Article

New insights into the dimerization and site-specific cooperative interaction of Azure B with model transport proteins by spectroscopic and computational studies

Authors:
To read the full-text of this research, you can request a copy directly from the authors.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

... Due to their high redox potential and effective interaction with Visible light, these dyes show great potential for pathogens in fresh plasma [35]. Azure B (AB) also belongs to the class of cationic phenothiazinium dyes and exhibit high reactivity towards DNA and proteins [36]. AB is more stable in aqueous solution compared to its analogue methylene blue (MB) (FDA approved PS agent) and form promising PS agents for PDT [37]. ...
... The studies of binding nature and dynamics of drugs or nanostructured materials with biological macromolecules have always been considered as an important area of research, as these provide insights into their physiological responses as well as their potential adverse effects. Serum albumins are the most abundant proteins present in circulatory system of various organisms, which regulate many physiological functions like pH buffering, and controlling osmotic pressure of serum etc. Serum albumins offer reversible binding interactions with wide range of endogenous and exogenous drugs, which make them potential vehicle for transportation and selective delivery of therapeutic agents to their cellular targets [36]. DNA is a vital cellular component that regulates all chemical processes that occur in cells. ...
... Therefore, to determine the mechanism of quenching of fluorescence of AB@CI-Au NPs due to Ct-DNA interactions, the spectra were recorded on four different temperatures 293 K, 298 K, 303 K and 308 K and spectral data was analysed using Stern Volmer equation [eq. (6)] [36] given below: (6) where F o and F represent the steady-state fluorescence intensities of control solution in the absence and presence of quencher, respectively; K sv denotes the Stern Volmer constant, K q represents quenching rate constant, [Q] is the concentration of quencher (AB@CI-Au NPs) and τ o is the average life time for Ct-DNA i.e. 10 −8 s. Fig. 9 (b) displays the Stern Volmer plot for the quenching of fluorescence of AB@CI-Au NPs by Ct-DNA at different temperatures. ...
Article
Multimodal or combination therapy has been considered as a powerful approach for treatment of complex diseases like cancer. The fascinating physicochemical and optoelectronic properties of gold nanoparticles make them potential candidate for cancer therapeutic and diagnostic applications. Herein, we design a multifunctional nanosystem by conjugating a photosensitizer, Azure B (AB) with citrate reduced gold nanoparticles (CI-Au NPs) through non-covalent interactions. The conjugation of AB with CI-Au NPs was confirmed through UV–Visible absorption spectroscopy and Fourier Transform Infra-red (FT-IR) spectroscopy. The morphology, size, and charge of the prepared nano-conjugates ([email protected] NPs) were determined by transmission electron microscopy (TEM), Dynamic light scattering (DLS), and zeta potential measurements. The proficiency of [email protected] NPs for cancer photo-therapies was demonstrated by evaluating their potential for photothermal heating and singlet oxygen generation in solution upon Visible laser (635 nm, 500 mW/cm²) irradiation. The [email protected] NPs display superior heating efficiency than CI-Au NPs alone or free AB, and offer better photostability as well as singlet oxygen generation rate compared to free photosensitizer. The interaction of [email protected] NPs with calf thymus DNA (Ct-DNA) and transport protein bovine serum albumin (BSA) were studied using various biophysical techniques including Circular dichroism, UV–Visible and fluorescence spectroscopic studies. [email protected] NPs show intercalative binding with Ct-DNA by inducing local perturbations in double helical structure and hence can exert chemotherapeutic action by targeting DNA. [email protected] NPs also display moderate binding with BSA with no adverse effect on BSA structure, which is desirable for significant biodistribution and pharmacokinetics of [email protected] NPs. Also, in vitro cytotoxicity of the [email protected] NPs with and without laser irradiation (635 nm, 500 mW/cm²) was demonstrated using the hepatocellular carcinoma (HepG2) cell lines. Our findings through photophysical and biophysical studies strongly recommend the exploitation of [email protected] NPs nanoconjugates as a multifunctional probe for trimodal anticancer therapy.
... In recent past, organic dye (Azure B) was increasingly employed in medical and clinical fields [14]. In particular, cationic PHZ dyes were used in the photodynamic therapy for the diagnostics of various fungal, bacterial and viral infections [14]. ...
... In recent past, organic dye (Azure B) was increasingly employed in medical and clinical fields [14]. In particular, cationic PHZ dyes were used in the photodynamic therapy for the diagnostics of various fungal, bacterial and viral infections [14]. These PHZ dyes have the skill to inactivate several forms of pathogenic agents in fresh plasma due to their more redox potential and their ability to effectively co-operate with visible light [14]. ...
... In particular, cationic PHZ dyes were used in the photodynamic therapy for the diagnostics of various fungal, bacterial and viral infections [14]. These PHZ dyes have the skill to inactivate several forms of pathogenic agents in fresh plasma due to their more redox potential and their ability to effectively co-operate with visible light [14]. AZA (Scheme 1(a)) is a blue colored, synthetic cationic dye belonging to the PHZ group [15]. ...
... Bond distance between -O-atom and ASN 318 residue was observed to be 1.99 Å (Fig. 9B). Formation of stabilized complex, DAS-HSA in turn confirmed the interaction of DAS with HSA [57,58]. All these results of molecular docking provided sophisticated platform to reveal the structural changes in HSA upon binding of DAS. ...
... In recent years, only the interactions of phenothiazine dyes with bovine hemoglobin and serum albumins, bovine (BSA) and human (HSA), have been studied [17][18][19][20][21][22]. Only two of these works investigated the formation thermodynamics of these complexes. ...
Article
The complexation between protein and polyphenol affects their biological functions. A complete understanding of such interactions requires comprehensive thermodynamic and kinetic characterizations. Surface plasmon resonance (SPR)and fluorescence spectroscopy (FS)described similarly the thermodynamic of interaction between bovine lactoferrin (bLF)and epigallocatechin-3-gallate (EGCG). The formation of the bLF-EGCG complex is spontaneous (ΔG SPRo ≈ -29.00 kJ mol ⁻¹ , ΔG FSo ≈ -26.00 kJ mol ⁻¹ )and entropically driven (ΔH SPRo = 14.26, ΔH FSo = 10.20 kJ mol ⁻¹ and TΔS SPRo ≈ 43.00, TΔS FSo ≈ 36.00 kJ mol ⁻¹ ). The kinetic parameters obtained by SPR showed that the reaction occurs through an activated complex, whose energetic formation parameters from the association of free molecules (E act(a) = 49.5 kJ mol ⁻¹ , ΔH a‡ = 47.0 kJ mol ⁻¹ , and TΔS a‡ = −2.10 kJ mol ⁻¹ )were higher than those in the opposite direction (namely the dissociation of the stable complex, E act(d) = 17.4 kJ mol ⁻¹ , ΔH d‡ = 32.8 kJ mol ⁻¹ , and TΔS d‡ = −45.10 kJ mol ⁻¹ ), except for ΔG ‡ (ΔG a‡ = 49.1 kJ mol ⁻¹ and ΔG d‡ = 77.9 kJ mol ⁻¹ ). This study provides useful information for optimizing the use of bLF–EGCGcomplex as a bioactive compound in different systems, such as medical, food, cosmetic, and pharmaceutical formulations.
... In recent years, only the interactions of phenothiazine dyes with bovine hemoglobin and serum albumins, bovine (BSA) and human (HSA), have been studied [17][18][19][20][21][22]. Only two of these works investigated the formation thermodynamics of these complexes. ...
Article
Life manifestation is mainly based on biopolymer-ligand molecular recognition; therefore, the elucidation of energy and speed associated with protein-ligand binding is strategic in understanding and modulating biological systems. In this study, the interactions between methylene blue (MB) or azure A (AZA) dyes and bovine lactoferrin (BLF) were investigated by surface plasmon resonance, fluorescence spectroscopy, and isothermal titration microcalorimetry. Despite the molecular similarities between the dyes, the BLF-AZA binding thermodynamic parameters (ΔGAZAo = −30.50 and ΔHAZAo = 10.8 (kJ·mol−1)) were higher in magnitude than those of the BLF-MB systems (ΔGMBo = −27.3 and ΔHMBo = 5.72 (kJ·mol−1)). To increase the systems' entropy (TΔSAZAo = 41.3 and TΔSMBo = 33.0 (kJ·mol−1)), the hydrophobic interactions must outweigh the electrostatic repulsion, thereby promoting BLF-dye binding. The activation complex formation (Eac, aMB = 33, Eac, aAZA = 32, ∆Ha, MB‡ = 31, ∆Ha, AZA‡ = 30, ∆Ga, MB‡ = 51.84, ∆Ga, AZA‡ = 50.7, T∆Sa, MB‡ = −21, T∆Sa, AZA‡ = −21 (kJ·mol−1)), owing to free BLF and MB (or AZA) associations, was not affected by the dye chemical structure, while for the thermodynamically stable BLF-dye complex dissociation, the same energetic parameters (Eac, dMB = 16, Eac, dAZA = 6.4, ∆Hd, MB‡ = 14, ∆Hd, AZA‡ = 3.9, ∆Gd, MB‡ = 81.4, ∆Gd, AZA‡ = 74.93, T∆Sd, MB‡ = −68, T∆Sd, AZA‡ = −71.0 (kJ·mol−1)) were considerably affected by the number of methyl groups. Our results may be very useful to determine binding processes controlled by kinetic parameters, as well as to optimize the application of these photosensitive dyes in biological systems.
