated physiological conditions (pH 7.4) by means of fluorescence spectroscopy, UV absorption spectroscopy, Fourier transform infrared spectroscopy (FT-IR), and molecular modeling studies. The steady-state fluorescence spectra manifested that static type, due to formation of the atenolol-BSA complex, was the dominant mechanism for fluorescence quenching. The characteristic information about the binding interaction of atenolol with BSA in terms of binding constant (K_b) were determined by the UV–vis absorption titration, and were found to be in the order of 10³ M^?1 at different temperatures, indicating the existence of a weak binding in this system. Thermodynamic analysis revealed that the binding process was primarily mediated by van der Waals force and hydrogen bonds due to the negative sign for enthalpy change (ΔH⁰), entropy change (ΔS⁰). The molecular docking results elucidated that atenolol preferred binding on the site II of BSA according to the findings observed in competitive binding experiments. Moreover, via alterations in synchronous fluorescence, three-dimensional fluorescence and FT-IR spectral properties, it was concluded that atenolol could arouse slight configurational and micro-environmental changes of BSA.     

Inhibitory effects of deferasirox on the structure and function of bovine liver catalase: a spectroscopic and theoretical study

Deferasirox (DFX), as an oral chelator, is used for treatment of transfusional iron overload. In this study, we have investigated the effects of DFX as an iron chelator, on the function and structure of bovine liver catalase (BLC) by different spectroscopic methods of UV–visible, fluorescence, and circular dichroism (CD) at two temperatures of 25 and 37 °C. In vitro kinetic studies showed that DFX can inhibit the enzymatic activity in a competitive manner. K _I value was calculated 39 nM according to the Lineweaver–Burk plot indicating a high rate of inhibition of the enzyme. Intrinsic fluorescence data showed that increasing the drug concentrations leads to a significant decrease in the intrinsic emission of the enzyme indicating a significant change in the three-dimensional environment around the chromophores of the enzyme structure. By analyzing the fluorescence quenching data, it was found that the BLC has two binding sites for DFX and the values of binding constant at 25 and 37 °C were calculated 1.7 × 10⁷ and 3 × 10⁷ M^?1, respectively. The static type of quenching mechanism is involved in the quenching of intrinsic emission of enzyme. The thermodynamic data suggest that hydrophobic interactions play a major role in the binding reaction. UV–vis spectroscopy results represented the changes in tryptophan (Trp) absorption and Soret band spectra, which indicated changes in Trp and heme group position caused by the drug binding. Also, CD data represented that high concentrations of DFX lead to a significant decreasing in the content of β-sheet and random coil accompanied an increasing in α-helical content of the protein. The molecular docking results indicate that docking may be an appropriate method for prediction and confirmation of experimental results and also useful for determining the binding mechanism of proteins and drugs. According to above results, it can be concluded that the DFX can chelate the Fe(III) on the enzyme active site leading to changes in the function and structure of catalase which can be considered as a side effect of this drug and consequently has an important role in hepatic complications and fibrosis.     

Study on the interaction between anti‐tuberculosis drug ethambutol and bovine serum albumin: multispectroscopic and cyclic voltammetric approaches

The binding of bovine serum albumin (BSA) to ethambutol (EMB) was investigated using spectroscopic methods, viz., fluorescence, Fourier transform infrared (FTIR), ultraviolet (UV)/vis absorption and cyclic voltammetry techniques. Spectroscopic analysis of the emission quenching at different temperatures revealed that the quenching mechanism of serum albumin by EMB is static, which was also confirmed by lifetime measurements. The number of binding sites, n, and binding constant, K, were obtained at various temperatures. The distance, r, between EMB and the protein was evaluated according to the Förster energy transfer theory. Based on displacement experiments using site probes, viz., warfarin, ibuprofen and digitoxin, the site of binding of EMB in BSA was proposed to be Sudlow's site I. The effect of EMB on the conformation of BSA was analyzed by using synchronous fluorescence spectra (SFS) and 3D fluorescence spectra. The results of fluorescence, UV/vis absorption and FTIR spectra showed that the conformation of BSA was changed in the presence of EMB. The thermodynamic parameters including enthalpy change (ΔH⁰), entropy change (ΔS⁰) and free energy change (ΔG⁰) for BSA–EMB were calculated according to the van't Hoff equation and are discussed.     

