Investigation on the effect of fluorescence quenching of bovine serum albumin by cefoxitin sodium using fluorescence spectroscopy and synchronous fluorescence spectroscopy

The reaction mechanism of cefoxitin sodium with bovine serum albumin was investigated using fluorescence spectroscopy and synchronous fluorescence spectroscopy at different temperatures. The results showed that the change of binding constant of the synchronous fluorescence method with increasing temperature could be used to estimate the types of quenching mechanisms of drugs with protein and was consistent with one of fluorescence quenching method. In addition, the number of binding sites, type of interaction force, cooperativity between drug and protein and energy‐transfer parameters of cefoxitin sodium and bovine serum albumin obtained from two methods using the same equation were consistent. Electrostatic force played a major role in the conjugation reaction between bovine serum albumin and cefoxitin sodium, and the type of quenching was static quenching. The primary binding site for cefoxitin sodium was sub‐hydrophobic domain IIA, and the number of binding sites was 1. The value of Hill's coefficients (n_H) was approximately equal to 1, which suggested no cooperativity in the bovine serum albumin–cefoxitin sodium system. The donor‐to‐acceptor distance r < 7 nm indicated that static fluorescence quenching of bovine serum albumin by cefoxitin sodium was also a non‐radiation energy‐transfer process. The results indicated that synchronous fluorescence spectrometry could be used to study the reaction mechanism between drug and protein, and was a useful supplement to the conventional method. Copyright © 2015 John Wiley & Sons, Ltd.     

Identification of the antitumoral drug emodin binding sites in bovine serum albumin by spectroscopic methods

Using SERS, fluorescence, circular dichroism and stopped-flow, we have unequivocally characterized the binding sites of emodin in bovine serum albumin. Emodin interacts with protein through two different binding sites corresponding to Sudlow's sites 1 and 2. Site 2, where the binding drug presents, in the cavity, a form between neutral and mono-anionic species slightly displaced to the neutral one, is the primary interaction site, with higher association binding constant, and hence, higher affinity than the other binding site. This interaction changes considerably the alpha-helical content of the protein and it occurs mainly within the interval [emodin]/[protein] < or = 2.0. The process involves a fast reaction and the observed rate constant increases when increasing the [emodin]/[protein] ratio. The secondary emodin interaction site corresponds to the Sudlow's site 1, where the drug shows a similar form to that deduced for site 2, but in this case, it is more displaced to mono-anionic species. This interaction does not change the alpha-helical content of bovine serum albumin, and it occurs mainly for [emodin]/[protein] > 2.0 ratios, the process implies a slower reaction than the union process to the site 2, with an observed rate constant that is invariable within the studied interval.     

Binding characteristics of a major thyroid hormone metabolite, 3,3',5'-triiodo-L-thyronine, to bovine serum albumin as measured by fluorescence

The interaction between a major thyroid hormone metabolite, 3,3',5'-triiodo-L-thyronine and bovine serum albumin was investigated by fluorescence measurements. The apparent binding constants were obtained at various pHs assuming the equivalence and independence of the interaction sites on the protein from the fluorescence titration curves. The maximum binding was attained at pH 8.0, and the apparent binding constant was (5.28 +/- 0.13).10(5) M-1 with one binding site per albumin molecule. Thermodynamic parameters were also determined from the van't Hoff plot of the apparent binding constants at pH 7.5. The free energy change, enthalpy change and entropy change were -7.70 +/- 0.09 kcal.mol-1, -4.59 kcal.mol-1 and 10.2 e.u., respectively.     

Association equilibrium of d-methamphetamine and l-methamphetamine with serum albumin

The association equilibria for complex formation between serum albumin (bovine, rat) and the optical isomers of methamphetamine (MAMP) was determined using an ultrafiltration method. It was found that serum albumin/d-MAMP and serum albumin/l-MAMP complexes had distinctly different Scatchard plots with bovine and rat albumin. The binding parameters of each association equilibrium were estimated from the Scatchard plots by Rosenthal's graphic method. This distinguished two kinds of specific binding sites in terms of the association equilibrium between bovine serum albumin and d-MAMP, and one binding site for rat serum albumin and d-MAMP. One specific binding site was found between serum albumin and l-MAMP in both bovine and rat. Molar ellipticities, [θ], of peaks were decreased in the CD spectra of the complexes formed between bovine serum albumin and d-MAMP or l-MAMP when compared with the CD spectrum of bovine serum albumin alone. However, no difference in [θ] was found between the CD spectra of the enantiomers of MAMP in the measured wavelength range. The non-specific binding site was distinct from the specific binding site and resulting from altered tertiary structure of the albumin molecule. Chirality 10:742–746, 1998. © 1998 Wiley-Liss, Inc.     

Comparative studies on the interaction of cefixime with bovine serum albumin by fluorescence quenching spectroscopy and synchronous fluorescence spectroscopy

Under simulated physiological conditions, the reaction mechanism between cefixime and bovine serum albumin at different temperatures (293, 303 and 310 K) was investigated using a fluorescence quenching method and synchronous fluorescence method, respectively. The results indicated that the fluorescence intensity and synchronous fluorescence intensity of bovine serum albumin decreased regularly on the addition of cefixime. In addition, the quenching mechanism, binding constants, number of binding sites, type of interaction force and energy‐transfer parameters of cefixime with bovine serum albumin obtained from two methods using the same equation were consistent. The results indicated that the synchronous fluorescence spectrometry could be used to study the binding mechanism between drug and protein, and was a useful supplement to the conventional method. Copyright © 2014 John Wiley & Sons, Ltd.     