... One of the most unique features of the amino acid composition of albumins is the low content of tryptophan (Trp), only one or two residues per molecule in mammalian albumins, HSA has just one Trp-214, while BSA presents two Trp-134 and Trp-213. Both HSA Trp-214 and BSA Trp-213 are located in a similar hydrophobic pocket in subdomain IIA and can be monitored for obtaining information about binder molecules [30][31][32][33][34][35][36][37][38]. ...
... However, hydrophobic PSs can completely or partially dissociate from aggregates due to their interaction with plasma proteins [12]. Human serum albumin (HSA) is the most abundant protein in blood plasma, having multiple binding sites and being an efficient carrier for amphiphilic and hydrophilic photosensitizers [12][13][14][15][16][17]. ...
Article
Recently, a new type of spin labels based on photoexcited triplet molecules was proposed for nanometer scale distance measurements by pulsed dipolar electron paramagnetic resonance (PD EPR). However, such molecules are also actively used within biological complexes as photosensitizers for photodynamic therapy (PDT) of cancer. Up to date, the idea of using the photoexcited triplets simultaneously as PDT agents and as spin labels for PD EPR has never been employed. In this work, we demonstrate that PD EPR in conjunction with other methods provides valuable information on the structure and function of PDT candidate complexes, exemplified here with porphyrins bound to human serum albumin (HSA). Two distinct porphyrins with different properties were used: amphiphilic meso-tetrakis(4-hydroxyphenyl)porphyrin (mTHPP) and water soluble cationic meso-tetrakis(N-methyl-4-pyridyl)porphyrin (TMPyP4); HSA was singly nitroxide-labeled to provide a second tag for PD EPR measurements. We found that TMPyP4 locates in a cavity at the center of the four-helix bundle of HSA subdomain IB, close to the interface with solvent, thus being readily accessible to oxygen. As a result, the photolysis of the complex leads to photooxidation of HSA by generated singlet oxygen and causes structural perturbation of the protein. Contrary, in case of mTHPP porphyrin, the binding occurs at the proton-rich pocket of HSA subdomain IIIA, where the access of oxygen to a photosensitizer is hindered. Structural data of PD EPR were supported by other EPR techniques, laser flash photolysis and protein photocleavage studies. Therefore, pulsed EPR on complexes of proteins with photoexcited triplets is a promising approach for gaining structural and functional insights into such PDT agents.
... The complex formed between HSA and different chemical compounds has been widely investigated in many fields by means of physicochemical, biochemical and biophysical studies [2,91] Several HSA-ligand binding experiments revealed the affinity with binding constant (Kb) in the range of~10 2 to 10 6 M −1 [9,92,93]. In so far, many studies have described the interaction between HSA and environmental pollutants as well as HSA [94] and food colorants, in the fields of food chemistry [95], as well as vitamins [92,96], histological dyes [97] and organic dyes [98]. ...
Article
Full-text available
Albumin is an abundant protein in nature with several biological functions. In the human body, both in health and in illness, its transport function is highlighted by the binding to medicinal drugs and consequent distribution in the bloodstream to the site of action. This is particularly relevant for anticancer treatments, since this protein accumulates in the tumor microenvironment to supply the energetic demands of cancer cells. Different spectroscopy, thermodynamic and in silico studies techniques can be employed to verify how albumin binds to ligands by using either human serum albumin (HSA) or bovine serum albumin (BSA) due to their structural similarity. There is an increasing tendency to use albumin in analyses of absorption, distribution, metabolism and excretion (ADME) properties of anticancer molecules, which further demonstrated the promising character of this investigation for proposing new drugs.
Article
In the present investigation, a detailed spectroscopic analysis on the interaction mechanism of 1-Butyl-2,3-dimethylimidazolium tetrafluoroborate [Bdmim][BF4] with human serum albumin (HSA) and bovine serum albumin (BSA) has been carried out by multispectroscopic studies. The results obtained from the fluorescence titration experiments indicated the existence of a weak interaction between HSA/BSA and [Bdmim][BF4]. The binding parameters revealed that [Bdmim][BF4] binds to HSA by cooperative process, while in the case of BSA the binding of [Bdmim][BF4] is found to be independent to the binding sites having the similar affinity. The outcome of the CD and absorbance spectral experiments of HSA/BSA-[Bdmim][BF4] systems revealed the occurrence of very little modifications in the micro-environmental and secondary structural conformations of HSA/BSA. Autodocking and molecular dynamics simulation studies indicated that the positively charged imidazolium moieties of ionic liquid (IL) is located in the vicinity of subdomain IIA and subdomain IB in HSA and BSA, respectively. The enzyme-like activity of HSA/BSA is inhibited slightly upon complexation with [Bdmim][BF4].
Article
Full-text available
The spectral study of binding of oxazine (NB) and thiazine (AA, AB, AC, MB and TB O) dyes with cationic cetyltrimethylammonium bromide (CTAB) and non ionic tween 80, surfactant has been done in alkaline medium at lab temperature. All dyes show blue shifting in their λmax value in presence of both CTAB and tween 80. Since, the dye which shows less blue shifting in its λmax value with CTAB/tween 80 is considered more stable. Therefore, the dyes MB, TB O and AA (less blue shifting) are more stable than AB, AC and NB (more blue shifting) in presence of CTAB/tween 80. But, the magnitude of difference between shifted wavelength and its λmax value of all dye are higher with tween 80 in comparison to CTAB. It means the dyes may be more stable with CTAB than tween 80 and the stability of dye-CTAB/tween 80 is directly proportional to the electrical output in photogalvanic cells. The order of stability of these dyes on the basis of blue shifting with CTAB/tween 80 is: MB > AA > TB O > AB> AC> NB. Hence, this type of study may be helpful for choosing a stable dye-surfactant combination and to understand the reason for stability and higher electrical output of the photogalvanic cell for solar energy conversion and storage.
Article
The vibronic absorption spectrum of Azure B (AB) in an aqueous solution is calculated using the time-dependent density functional theory (TD-DFT). The results of calculations are analyzed using all hybrid functionals supported by Gaussian16, the 6-31++G(d,p) basis set, and the IEFPCM and SMD solvent models. The solvent model IEFPCM gave significantly underestimated values of λmax in comparison with the experiment. This is a manifestation of the TD-DFT "cyanine failure". However, the SMD model made it possible to obtain good agreement between the calculation results and experimental data. The best fit was achieved using the X3LYP functional. According to our calculations, the shoulder in the visible absorption spectrum of AB has a vibronic origin. However, the calculated shoulder is weaker than the experimental one. Explicit assignment of two water molecules, which form strong hydrogen bonds with a dye molecule, leads to a shift of the calculated absorption spectrum to longer wavelengths by approximately 17 nm but does not lead to an improvement in its shape. Comparative analysis of the calculated vibronic absorption spectra of Azure B with those obtained earlier for Azure A and methylene blue showed that the presence and intensity of the short-wavelength shoulder are determined by the location of the bands of higher vibronic transitions relative to the band of the 00→0⁰ main transitions. Photoexcitation leads to an increase in the dipole moment of the dye molecule. An insignificant photoinduced electron transfer was found in the central ring of the chromophore of the dye molecule.
Article
Full-text available
In the present work, we have studied the interaction of synthesized indole derivative (ID) with a model protein, human serum albumin (HSA). Various spectroscopic and molecular dynamic simulation methods were employed to characterize the binding between ID and HSA. ID showed strong binding affinity towards HSA (1.86x10⁶ M⁻¹) and quenched the fluorescence intensity of HSA by dynamic quenching mechanism. The existence of dynamic quenching mechanism in ID‐HSA interaction was further confirmed by lifetime measurement study. ID‐HSA interaction was favored by hydrophobic forces. The binding site for ID on HSA was examined by site probe competitive experiments and molecular docking studies. The results revealed that ID was bound to HSA primarily at site I of subdomain IIA. Absorption, 3D fluorescence and circular dichroism (CD) studies provided information on the conformational and microenvironmental changes in HSA upon binding to ID.
Article
This work aimed to develop a simple and low-cost method to obtain Human serum albumin (HSA) and its consequent application for in vitro drug interaction assays. The HSA was purified by classic principles of plasma precipitation and thermocoagulation, using a multiple-stage fractionation. The quality of the final product was assessed by electrophoresis, protein dosage by the Lowry method and the pharmacopeial thermal stability. At the end, an isotonic solution of HSA with a total protein concentration of 2.7 mg·mL-1 was obtained, which was visualized as a single band corresponding to the molecular weight of 66 kDa. After the thermal stability test, there was no indication of turbidity or color change of the solution. Finally, the HSA was useful for interactions assays with indole-thiazole and indole-thiazolidinone derivatives through UV-vis absorption and fluorescence spectroscopic studies, as well as by docking molecular analysis. Derivatives quenched the intrinsic fluorescence of HSA, disrupted the tryptophan residues microenvironment, and probably bind at Sudlow's site I. Therefore, the simplified methodology developed in this work proved to be effective in obtaining HSA that can be applied to research goals including drug interaction assays.