Analyzing the interaction between porcine serum albumin (PSA) and ester-functionalized cationic gemini surfactants

Green gemini surfactants (m-E2-m) containing cleavable ester functionalities were allowed to interact with porcine serum albumin (PSA) and the binding mechanism along with concomitant physicochemical changes were analyzed using a multi-technique approach. Intrinsic fluorescence study reveals that the concerned gemini surfactants have a substantial affinity for PSA at a pH well above its isoelectric point. Static quenching is indicated by the relevant value of bimolecular quenching constant (k_q). Synchronous, three-dimensional and pyrene fluorescence data depict the PSA−m-E2-m complex formation together with significant conformational changes induced in PSA. UV–vis studies are also indicative of ground state complexation involving the biomolecule and the amphiphile. Far-UV CD spectra indicate the stabilization of protein structure as deciphered by the increased α-helical content, whereas near-UV CD spectra signify a tertiary structure close to that of the native state. In order to validate the results obtained by fluorescence, cyclic voltammetry is employed, which unveils the formation of electrochemically inactive PSA−m-E2-m complex. Moreover, it is revealed that gemini with longer chain interacts more efficiently than the one with shorter chain owing to stronger hydrophobic forces. All these findings suggest that in future, the m-E2-m geminis may be potentially utilized as excipients in drug, skincare and immunoassay reagent formulations. Furthermore, results of this research work will also contribute in understanding the physicochemical effect of surfactant architecture on its interaction with biomacromolecules.     

Interactions between m‐phenylenediamine and bovine serum albumin measured by spectroscopy

This study explored interactions between m‐phenylenediamine (MPD) and bovine serum albumin (BSA) by spectrophotometry. The Stern‐Volmer equation and UV‐vis spectra examination at different temperatures and pH were used to explore different quenching mechanisms. Under simulated physiological conditions, the binding distance between MPD and BSA was 5.18 nm with a ratio of 1:1. The quenching effect of MPD on BSA intrinsic fluorescence depended strongly on pH, and maximum quenching was observed at alkaline pH. Moreover, the thermodynamic parameters of the MPD‐BSA system showed that the predominant acting force between MPD and BSA was a hydrophobic force. The impact of MPD on the conformation of BSA and the effects of co‐ions on binding interactions were also examined. Copyright © 2012 John Wiley & Sons, Ltd.     

Combined spectroscopies and molecular docking approach to characterizing the binding interaction of enalapril with bovine serum albumin

The binding interaction between bovine serum albumin (BSA) and enalapril (ENPL) at the imitated physiological conditions (pH = 7.4) was investigated using UV–vis absorption spectroscopy (UV–vis), fluorescence emission spectroscopy (FES), synchronous fluorescence spectroscopy (SFS), Fourier transform infrared spectroscopy (FT‐IR), circular dichroism (CD) and molecular docking methods. It can be deduced from the experimental results from the steady‐state fluorescence spectroscopic titration that the intrinsic BSA fluorescence quenching mechanism induced by ENPL is static quenching, based on the decrease in the BSA quenching constants in the presence of ENPL with increase in temperature and BSA quenching rates >10¹⁰ L mol^?1 sec^?1. This result indicates that the ENPL–BSA complex is formed through an intermolecular interaction of ENPL with BSA. The main bonding forces for interaction of BSA and ENPL are van der Waal's forces and hydrogen bonding interaction based on negative values of Gibbs free energy change (ΔG ⁰), enthalpic change (ΔH ⁰) and entropic change (ΔS ⁰). The binding of ENPL with BSA is an enthalpy‐driven process due to |ΔH °| > |T ΔS °| in the binding process. The results of competitive binding experiments and molecular docking confirm that ENPL binds in BSA sub‐domain IIA (site I) and results in a slight change in BSA conformation, but BSA still retains its α‐helical secondary structure.     

The investigation of the interaction between ribavirin and bovine serum albumin by spectroscopic methods

The interaction between ribavirin (RIB) with bovine serum albumin (BSA) has been investigated by fluorescence quenching technique in combination with UV–vis absorption and circular dichroism (CD) spectroscopies under the simulative physiological conditions. The quenching of BSA fluorescence by RIB was found to be a result of the formation of RIB–BSA complex. The binding constants and the number of binding sites were calculated at three different temperatures. The values of thermodynamic parameters ?H, ?S, ?G at different temperatures indicate that hydrophobic and hydrogen bonds played important roles for RIB–BSA association. The binding distance r was obtained according to the theory of FÖrster’s non–radiation energy transfer. The displacement experiments was performed for identifying the location of the binding site of RIB on BSA. The effects of common ions on the binding constant of RIB and BSA were also examined. Finally, the conformational changes of BSA in the presence of RIB were also analyzed by CD spectra and Synchronous fluorescence spectra.     

Investigation of the interactions of quercetin and morin with trypsin

The interactions of quercetin and morin with trypsin were investigated by UV–vis absorption, fluorescence, synchronous fluorescence and three‐dimensional fluorescence spectra techniques under physiological pH 7.40. Quercetin and morin effectively quenched the intrinsic fluorescence of trypsin via static quenching. The process of binding quercetin and morin on trypsin was a spontaneous molecular interaction procedure. The binding constants and thermodynamic parameters at two different temperatures, the binding locality and the binding power were obtained. The conformation of trypsin was discussed by synchronous and three‐dimensional fluorescence techniques. Copyright © 2009 John Wiley & Sons, Ltd.     