Article
Full-text available
The photochemistry of dye is playing a significant role for understanding the mechanism of electron transfer reactions in photoelectrochemical devices such as photogalvanic cells, DSSC, semiconductor photo-catalysis, photoconductors, etc. Oxazines (Brilliant Cresyl Blue and Nile Blue O) and thiazines (Azur A, Azur B, Azur C, Methylene Blue and Toluidine Blue O) dyes have been used widely as a photosensitizer with and without surfactants in the photogalvanic cells for solar power conversion and storage. Since, the stability and solubility of photosensitizers (dyes) are increased in the presence of surfactant and these properties lead to enhance the electrical output of the photogalvanic cells. Therefore, here we have studied the extent of interaction of different dyes with sodium dodecyl sulphate (SDS), find out the order of stability of dye–SDS on the basis of magnitudes of shifting in λmax of dye monomer and try to correlate order of dye–SDS interaction with already reported electrical output data of photogalvanic cells. Brilliant Cresyl Blue, Nile Blue O, Azur A and TB O have shown red shifting while Azur B, Azur C and MB have shown blue shifting in their λmax value with SDS, which indicates formation of dye–surfactant complex. But, the extent of formation of complex for different dyes with SDS was different due to change in their alkyl groups. Dyes with red shifting have greater stability in excited state as well as higher electrical output data of the cell than dye with blue shifting. On the basis of both red and blue shifting, order of stability of dyes–SDS complex was found as: Brilliant Cresyl Blue > Toluidine Blue O > Azur A > Nile Blue > Azur B > Methylene Blue > Azur C. The order of electrical output values of these dyes in photogalvanic cells have also been supported by literature data in the presence of SDS. Hence, the dye–surfactant complex which would have greater stability in excited state might be more useful for improvement of conversion efficiency and storage capacity of photogalvanic cells in the future.
Article
The interaction between a synthesized dye with proteins, bovine and human serum albumin (BSA, HSA respectively) under physiological conditions has been characterized in details, by means of steady state and time resolved fluorescence, UV-Vis absorption and circular dichroism (CD) techniques. An extensive time-resolved fluorescence spectroscopic characterization of the quenching process has been undertaken in conjugation with temperature-dependent fluorescence quenching studies to divulge the actual quenching mechanism. From the thermodynamic observations, it is clear that the binding process is a spontaneous molecular interaction, in which van der Waals and hydrogen bonding interactions play the major roles. The UV-Vis absorption and CD results confirm that the dye can induce conformational and micro environmental changes of both the proteins. In addition, the dye binding provoke the functionality of the native proteins in terms of esterase-like activity. The average binding distance (r) between proteins and dye has been calculated using FRET. Cytotoxicity and antiviral effect of the dye has been found using Vero cell and HSV-1F virus by performing MTT assay. The AutoDock-based docking simulation reveals the probable binding location of dye within the sub domain IIA of HSA and IB of BSA.
Article
Two novel red BODIPY-Triphenylamine with conjugated pyridines and one quaternary pyridium salt were synthesized. The long wavelength peaks of POPA-BODIPY and PPPA-BODIPY which were located at 643 nm, 631 nm were predominant and the quantum yield of them was 0.16, 0.05, respectively, so both of them showed red fluorescence in THF. PPSPA-BODIPY had a red emission peak at 624 nm and the quantum yield was 0.10. All the three compounds exhibited good aggregation-induced emission property. Furthermore, the fluorescence intensity of these dyes was strengthened continuously with increasing concentration of bovine serum albumin (BSA) which showed that these dyes could be used as BSA probes. Meanwhile, the detection limit of POPA-BODIPY, PPPA-BODIPY and PPSPA-BODIPY for BSA was calculated to be 1.44 × 10⁻⁷ M, 1.52 × 10⁻⁷ M, 5.37 × 10⁻⁸ M,respectively. POPA-BODIPY was applied to cell imaging and showed a good uptake by SK-BR-3 cells which suggested its promising application for biosensors.
Article
This study focuses on the understanding of the interaction of phenothiazinium dyes methylene blue (MB), new methylene blue (NMB), azure A (AZA) and azure B (AZB) with tRNA(Phe) with particular emphasis on deciphering the mode and energetics of the binding. Strong intercalative binding to tRNA(Phe) was observed for MB, NMB and AZB, bound by a partial intercalative mode. AZA has shown groove binding characteristics. From spectroscopic studies binding affinity values of the order of 10(5) M(-1) were deduced for these dyes; the trend varied as MB > NMB > AZB > AZA. The binding was characterized by an increase of thermal melting temperatures and perturbation in the circular dichroism spectrum of tRNA. All the dyes acquired optical activity upon binding to tRNA. The binding was predominantly entropy driven with a favorable enthalpy term that increased with temperature in all the cases. Dissection of the Gibbs energy to polyelectrolytic and non-polyelectrolytic terms revealed a major role of the non-electrostatic forces in the binding. The small but significant heat capacity changes and the observed enthalpy-entropy compensation phenomenon confirmed the involvement of multiple weak non-covalent forces driving the interaction. The mode of binding was confirmed from quenching, viscosity and cyclic voltammetric results. Using density functional theory, ground state optimized structures of the dyes were calculated to provide insight into theoretical docking studies to correlate the experimental approaches. The modeling results verified the binding location as well as the binding energy of complexation. The results may provide new insights into the structure-activity relationship useful in the design of effective RNA targeted therapeutic agents.
Article
Full-text available
The absorption and emission spectral properties of toluidine blue O (TBO) and Meldola's blue (MDB) in the presence of cyclodextrins (CDs) were studied. Formation of 1 : 1 and 1 : 2 (CD-Dye) inclusion complexes were observed with β-CD and γ-CD, respectively. An increase in the emission intensity was noticed in the presence of β-CD due to the formation of 1 : 1 (CD-Dye) inclusion complexes. However, a decrease in the emission intensity was observed in the presence of γ-CD due to the formation of 1 : 2 (CD-Dye) inclusion complexes. Dimerization of dye molecules in the presence of γ-CD lead to a decrease in the emission intensity. The formation constants for the 1 : 1 and 1 : 2 (CD-Dye) inclusion complexes were calculated.
Article
Full-text available
The molecule, 1,2-Bis(2-benzimidazolyl)-1,2-ethanediol (BBE) is known to act as a selective inhibitor of poliovirus, rhinovirus, Candida albicans, several bacterial species, and is easily synthesized by Phillips reaction. The interaction of BBE with BSA and the effects of its binding on the conformation and unfolding/refolding pathways of the protein were investigated using multispectroscopic techniques and molecular modeling. The binding studies indicate that BSA has one high affinity BBE binding site with association constant 6.0260.05610 4 M 21 at 298 K. By measuring binding at different temperatures, we determined the changes in enthalpy (DH = 215.1362.15 kJ mol 21), entropy (DS = 40.8767.25 J mol 21 K 21) and free energy (DG = 26.7861.02) of interaction, which indicate that the binding was spontaneous and both enthalpically and entropically driven. Based on molecular modeling and thermodynamic parameters, we proposed that the complex formation involved mainly hydrophilic interaction such as hydrogen bonding between hydroxyl groups of ethane-1,2-diol fragment with Tyr410 and benzimidazole sp 2 nitrogen atom with Ser488 and hydrophobic interaction between phenyl ring of one benzimidazole of the ligand and hydrophobic residues namely, Ile387, Cys391, Phe402, Val432 and Cys437. The sequential unfolding mechanism of BSA, site-specific marker displacement experiments and molecular modeling showed that the molecule preferably binds in subdomain IIIA. The BBE binding to BSA was found to cause both secondary and tertiary structural alterations in the protein as studied by intrinsic fluorescence, near-UV and far-UV circular dichroism results. The unfolding/ refolding study showed that BBE stabilized native to intermediate states (N=I) transition of the protein by ,2 kJ mol 21 without affecting the intermediate to unfolded states (I=U) transition and general mechanism of unfolding of BSA.
Article
Full-text available
RESUMEN Los estándares de seguridad de la sangre y sus productos han alcanzado un elevado nivel en la actualidad debido a la normatividad internacional vigente con la que funcionan los Bancos de Sangre. Ésta implica el control de donadores, los avances en la inmunohematología y la implementación de pruebas de tamizaje modernas. La radiación con rayos gamma y la filtración han minimizado la transmisión de agentes virales (hepatitis B y C, virus de la inmunodeficiencia humana, cito-megalovirus) y las reacciones no hemolíticas. Las pruebas de ácidos nucleicos (NAT, acid nucleic tests) han sido las principales en aumentar la seguridad de productos sanguíneos. Sin embargo, siguen persistiendo riesgos en la transfu-sión sanguínea debido a la falta de referencia de estos factores por parte de los donadores, donadores infectados que se presentan en el período de ventana diagnóstica, falta de pruebas de tamizaje para otros virus, bacterias y parásitos, lo cual se ha convertido en un problema a partir del aumento de la migración y el turismo "ecológico", así como de la apari-ción de nuevos patógenos como por ejemplo los virus del Nilo, de la neuroencefalitis espongiforme y el de la insuficiencia respiratoria aguda. Por lo anterior, se han desarrollado métodos para reducir o eliminar a los agentes patógenos transmi-sibles por la transfusión, que a su vez no alteren las funciones de las células transfundidas; sin embargo la mayoría de tales métodos se encuentra en proceso de validación. El presente artículo presenta una revisión sobre el estado actual de los métodos fotoinactivadores (fotoquímicos y fotosensibilizadores) de patógenos para productos sanguíneos. Palabras clave: Transfusión, banco de sangre, inactivación de patógenos. ABSTRACT Improvements of blood donor selection, blood processing, histocompatibility, immunohematology, serologic screening for infectious markers, and more recently, the introduction of nucleic acid testing (NAT-tests) for different virus genoms in-creased the safety of blood transfusion. To date, the transfusion-associated risk of HIV, HBV or HCV (human immunode-ficiency virus, hepatitis B or hepatitis C-virus) infection has become negligible in Western communities. However, blood transfusion is not without risk. Transfusion-transmitted infections based on emerging viruses, parasites or mainly bacterial contamination still exist and can lead to severe morbidity and death of the recipient. Techniques with the potential to direct-ly target the possible pathogen, are expected to diminish this residual risk of blood transfusion. Only recently, procedures for pathogen inactivation (PI) of cellular blood components targeting a broad variety of enveloped and non-enveloped virus-es, bacteria, parasites as well as leucocytes by targeting nucleic acids have been developed. The mechanism is based on activation of a so-called photosensitizer with defined light sources. The most intensively studied dyes with photodynam-ic properties are phenothiazines, porphyrins, cyanins, and riboflavin (vitamin B 2). Psoralens like S-59 (amotosalen-HCl) have photochemical properties. Other compounds interfere with nucleic acids without an external energy sources (ethyl-ene imine PEN 110) or upon pH-shift (S-303). Because of the possibility to use visible or UV light for treatment, studies on PI of platelet concentrates and plasma are more advanced than with red blood cell concentrates strongly absorbing light of this energy. Other drawbacks of PI procedures for red blood cell concentrates are cellular damage increasing during pro-longed storage in case of porphyrins (partly prevented by the addition of oxygen scavengers) and cyanins, the necessity of an integrated, time consuming washing step in addition to unresolved toxicologic questions in case of PEN 110, and an-tibody formation in case of S-303. PI techniques may become available for blood services and clinicians in the near future. Before implementation, open questions on safety profile of rest substances, cost effectiveness, and the real extent to which PI procedures affect cell function should be answered.
Article
Full-text available
Background and Objectives Photodynamic therapy (PDT) appears to be endowed with several favorable features for the treatment of infections originated by microbial pathogens, including a broad spectrum of action, the efficient inactivation of antibiotic-resistant strains, the low mutagenic potential, and the lack of selection of photoresistant microbial cells. Therefore, intensive studies are being pursued in order to define the scope and field of application of this approach.ResultsOptimal cytocidal activity against a large variety of bacterial, fungal, and protozoan pathogens has been found to be typical of photosensitizers that are positively charged at physiological pH values (e.g., for the presence of quaternarized amino groups or the association with polylysine moieties) and are characterized by a moderate hydrophobicity (n-octanol/water partition coefficient around 10). These photosensitizers in a micromolar concentration can induce a >4–5 log decrease in the microbial population after incubation times as short as 5–10 minutes and irradiation under mild experimental conditions, such as fluence-rates around 50 mW/cm2 and irradiation times shorter than 15 minutes.ConclusionsPDT appears to represent an efficacious alternative modality for the treatment of localized microbial infections through the in situ application of the photosensitizer followed by irradiation of the photosensitizer-loaded infected area. Proposed clinical fields of interest of antimicrobial PDT include the treatment of chronic ulcers, infected burns, acne vulgaris, and a variety of oral infections. Lasers Surg. Med. 38:468–481, 2006. © 2006 Wiley-Liss, Inc.
Article
Full-text available
The present work demonstrates a detailed characterization of the interaction of a bio-active drug molecule 3,5-dichlorosalicyclic acid (3,5DCSA) with a model transport protein Bovine Serum Albumin (BSA). The drug molecule is a potential candidate exhibiting Excited-State Intramolecular Proton Transfer (ESIPT) reaction and the modulation of ESIPT photophysics within the bio-environment of the protein has been exploited spectroscopically to monitor the drug-protein binding interaction. Apart from evaluating the binding constant (K (±10%) = 394 M(-1)) the probable location of the neutral drug molecule within the protein cavity (hydrophobic subdomain IIA) is explored by AutoDock-based blind docking simulation. The rotational relaxation dynamics of the drug within the protein has been interpreted on the lexicon of the two-step and wobbling-in-cone model. Circular dichroism (CD) spectroscopy delineates the effect of drug binding on the protein secondary structure in terms of decrease of α-helical content of BSA with increasing drug concentration. Also the esterase activity of the drug:protein conjugate system is found to be reduced in comparison to the native protein.
Article
Full-text available
α-Synuclein is a protein that is intrinsically disordered in vitro and prone to aggregation, particularly at high temperatures. In this work, we examined the ability of curcumin, a compound found in turmeric, to prevent aggregation of the protein. We found strong binding of curcumin to α-synuclein in the hydrophobic non-amyloid-β component region and complete inhibition of oligomers or fibrils. We also found that the reconfiguration rate within the unfolded protein was significantly increased at high temperatures. We conclude that α-synuclein is prone to aggregation because its reconfiguration rate is slow enough to expose hydrophobic residues on the same time scale that bimolecular association occurs. Curcumin rescues the protein from aggregation by increasing the reconfiguration rate into a faster regime.
Article
Full-text available
Bovine serum albumin (BSA) nonspecifically binds to well-dispersed multiwalled carbon nanotubes (MWCNTs), forming a stable bioconjugate. After accounting for the inner filter effect, we found the fluorescence intensity of BSA was quenched by MWCNTs in static mode, which was authenticated by lifetime measurements and Stern-Volmer calculations. The thermodynamic parameters DeltaG(o), DeltaS(o), and DeltaH(o) were -9.67 x 10(3) + 2.48 x 10(3) ln lambda J x mol(-1), 41.0 - 0.828 ln lambda J x mol(-1) x K(-1), and 7.30 x 10(3) + 2.23 x 10(3) ln lambda J x mol(-1) (lambda < 1 x 10(-4)), respectively, which shows a spontaneous and electrostatic interaction. Scatchard analysis and UV-visible results provide statistical data concerning changes in the microenvironment of amide moieties in response to different doses of MWCNTs, revealing different behavior of the BSA molecules. The absorption spectra also show that the tertiary structure of the protein was partially destroyed. The content of secondary structure elements of BSA was changed by the tubes. This work elucidates the interaction mechanism of BSA and MWCNTs from a spectroscopic angle.
Article
Sofosbuvir (SOF), an oral nucleotide inhibitor of the nonstructural protein 5B RNA of the hepatitis C virus (HCV), was approved for treating chronic HCV infection by the Food and Drug Administration in 2013. Understanding drug–protein interactions is a crucial factor in determining the pharmacokinetics and pharmacodynamics of a drug. In this study, the interaction between SOF and human serum albumin (HSA) was investigated using 1H nuclear magnetic resonance (NMR) spectroscopy, the molecular docking method, fluorescence, Fourier transform infrared (FT-IR) and circular dichroism (CD) spectroscopy. The analysis of saturation transfer difference (STD) and WaterLOGSY data indicated that SOF was bound to HSA, and the STD signals showed that the methyl and aromatic protons of the hydrophobic components of SOF have the most intimate contact with protein. The negative free energies (−7.17 kcal mol−1 and −6.18 kcal mol−1) obtained from molecular docking and fluorescence studies clearly suggested the spontaneity of the interaction of the SOF–HSA complex. STD, WaterLOGSY and fluorescence displacement experiments demonstrated that SOF was preferentially bound to site I of HSA, and this finding was supported by the docking results. In addition, synchronous and three-dimensional (3D) fluorescence, FT-IR, and CD spectrocopy provided complementary informations on the micro-environmental and conformational changes of HSA with the addition of SOF. The combination of 1H NMR and conventional methods provided useful information to elucidate further the binding mechanisms of antiviral drugs with HSA.
Article
DAPK3 belongs to family of DAPK (death-associated protein kinases) and is involved in the regulation of progression of the cell cycle, cell proliferation, apoptosis and autophagy. It is considered as a tumor suppressor kinase, suggesting the loss of its function in case of certain specific mutations. The T112M, D161N and P216S mutations in DAPK3 have been observed in cancer patients. These DAPK3 mutants have been associated with very low kinase activity, which results in the cellular progression towards cancer. However, a clear understanding of the structural and biophysical variations that occur in DAPK3 with these mutations, resulting in the decreased kinase activity has yet not been deciphered. We performed a molecular dynamic simulation study to investigate such structural variations. Our results revealed that mutations caused a significant structural variation in DAPK3, majorly concentrated in the flexible loops that form part of the ATP binding pocket. Interestingly, D161N and P216S mutations collapsed the ATP binding pocket through flexible loops invasion, hindering ATP binding which resulted in very low kinase activity. On the contrary, T112M mutant DAPK3 reduces ATP binding potential through outward distortion of flexible loops. In addition, the mutant lacked characteristic features of the active protein kinase including proper interaction between HR/FD and DFG motifs, well structured hydrophobic spine and Lys42-Glu64 salt bridge interaction. These observations could possibly explain the underlying mechanism associated with the loss of kinase activity with T112M, D161N and P216S mutation in DAPK3.