Molecular interactions of thymol with bovine serum albumin: Spectroscopic and molecular docking studies

Thymol is the main monoterpene phenol present in the essential oils which is used in the food industry as flavoring and preservative agent. In this study, the interaction of thymol with the concentration range of 1 to 6 μM and bovine serum albumin (BSA) at fixed concentration of 1 μM was investigated by fluorescence, UV‐vis, and molecular docking methods under physiological‐like condition. Fluorescence experiments were performed at 5 different temperatures, and the results showed that the fluorescence quenching of BSA by thymol was because of a static quenching mechanism. The obtained binding parameters, K, were in the order of 10⁴ M^?1, and the binding number, n, was approximately equal to unity indicating that there is 1 binding site for thymol on BSA. Calculated thermodynamic parameters for enthalpy (ΔH), entropy (ΔS), and Gibb's free energy (ΔG) showed that the reaction was spontaneous and hydrophobic interactions were the main forces in the binding of thymol to BSA. The results of UV‐vis spectroscopy and Arrhenius' theory showed the complex formation in the interaction of thymol and BSA. Negligible conformational changes in BSA by thymol were observed in fluorescence experiments, and the same results were also obtained from UV‐vis studies. Results of molecular docking indicated that the subdomain IA of BSA was the binding site for thymol.     

Comparative studies on the interactions of dihydroartemisinin and artemisinin with bovine serum albumin using spectroscopic methods

The interactions of dihydroartemisinin (DHA) and artemisinin (ART) with bovine serum albumin (BSA) have been investigated using fluorescence, UV/vis absorption and Fourier transform infrared (FTIR) spectra under simulated physiological conditions. The binding characteristics of DHA/ART and BSA were determined by fluorescence emission and resonance light scattering (RLS) spectra. The quenching mechanism between BSA and DHA/ART is static. The binding constants and binding sites of DHA/ART–BSA systems were calculated at different temperatures (293, 298, 304 and 310 K). According to Förster non‐radiative energy transfer theory, the binding distance of BSA to DHA/ART was calculated to be 1.54/1.65 nm. The effect of DHA/ART on the secondary structure of BSA was analyzed using UV/vis absorption, FTIR, synchronous fluorescence and 3D fluorescence spectra. In addition, the effects of common ions on the binding constants of BSA–DHA and BSA–ART systems were also discussed. Copyright © 2014 John Wiley & Sons, Ltd.     

Study of characterization and application on the binding between 5-iodouridine with HSA by spectroscopic and modeling

The binding of 5-iodouridine with human serum albumin was investigated under the simulative physiological conditions. The fluorescence spectra in combination with UV absorption and modeling method were used in the present work. A strong fluorescence quenching reaction of 5-iodouridine to HSA was observed and the quenching mechanism was suggested as static quenching procedure. The binding constants (K) at different temperatures as well as thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS), were calculated. It showed that the hydrophobic interaction was a predominant intermolecular force in order to stabilize the complex, which was in agreement with the result of modeling study. The binding distance between 5-iodouridine and HSA was calculated on the basis of the theory of Föster energy transfer. The effects of other ions on the binding constants were also discussed. Synchronous fluorescence spectroscopy (SFS) technique were successfully applied to determine protein in the biological samples.     

Comparison of the binding behavior of FCCP with HSA and HTF as determined by spectroscopic and molecular modeling techniques

The interaction of carbonylcyanide p‐(trifluoromethoxy) phenylhydrazone (FCCP) with human serum albumin (HSA) and human transferrin (HTF) was investigated using multiple spectroscopy, molecular modeling, zeta‐potential and conductometry measurements of aqueous solutions at pH 7.4. The fluorescence, UV/vis and polarization fluorescence spectroscopy data disclosed that the drug–protein complex formation occurred through a remarkable static quenching. Based on the fluorescence quenching, two sets of binding sites with distinct affinities for FCCP existed in the two proteins. Steady‐state and polarization fluorescence analysis showed that there were more affinities between FCCP and HSA than HTF. Far UV‐CD and synchronous fluorescence studies indicated that FCCP induced more structural changes on HSA. The resonance light scattering (RLS) and zeta‐potential measurements suggested that HTF had a greater resistance to drug aggregation, whereas conductometry measurements expressed the presence of free ions improving the resistance of HSA to aggregation. Thermodynamic measurements implied that a combination of electrostatic and hydrophobic forces was involved in the interaction between FCCP with both proteins. The phase diagram plots indicated that the presence of second binding site on HSA and HTF was due to the existence of intermediate structures. Site marker competitive experiments demonstrated that FCCP had two distinct binding sites in HSA which were located in sub‐domains IIA and IIIA and one binding site in the C‐lobe of HTF as confirmed by molecular modeling. The obtained results suggested that both proteins could act as drug carriers, but that the HSA potentially had a higher capacity for delivering FCCP to cancerous tissues. Copyright © 2013 John Wiley & Sons, Ltd.