Article
The three-dimensional structure of human serum albumin has been determined crystallographically to a resolution of 2.8 Å. It comprises three homologous domains that assemble to form a heart-shaped molecule. Each domain is a product of two subdomains that possess common structural motifs. The principal regions of ligand binding to human serum albumin are located in hydrophobic cavities in subdomains IIA and IIIA, which exhibit similar chemistry. The structure explains numerous physical phenomena and should provide insight into future pharmacokinetic and genetically engineered therapeutic applications of serum albumin.
Article
This manuscript presents spectroscopic characterization of the interaction of two phenothiazinium dyes, azure A and azure B with double stranded (ds) ribonucleic acids, poly(A).poly(U), poly(C).poly(G) and poly(I).poly(C). Absorbance and fluorescence studies revealed that these dyes bind to the RNAs with binding affinities of the order 10(6)M(-1) to poly(A).poly(U), and 10(5)M(-1) to poly(C).poly(G) and poly(I).poly(C), respectively. Fluorescence quenching and viscosity data gave conclusive evidence for the intercalation of the dyes to these RNA duplexes. Circular dichroism results suggested that the conformation of the RNAs was perturbed on interaction and the dyes acquired strong induced optical activity on binding. Azure B bound to all the three RNAs stronger than azure A and the binding affinity varied as poly(A).poly(U)>poly(C).poly(G)>poly(I).poly(C) for both dyes. Copyright © 2015. Published by Elsevier B.V.
Article
In the present investigation, we have systematically studied the binding mechanism of model protein human serum albumin (HSA) with Gold/Silver alloy nanoparticles (Au/Ag NPs) using multi spectroscopic techniques. Absorption spectral studies of Au/Ag NPs in the presence of increasing concentrations of HSA resulted in slight red shift of Au/Ag NPs surface plasmon resonance band (SPR), suggesting changes in the refractive index around the nanoparticle surface owing to the adsorption of HSA. The results from high resolution transmission electron microscopy (HR-TEM), dynamic light scattering (DLS) together with zeta potential analysis substantiated the formation of dense layer of HSA on the surface of Au/Ag NPs. The formation of ground state complex between HSA and Au/Ag NPs was evident from the outcome of the steady state emission titration experiments of HSA-Au/Ag NPs system. The binding parameters computed from corrected emission quenching data revealed that HSA exhibited a significant binding affinity towards Au/Ag NPs. The identical fluorescence life time values of HSA and HSA-Au/Ag NPs from time resolved fluorescence spectroscopic analysis further authenticated the findings of steady state emission measurements. The formation of HSA corona on Au/Ag NPs surface was established on the basis of experimental quenching data and theoretical values. The occurrence of partial unfolding of HSA upon its interaction with Au/Ag NPs surface was established by using an extrinsic fluorophore 1-anilino-8-naphthalenesulfonic acid (ANS). Absorption, Fourier transform infra red (FT-IR), Raman, circular dichroism (CD) and excitation-emission matrix (3D) spectral studies were also carried out to explore Au/Ag NPs induced tertiary and secondary conformational changes of HSA. The influence of Au/Ag NPs on the esterase like activity of HSA was established by probing the hydrolysis of p-nitrophenyl acetate.
Article
Proanthocyanidins are a mixture of monomers, oligomers, and polymers of flavan-3-ols that are widely distributed in the plant kingdom. One of the most widely studied proanthocyanidins is procyanidin B3. In this study, the interaction between procyanidin B3 and bovine serum albumin (BSA) was investigated using isothermal titration calorimetry (ITC), in combination with fluorescence spectroscopy, UV-vis absorption spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, circular dichroism (CD) spectroscopy and molecular docking. Thermodynamic investigations reveal that the electrostatic interaction and hydrophobic interaction are the major binding forces in the binding of procyanidin B3 to BSA. The binding of procyanidin B3 to BSA is synergistically driven by enthalpy and entropy. Fluorescence experiments suggest that procyanidin B3 can quench the fluorescence of BSA through a static quenching mechanism. The obtained binding constants and the equilibrium fraction of unbound procyanidin B3 show that procyanidin B3 can be stored and transported from the circulatory system to reach its target organ. Binding site I is found to be the primary binding site for procyanidin B3, which is consistent with the result of molecular docking studies. Additionally, as shown by the UV-vis absorption, synchronous fluorescence spectroscopy, FT-IR and CD, procyanidin B3 may induce conformational and microenvironmental changes of BSA.
Article
This study explores the binding interaction of thionine (TH) with bovine serum albumin (BSA) under physiological conditions (pH 7.40) using absorption, emission, synchronous emission, circular dichroism (CD) and three-dimensional (3D) emission spectral studies. The results of emission titration experiments revealed that TH strongly quenches the intrinsic emission of BSA via a static quenching mechanism. The apparent binding constant (K) and number of binding sites (n) were calculated as 2.09 × 105 dm3/mol and n~1, respectively. The negative free energy change value for the BSA–TH system suggested that the binding interaction was spontaneous and energetically favourable. The results from absorption, synchronous emission, CD and 3D emission spectral studies demonstrated that TH induces changes in the microenvironment and secondary structure in BSA. Site marker competitive binding experiments revealed that the binding site of TH was located in subdomain IIA (Sudlow site I) of BSA. The molecular docking study further substantiates Sudlow site I as the preferable binding site of TH in BSA. Copyright © 2014 John Wiley & Sons, Ltd.
Article
In the present study the interaction of chemotherapeutic agent, Azure A (AZA) with Human serum albumin (HSA) and Bovine serum albumin (BSA) was investigated by multi spectroscopic and molecular docking methods. The influence of inner filter effect (IFE) on the emission quenching of HSA/BSA at low concentration of AZA (absorption value < 0.1) suggested the need to employ IFE correction factor even for low concentration regime. The emission titration experiments of HSA/BSA with AZA revealed the formation of AZA-HSA/BSA complexes. The binding parameters calculated from corrected emission intensities showed that AZA binds to HSA/BSA with moderately strong binding affinities. The negative free energy obtained for the binding of AZA with HSA/BSA indicated that the complexation process is spontaneous. The results from site maker competitive experiments with specific site markers revealed that the probable binding location of AZA is located near site I of HSA/BSA. AutoDock based molecular docking approach was utilized to characterize the binding models of AZA-HSA/BSA complexes. The free energy calculations for the most stable conformer from molecular docking studies were utilized to examine the energy contributions and the role of various amino acid residues of HSA/BSA in AZA binding. The results of site-competitive replacement experiments with specific site markers and molecular docking simulation studies unambiguously helped us to conclude that AZA binds to site I of HSA/BSA. Constant wavelength synchronous emission, excitation–emission matrix (three-dimensional) emission, absorption and circular dichroism spectroscopic techniques have been exploited to unravel AZA induced tertiary and secondary conformational changes of HSA/BSA.
Article
The protein–gold nanoparticle bioconjugates are playing an important role in the studies of biological systems. The nature of the interaction and the magnitude of the binding affinity together with the conformational changes in the protein upon binding are the most addressed topics in relation to the uses of the bioconjugates in different organisms. In this work, we study the human serum albumin (HSA) protein–gold nanoparticle (AuNP) interactions focusing on the nature of the gold nanoparticle surface modification. We have found that the interactions of the HSA with the AuNPs are mainly electrostatic and that the concentration of protein necessary to stabilize the conjugates decreases when the overall negative charge on the nanoparticle surface increases. The changes in the localized surface plasmon resonance (LSPR) signals of the gold nanoparticles (13 nm diameter) are used to determine the number of protein molecules necessary to stabilize the conjugates in a high ionic strength medium. Fluorescence spectroscopy (stationary and time-resolved) is used to characterize the different bioconjugates and determine the binding constants under different experimental conditions. Moreover, the use of an extrinsic fluorescence probe (1-anilino-8-naphthalenesulfonic acid, ANS) gives us some information about the existence of partial unfolding of the protein upon binding to the nanoparticle.
Article
The mechanism of interaction between imipenem and HSA was investigated by various techniques like fluorescence, UV-Vis absorbance, FRET, circular dichroism, urea denaturation, enzyme kinetics, ITC and molecular docking. We found that imipenem binds to HSA at a high affinity site located in sub-domain IIIA (Sudlow's site I) and a low affinity site located in sub-domain IIA-IIB. Electrostatic interactions played a vital role along with hydrogen bonding and hydrophobic interactions in stabilizing imipenem-HSA complex at sub-domain IIIA, while only electrostatic and hydrophobic interactions were present at sub-domain IIA-IIB. The binding and thermodynamic parameters obtained by ITC showed that the binding of imipenem to HSA was a spontaneous process ( = -32.31 kJ mol-1 for high affinity site and = -23.02 kJ mol-1 for low affinity site) with binding constants in the range of 104-105 M-1. Spectroscopic investigation revealed only one binding site of imipenem on HSA (Ka~104 M-1). FRET analysis showed that the binding distance between imipenem and HSA (Trp-214) was optimal (r = 4.32 nm) for quenching to occur. Decrease in esterase-like activity of HSA in the presence of imipenem showed that Arg-410 and Tyr-411 of sub-domain IIIA (Sudlow's site II) were directly involved in the binding process. CD spectral analysis showed altered conformation of HSA upon imipenem binding. Moreover, the binding of imipenem to sub-domain IIIA (Sudlow's site II) of HSA also affected its folding pathway as clear from urea-induced denaturation studies.
Article
In the present investigation the interaction of a biologically active photodynamic therapeutic agent Toluidine blue O (TBO) with Serum albumins viz Human serum albumin (HSA) and Bovine serum albumin (BSA) was studied using absorption, emission, circular dichroism spectroscopy and molecular docking experiments. The emission titration experiments between HSA/BSA and TBO revealed the existence of strong interactions between TBO and the proteins. The site competitive experiment of HSA and BSA showed that the primary binding site of TBO is located in site I of HSA/BSA involving hydrophobic, hydrogen bonding and electrostatic interaction. To ascertain the results of site competitive experiments, molecular docking was utilized to characterize the binding models of TBO–HSA/BSA complexes. From the molecular docking studies, free energy calculations were undertaken to examine the energy contributions and the role of various amino acid residues of HSA/BSA in TBO binding. The existence of Forster Resonance Energy Transfer (FRET) between the ligand and the protein was utilized to calculate the donor–acceptor distance of TBO and protein. The TBO induced conformational changes of HSA/BSA was established using synchronous emission, three dimensional emission and circular dichroism studies.
Article
Binding of ascorbic acid (water-soluble antioxidant) and α-tocopherol (lipid-soluble antioxidant) to bovine serum albumin (BSA) has been studied using isothermal titration calorimetry (ITC), in combination with fluorescence spectroscopy, UV-vis absorption spectroscopy and Fourier transform infrared (FT-IR) spectroscopy. Thermodynamic investigations reveal that ascorbic acid/α-tocopherol binding to BSA is driven by favorable enthalpy and unfavorable entropy, and the major driving forces are hydrogen bonding and van der Waals forces. For ascorbic acid, the interaction is characterized by a high number of binding sites, which suggests that binding occurs by a surface adsorption mechanism that leads to coating of the protein surface. For α-tocopherol, one molecule of α-tocopherol combines with one molecule of BSA and no more α-tocopherol binding to BSA occurs at concentration ranges used in this study. Fluorescence experiments suggest that ascorbic acid has predominantly a "sphere of action" quenching mechanism, whereas, for α-tocopherol, the quenching mechanism is "static quenching" and due to the formation of a ground state complex. Additionally, as shown by the UV-vis absorption, synchronous fluorescence spectroscopy, and FT-IR, ascorbic acid and α-tocopherol may induce conformational and microenvironmental changes of BSA.
Article
Investigating the adsorption process of proteins on nanoparticle surfaces is essential to understand how to control the biological interactions of functionalized nanoparticles. In this work, a library of spherical and rod-shaped gold nanoparticles (GNPs) was used to evaluate the process of protein adsorption to their surfaces. The binding of a model protein (bovine serum albumin, BSA) to GNPs as a function of particle shape, size, and surface charge was investigated. Two independent comparative analytical methods were used to evaluate the adsorption process: steady-state fluorescence quenching titration and affinity capillary electrophoresis (ACE). Although under favorable electrostatic conditions, kinetic analysis showed a faster adsorption of BSA to the surface of cationic GNPs, equilibrium binding constant determinations indicated that BSA has a comparable binding affinity to all the GNPs tested, regardless of surface charge. BSA was even found to adsorb strongly to GNPs with a pegylated/neutral surface. However, these fluorescence titrations suffer from significant interference from the strong light absorption of the GNPs. The BSA-GNP equilibrium binding constants, as determined by the ACE method, were 105 times lower than values determined using spectroscopic titrations. While both analytical methods could be suitable to determine the binding constants for protein adsorption to NP surfaces, both methods have limitations that complicate the determination of protein-GNP binding constants. The optical properties of GNPs interfere with Ka determinations by static fluorescence quenching analysis. ACE, in contrast, suffers from material compatibility issues, as positively-charged GNPs adhere to the walls of the capillary during analysis. Researchers seeking to determine equilibrium binding constants for protein-GNP interactions should therefore utilize as many orthogonal techniques as possible to study a protein-GNP system.
Article
Vitamin B12 deficiency is a problem for many elderly people around the world caused by food-cobalamin malabsorption syndrome due to gastrointestinal problems. In this study, the molecular interactions of VB12 with two major soy protein fractions, β-conglycinin (7S) and glycinin (11S) were studied using fluorescence and far-UV circular dichroism spectroscopic techniques. The results show that the fluorescence of 7S and 11S was quenched by VB12 through binding-related quenching after correcting for the inner-filter effect. Both 7S and 11S had a good affinity to VB12 as indicated by their high binding constant 1.252 (± 0.085) × 104 M-1 for 7S and 0.952 (± 0.04) × 104 M-1 for 11S at 292 K, respectively. Such binding induced a more organized protein confirmation with increased β-sheet and β-turn structure components and a more folded tertiary structure. It is deduced that VB12 was bound in the interior of protein three-dimensional network mainly via hydrophobic interactions to form 7S-VB12 and 11S-VB12 complexes. The results suggest that soy protein has potential to be used as carrier of VB12.
Chapter
This chapter focuses on amino acid sequence. It reviews the primary structures of bovine and human serum albumin. Elucidation of the amino acid sequence of serum albumin has led to much greater insights into the structure, function, and evolution of this protein than was anticipated. The application of the three-dimensional models of albumin are supported by a variety of evidence, namely, helix content, properties of fragments, reactive groups, binding properties, protein dimensions, and similarity to the G-H region of globin structure. The models are specific enough that details can be tested and refined by further experiments, such as affinity labeling, crosslinking, and further preparation and analysis of properties of fragments.
Article
Guidelines for submitting commentsPolicy: Comments that contribute to the discussion of the article will be posted within approximately three business days. We do not accept anonymous comments. Please include your email address; the address will not be displayed in the posted comment. Cell Press Editors will screen the comments to ensure that they are relevant and appropriate but comments will not be edited. The ultimate decision on publication of an online comment is at the Editors' discretion. Formatting: Please include a title for the comment and your affiliation. Note that symbols (e.g. Greek letters) may not transmit properly in this form due to potential software compatibility issues. Please spell out the words in place of the symbols (e.g. replace “α” with “alpha”). Comments should be no more than 8,000 characters (including spaces ) in length. References may be included when necessary but should be kept to a minimum. Be careful if copying and pasting from a Word document. Smart quotes can cause problems in the form. If you experience difficulties, please convert to a plain text file and then copy and paste into the form.
Article
Thionine exhibits a much higher solubility in water than could be expected based on previously published data. Concentrated solutions of thionine up to 0.1 M have been prepared and are red whereas dilute solutions are blue. The absorption spectra of the thionine solutions from 1.0 × 10-5 to 3.5 × 10-2 M have been obtained. The absorption maxima are shifted gradually and continuously to shorter wavelengths as the concentration of thionine is increased. A previously employed monomer-dimer model is not sufficient to explain the experimental results and it is necessary to invoke the formation of higher aggregates (H aggregate) to explain the experimental results. An approach based on the McRae-Kasha exciton model was employed to obtain the sizes of the aggregates in the solutions. Aggregates ranging in size from dimers to 9-mers have been identified. The apparent aggregation number (na) increases with increasing thionine concentration (C, mol/L) above 0.002 M as na = 1.0 + 225C. The stepwise formation constants of the lower aggregates have been calculated as K2 = 4 × 103 M-1 and K3 ≃ K4 ≃ 1.7 × 103 M-1. Increasing temperature causes the deaggregation of the larger aggregates into monomers and smaller aggregates. At a thionine concentration of 0.025 M, the apparent aggregation number in the temperature range 7-40°C is given by na = 11.6-0.16T.
Article
The interaction of human serum albumin with six long-chain fatty acid anions has been studied by the technique of partition analysis, in which varying quantities of each fatty acid were equilibrated between two phases. The phases employed were n-heptane and an aqueous solution of serum albumin in phosphate buffer, pH 7.45, ionic strength 0.16, at 23°. The concentration of unbound fatty acid in each aqueous phase was determined from the concentration in heptane, using the results of identical distribution experiments performed in the absence of albumin. The fatty acids studied included lauric, myristic, palmitic, stearic, oleic and linoleic acids. The human serum albumin was a special preparation with a fatty acid content of 0.1 mole/mole or less. The data obtained have been analyzed in terms of three classes of binding sites and the apparent association constants determined. The first class consists of 2 sites, the second of 5, and the third of a larger number, arbitrarily taken to be 20. The values of k1′ for the different fatty acids are: laurate 1.6 × 106; myristate 4.0 × 106; palmitate 6.0 × 107; stearate 8.0 × 107; oleate 1.1 × 108; linoleate 1.3 × 107. The relationship between the apparent and the intrinsic association constants is discussed in detail. The possible structural specificity of the different classes of binding sites is also discussed, as are some of the implications for metabolic studies.
Article
The consequences of multiple equilibria on the calculation of formation constants and other molecular constants for weak complexes are considered from a theoretical standpoint. A microscopic model is used to illustrate the averaging process involved in the assignment of formation constants for higher order complexes, and the slopes, and intercepts of various plotting forms usually applied to the evaluation of weak complexes are derived for a PX, PX2 system. It is shown that, except in the special case where all the microscopic constants of the system are closely similar, curved lines are to be expected on all of the usual plots. At very low saturation fractions, however, curvature is small, and in the limit as the concentration of the excess component approaches zero, the plotted curves are indistinguishable from straight lines. The slopes and intercepts of such lines have widely differing experimental meaning and cannot be interpreted simply as "K" or "K∈" except in cases of very little practical interest. From the shape of the theoretical curves, it appears that it is necessary to measure of the order of 75% of the entire saturation curve before a given stoichiometric model can be assigned to the fitted data.
Article
Some aspects of the measurement of formation constants and other constants for weak molecular complexes are considered from the standpoint of basic binding theory. The theoretical minimum errors in the formation constant k and the extinction coefficient ∈ are discussed as a function of the saturation fraction of the most dilute component, and it is shown in agreement with previous work that the most accurate values of either parameter are obtained when the saturation fraction lies between 0.2 and 0.8. Outside this region, the determined values become extremely uncertain, and in addition, insufficient data are available to adequately fit a given stoichiometric model. It is suggested that the criteria that the highest concentration of the excess component be not less than 0.1/k are insufficient to assign a given stoichiometric model to the data, but apply instead to the smallest range of saturation fraction required to show that a line of finite limiting slope and/or intercept exists. To develop criteria by which a given stoichiometric model can be considered in 1:1 correspondence with the phenomenological equation fitting the data, the principles of information theory are applied to the binding process. It is concluded that of the order of 75% of the saturation curve is required to show the correspondence between the equation of the model and the equation fitting the data. Also discussed are various plotting forms and the errors arising from various rearrangements of the basic binding equation.
Article
Spectrophotometry was employed to study binding of methylene blue (MB) dye to sodium montmorillonite (NaM) in dilute aqueous suspension. The adsorption isotherm may be expressed u = k1 + k2c1/n, the first term (k1 = 80 meq. MB/100 g. NaM) arising from ion exchange, the second from physical adsorption. Large spectral changes (metachromasy) were found to accompany changes in the coverage, u, at values well below k1. These are similar to the spectral shift accompanying dimerization of MB in aqueous solution and are attributed to dye-dye interactions. The spectral properties showed large aggregates were formed at moderate coverage. The spectra of free MB monomer and dimer were determined quantitatively. The peak molar absorbancy index of the monomer was found to be 9.5 × 104 at 6640 Å. The spectral variations up to concentrations of 2 × 10-4 M were interpreted quantitatively in terms of a monomer-dimer equilibrium, with a dimer dissociation constant of 1.7 × 10-4 at 25°. The dimer spectrum was found to contain a long wave length peak which can be explained by a sandwich structure having the monomer transition moments at a mean angle of about 13° to each other. Trimethylthionine, formed by base-catalyzed demethylation, was found chromatographically to be a common impurity in methylene blue; a spectral criterion for this impurity was introduced and an extraction procedure was developed for removing it.
Article
In recent years fluorescence quenching has become a popular tool to investigate various aspects of ligand binding. Unfortunately, various pitfalls are often overlooked in a large number of papers, published in many different journals. In this criticism we discuss a number of possible mistakes and show how they may affect the data and their analysis. Moreover, we point to problems in the understanding of the fundamentals of fluorescence quenching, and show direct contradictions within many of these papers. This review hopefully contributes to a re-appraisal of the published literature and to a more appropriate use of fluorescence quenching to study ligand binding.
Article
The interactions of methylene blue, azure B, and thionine with calf thymus DNA, [poly (dG-dC)]2, [poly(dA-dT)]2, and the constituent mononucleotides 2′-deoxyguanosine-5′-monophosphate(dGMP), 2′-deoxyadenosine-5′-monophosphate(dAMP), 2′-deoxycytidine-5′-monophosphate(dCMP), and thymidine-5′-monophosphate(dTMP) have been studied by steady-state absorption spectroscopy and with equilibrium dialysis. Scatchard plots for binding of the dyes to the nucleic acid polymers were convex downward at low binding ratios, characteristic of intercalation, and binding constants for this mode were calculated under conditions of varying ionic strength. For each of the dyes, binding constants with [poly(dG-dC)]2 and [poly(dA-dT)]2 were of the same order of magnitude, so that previously reported (G-C) preferentially is not very marked. At high binding ratios, the Scatchard plots did not return to the abscissa but curved upward, indicative of a weaker cooperative binding mode, occurring under conditions where the dye is in excess, which is suggested to be external stacking of the dye molecules promoted by the polyanion. The dependence of the absorption spectra on added salt demonstrated a shift in the strong binding mode for the three dyes with [poly(dA-dT)]2 with increasing ionic strength, while with [poly(dG-dC)]2 this does not occur. The dyes were found to bind to purine but not pyrimidine mononucleotides with dGMP and dAMP, 1:1 complexes were formed initially and also 1:2 dye/nucleotide complexes with increasing nucleotide concentrations. Under low salt conditions, binding to dAMP was slightly stronger than to dGMP for the three dyes studied, while at high ionic strength, when the binding constants are significantly lower, all binding constants become very similar. Binding to mononucleotides is suggested to be primarily stabilised by π-π stacking interactions between the planar dyes and the nucleobases: for thionine and azure B there also appears to be H-bonds between the exocyclic amines and the sugar–phosphates conferring extra stability. Neither increasing the number of phosphate groups on the nucleotides nor changing from deoxyribose to ribose sugars had any significant effect on the binding constants. © 1995 John Wiley & Sons, Inc.
Article
VMD is a molecular graphics program designed for the display and analysis of molecular assemblies, in particular biopolymers such as proteins and nucleic acids. VMD can simultaneously display any number of structures using a wide variety of rendering styles and coloring methods. Molecules are displayed as one or more "representations," in which each representation embodies a particular rendering method and coloring scheme for a selected subset of atoms. The atoms displayed in each representation are chosen using an extensive atom selection syntax, which includes Boolean operators and regular expressions. VMD provides a complete graphical user interface for program control, as well as a text interface using the Tcl embeddable parser to allow for complex scripts with variable substitution, control loops, and function calls. Full session logging is supported, which produces a VMD command script for later playback. High-resolution raster images of displayed molecules may be produced by generating input scripts for use by a number of photorealistic image-rendering applications. VMD has also been expressly designed with the ability to animate molecular dynamics (MD) simulation trajectories, imported either from files or from a direct connection to a running MD simulation. VMD is the visualization component of MDScope, a set of tools for interactive problem solving in structural biology, which also includes the parallel MD program NAMD, and the MDCOMM software used to connect the visualization and simulation programs. VMD is written in C++, using an object-oriented design; the program, including source code and extensive documentation, is freely available via anonymous ftp and through the World Wide Web.
Article
Non-covalent interactions between polymethine dyes of various types (cationic and anionic thiacarbocyanines as well as anionic oxonols and tetracyanopolymethines) and human serum albumin (HSA) were studied by means of absorption, fluorescence and circular dichroism (CD) spectroscopies. Complexation with the protein leads to a red shift of the dye absorption spectra and, in most cases, to a growth of the fluorescence quantum yield (Φf; for oxonols this growth is very small). The binding constants (K) obtained from changing the absorption spectra and Φf vary from 104 to (5–6)×107 M−1. K for the anionic dyes is much higher than for the cationic dyes (the highest K was found for oxonols). Interaction of meso-substituted anionic thiacarbocyanines with HSA results in cis→trans isomerization and, as a consequence, an appearance and a steep rise of dye fluorescence. Binding to HSA gives rise to dye CD signals and in many cases is accompanied by aggregation of the dyes. These aggregates often exhibit biphasic CD spectra. The aggregates formed by the dyes alone are decomposed in the presence of HSA.
Article
Based on the measurements of molecular absorption and resonance light scattering (RLS), the aggregation of Azur B (AB) was in a medium of pH ranging from 1.98 to 2.56 and ionic strength <0.12 M. The presence of double stranded DNA prompts the aggregation, resulting in enhanced RLS signals. Linear relationships were achieved between the enhanced RLS intensity at 359.7 nm and DNA concentration in the range of 0–4.5 μg ml−1 for both calf thymus DNA (ctDNA) and fish sperm DNA (fsDNA) if 3.0×10−5 M AB was employed. The 3σ limits of detection were 9.3 and 8.9 ng ml−1 for ctDNA and fsDNA, respectively. Five synthetic samples were analysed satisfactorily.
Article
The present work describes the interaction of a promising cancer cell photosensitizer, harmane (HM), with a model transport protein, Bovine Serum Albumin (BSA). The studied molecule of interest (HM) belongs to the family of naturally occurring fluorescent drug-binding alkaloids, the β-carbolines. A combined use of steady-state and time-resolved fluorescence techniques is applied to follow and characterize the binding interaction. The polarity-dependent prototropic activity of HM is found to be responsible for the commendable sensitivity of the probe to the protein environments and is distinctly reflected on the emission profile. Steady-state fluorescence anisotropy study reveals the impartation of a considerable degree of motional restriction on the drug molecule as a result of binding to the protein. Contrary to the single-exponential nature of fluorescence anisotropy decay of HM in aqueous buffer, they are found to be biexponential in the protein environment. The rotational relaxation dynamics of HM within the protein has been interpreted on the lexicon of the Two-Step and Wobbling-in-Cone model. The probable binding location for the cationic drug is found to be the hydrophilic binding zone of BSA, i.e., domain I (characterized by a net negative charge). The AutoDock-based blind docking simulation has been explored for evaluating an unbiased result of the probable interaction site of HM in the protein. To unfold the effect of binding of the drug on the secondary structural content of the protein, circular dichroism (CD) spectroscopy has been exploited to see that binding of the drug accompanies some decrease in α-helical content of BSA, and the effect gradually saturates toward a higher drug/protein molar ratio.
Article
Protein conformational changes may be associated with particular properties such as function, transportation, assembly, tendency to aggregate, and potential cytotoxicity. Protein misfolding, in particular, has been intimately related to protein-mediated diseases. In this study, the conformational structure changes of hemoglobin (Hb) induced by the assembly on gold nanoparticles (AuNPs) surface were studied in detail by a combination of electrochemical method and various spectroscopic techniques including UV-vis absorption, fluorescence, circular dichroism (CD), and Fourier transform infrared (FTIR) spectroscopy. The results indicated that Hb in the Hb-AuNPs bioconjugate system that was prepared by the assembly of Hb on the surface of AuNPs underwent substantial conformational changes both at secondary and tertiary structure level. The assembly of Hb on the boundary surface of AuNPs could result a disturbance of the structure of Hb and induce the exposure of the heme group and tryptophan (Trp) residues to the solvent, leading to the enhancement in the electron transfer rate of the protein. The calculation from quantitative second-derivative infrared and CD spectra of the Hb-AuNPs bioconjugate system showed that AuNPs could induce the conversion of α-helix to β-sheet structures and unfolding of the protein. Moreover, the effects of the concentration and the size of AuNPs on the conformational structure changes of Hb in the bioconjugate system were also demonstrated. The results obtained here not only provide the detailed conformational behavior of Hb molecules on nanoparticles, but also create a framework for analyzing the biosafety of nanoparticles in terms of the biological behavior of biomacromolecules.
Article
New parameter sets of the GROMOS biomolecular force field, 54A7 and 54B7, are introduced. These parameter sets summarise some previously published force field modifications: The 53A6 helical propensities are corrected through new φ/ψ torsional angle terms and a modification of the N-H, C=O repulsion, a new atom type for a charged -CH(3) in the choline moiety is added, the Na(+) and Cl(-) ions are modified to reproduce the free energy of hydration, and additional improper torsional angle types for free energy calculations involving a chirality change are introduced. The new helical propensity modification is tested using the benchmark proteins hen egg-white lysozyme, fox1 RNA binding domain, chorismate mutase and the GCN4-p1 peptide. The stability of the proteins is improved in comparison with the 53A6 force field, and good agreement with a range of primary experimental data is obtained.
Article
Hydrazone derivatives possess potential antitumor activities based on modulation of the iron metabolism in cancer cell. A novel hydrazone, N'-(2,4-dimethoxybenzylidene)-2-hydroxybenzohydrazide (DBH), has been synthesized and characterized, which is an analogue of 311 possessing potent anticancer activity. The interactions between DBH and bovine serum albumin (BSA) have been investigated systematically by fluorescence, molecular docking, circular dichroism (CD), UV-vis absorption, and electrochemical impedance spectroscopy (EIS) methods under physiological conditions. The fluorescence quenching observed is attributed to the formation of a complex between BSA and DBH, and the reverse temperature effect of the fluorescence quenching has been found and discussed. The primary binding pattern is determined by hydrophobic interaction occurring in Sudlow's site I of BSA. DBH could slightly change the secondary structure and induce unfolding of the polypeptides of protein. An average binding distance of ~4.0 nm has been determined on the basis of the Förster resonance energy theory (FRET). The effects of iron on the system of DBH-BSA have also been investigated. It is found that iron could compete against BSA to bind DBH. All of these results are supported by a docking study using a BSA crystal model. It is shown that DBH can efficiently bind with BSA and be transported to the focuses needed. Subsequent antitumor test and detailed anticancer mechanism are undergoing in our lab.
Article
Pharmaceutical interactions with human serum albumin (HSA) are of great interest, because HSA is a pharmacokinetic determinant and a good model for exploring the protein-ligand interactions. Due to their hydrophobic nature, naturally occurring flavones, which possess various pharmacological activities, bind to HSA in human plasma. Here, we have identified the binding modes of two representative flavones--baicalin (BLI) and its aglycon, baicalein (BLE)--to HSA using a combination of experimental and computational approaches. The association properties were measured by applying spectroscopic methods, and a higher affinity was found for BLE. As evidenced by displacement and chemical unfolding assays, both ligands bind at Sudlow site I. Furthermore, molecular docking was utilized to characterize the models of HSA-flavone complexes, and molecular dynamics (MD) simulations as well as free energy calculations were undertaken to examine the energy contributions and the roles of various amino acid residues of HSA in flavones binding; the mechanism whereby glycosylation affects the association was also discussed. The present work provides reasonable binding models for both flavones to HSA.
Article
Beta-sitosterol is a naturally occurring phytosterol that is widely used to cure atherosclerosis, diabetes, cancer, and inflammation and is also an antioxidant. Here, we studied the interaction of beta-sitosterol, isolated from the aerial roots of Ficus bengalensis, with human serum albumin (HSA) at physiological pH 7.2 by using fluorescence, circular dichroism (CD), molecular docking, and molecular dynamics simulation methods. The experimental results show that the intrinsic fluorescence of HSA is quenched by addition of beta-sitosterol through a static quenching mechanism. The binding constant of the compound to HSA, calculated from fluorescence data, was found to be K(beta-sitosterol) = 4.6 +/- 0.01 x 10(3) M(-1), which corresponds to -5.0 kcal M(-1) of free energy. Upon binding of beta-sitosterol to HSA, the protein secondary structure was partially unfolded. Specifically, the molecular dynamics study makes an important contribution to understanding the effect of the binding of beta-sitosterol on conformational changes of HSA and the stability of a protein-drug complex system in aqueous solution. Molecular docking studies revealed that the beta-sitosterol can bind in the large hydrophobic cavity of subdomain IIA, mainly by the hydrophobic interaction but also by hydrogen bond interactions between the hydroxyl (OH) group of carbon-3 of beta-sitosterol to Arg(257), Ser(287), and Ala(261) of HSA, with hydrogen bond distances of 1.9, 2.4, and 2.2 A, respectively.
Article
N-trans-p-coumaroyltyramine (CT) isolated from Physalis minima is a phenolic substance exhibiting many pharmacological activities like potent inhibition of acetyl cholinesterase, cell proliferation, platelet aggregation, and also antioxidant activity. Here, we have studied the binding of CT with HSA at physiological pH 7.2 by using fluorescence, circular dichroism spectroscopy, mass spectrometry, and molecular docking methods. From the fluorescence emission studies, the number of binding sites and binding constant were calculated to be 2 and (4.5 +/- 0.01) x 10(5) M(-1), respectively. The free energy change was calculated as -7.6 kcal M(-1) at 25 degrees C, which indicates the hydrophobic interactions of CT with HSA and is in well agreement with the computational calculations and molecular docking studies. The changes in the secondary structure of HSA after its complexation with the ligand were studied with CD spectroscopy, which indicated that the protein became partially unfolded. Also, temperature did not affect the HSA-CT complexes. The binding of CT with HSA was detected as 2 molecules bound to HSA was determined using micro TOF-Q mass spectrometry. Further, molecular docking studies revealed that CT was binding at subdomain IIA with hydrophobic interactions and also by hydrogen-bond interactions between the hydroxyl (OH) group of carbon-16 and carbon-2 of CT and Arg222, Ala291, Val293, and Met298 of HSA, with hydrogen-bond distances of 2.488, 2.811, 2.678, and 2.586 A, respectively.
Article
In this study, fluorescence spectroscopy in combination with UV-vis absorption spectroscopy and circular dichroism (CD) spectroscopy was employed to investigate the high affinity binding of palmatine to human serum albumin (HSA) under the physiological conditions. In the mechanism discussion it was proved that the fluorescence quenching of HSA by palmatine is a result of the formation of palmatine/HSA complex. Binding parameters calculating from Stern-Volmer method and Scatchard method showed that palmatine bind to HSA with the binding affinities of the order 10(4) L.mol(-1). The thermodynamic parameters studies revealed that the binding was characterized by negative enthalpy and positive entropy changes and the electrostatic interactions play a major role for palmatine-HSA association. Site marker competitive displacement experiments demonstrating that palmatine bind with high affinity to site I (subdomain IIA) of HSA. The specific binding distance r (2.91 nm) between donor (Trp-214) and acceptor (palmatine) was obtained according to fluorescence resonance energy transfer (FRET). Furthermore, the CD spectral result indicates that the secondary structure of HSA was changed in the presence of palmatine.
Article
Berberine is an important traditional medicinal herb, which has been effectively used in the treatment of dysentery, diarrhea, stomatitis, throat infections, and hepatitis in folk medicine. In this study, the interaction between Berberine and human serum albumin (HSA) was investigated by fluorescence spectroscopy and UV-vis absorbance spectroscopy. In the mechanism discussion, it was proved that the fluorescence quenching of HSA by berberine is a result of the formation of berberine-HSA complex. Fluorescence quenching constants were determined using the Stern-Volmer equation and Scatchard equation to provide a measure of the binding affinity between berberine and HSA. The results of thermodynamic parameters DeltaG, DeltaH, and DeltaS at different temperatures indicate that the electrostatic interactions play a major role for berberine-HSA association. Site marker competitive experiments indicated that the binding of berberine to HSA primarily took place in subdomain IIA. Furthermore, the distance r between donor (Trp-214) and acceptor (berberine) was obtained according to fluorescence resonance energy transfer (FRET).