The binding affinity of human serum albumin and paclitaxel through MMPBSA based on docked complex

The distribution, free concentration and metabolism of drugs can be significantly altered as a result of binding to albumin. At the same time, the conformational of serum albumin was also changed by interaction with low molecular weight drugs. In present work, we first equilibrated HSA in aqueous solution to obtain the solvated-HSA model. Further solvated-HSA was performed molecular docking with paclitaxel to find the binding sites. The two docking HSA-paclitaxel complexes were obtained and further equilibrated by a 12 ns MD simulation. Then, MMPBSA method was used to investigate the binding free energy of them. Finally, we correlated the fluctuations of residues with corresponding changes in the secondary structure by dssp method. Two binding sites of paclitaxel were found on HSA having considered the solvation effect. More hydrogen bonds were formed at site I respected to site II. A larger binding energy for primary binding also indicated that paclitaxel showed higher binding affinity mainly due to the stronger hydrogen bonding interactions. There was a significant difference between the two complexes on structure according to the dssp results. Moreover, structure of the binding sites exhibited more fluctuations after binding paclitaxel compared with other regions. Paclitaxel binding also induced distinct conformational changes in drug binding site even when it was empty and have contributed to a reduced binding capacity of HSA towards adriamycin.     

Bovine serum albumin. Study of the fatty acid and steroid binding sites using spin-labeled lipids.

Three spin-labeled derivatives of stearic acid and two derivatives of palmitic acid have been used to study the structure of the strong fatty acid binding site of bovine serum albumin. The steroid and indole binding sites have been studied using spin-labeled derivatives of androstol and indole, respectively. Paramagnetic resonance and fluorescence quenching data suggest that the fatty acid, steroid, and indole binding sites may be identical. The mobility of the nitroxyl group at C-8 of palmitic acid bound to albumin at a 1:1 molar ratio is unaffected when the carboxyl group is esterified. When the nitroxyl group is located at C-5 on this acid its motion is detectably increased by esterification of the carboxyl group but the magnitude of this change is small. This result suggests that the carboxyl group may play a minor role in the binding of fatty acids to the strongest fatty acid binding site of albumin. When stearic acid derivatives bearing the nitroxide at C-5, C-12, and C-16 are bound to albumin at a ligand to albumin ratio of 1, the order of mobility at 0-30 degrees is C-16 greater than C-12 congruent to C-5. Although motion at the methyl terminus is always greater than at the COOH terminus in the range 0-60 degrees, a simple monotonic increase in chain motion between the two termini is not observed. Arrhenius plots of the motion parameters for these bound fatty acids show two abrupt changes in slope. The temperature ranges for these changes are 15-23 degrees and 38-45 degrees. These results suggest that when one mole of spin-labeled fatty acid is bound to albumin, the protein undergoes a conformational change in each of these temperature ranges.     

Crystallographic analysis reveals common modes of binding of medium and long-chain fatty acids to human serum albumin

Human serum albumin (HSA) is an abundant plasma protein that is responsible for the transport of fatty acids. HSA also binds and perturbs the pharmacokinetics of a wide range of drug compounds. Binding studies have revealed significant interactions between fatty acid and drug-binding sites on albumin but high-resolution structural information on ligand binding to the protein has been lacking. We report here a crystallographic study of five HSA-fatty acid complexes formed using saturated medium-chain and long-chain fatty acids (C10:0, C12:0, C14:0, C16:0 and C18:0). A total of seven binding sites that are occupied by all medium-chain and long-chain fatty acids have been identified, although medium-chain fatty acids are found to bind at additional sites on the protein, yielding a total of 11 distinct binding locations. Comparison of the different complexes reveals key similarities and significant differences in the modes of binding, and serves to rationalise much of the biochemical data on fatty acid interactions with albumin. The two principal drug-binding sites, in sub-domains IIA and IIIA, are observed to be occupied by fatty acids and one of them (in IIIA) appears to coincide with a high-affinity long-chain fatty acid binding site.     

Interaction of ergosterol with bovine serum albumin and human serum albumin by spectroscopic analysis

This study was designed to examine the interactions of ergosterol with bovine serum albumin (BSA) and human serum albumin (HSA) under physiological conditions with the drug concentrations in the range of 2.99-105.88?μM and the concentration of proteins was fixed at 5.0?μM. The analysis of emission spectra quenching at different temperatures revealed that the quenching mechanism of HSA/BSA by ergosterol was the static quenching. The number of binding sites n and the binding constants K were obtained at various temperatures. The distance r between ergosterol and HSA/BSA was evaluated according to F?ster non-radioactive energy transfer theory. The results of synchronous fluorescence, 3D fluorescence, FT-IR, CD and UV-Vis absorption spectra showed that the conformations of HSA/BSA altered in the presence of ergosterol. The thermodynamic parameters, free energy change (ΔG), enthalpy change (ΔH) and entropy change (ΔS) for BSA-ergosterol and HSA-ergosterol systems were calculated by the van't Hoff equation and discussed. Besides, with the aid of three site markers (for example, phenylbutazone, ibuprofen and digitoxin), we have reported that ergosterol primarily binds to the tryptophan residues of BSA/HSA within site I (subdomain II A).     

Magnetic core/shell Fe3O4/Au nanoparticles for studies of quinolones binding to protein by fluorescence spectroscopy

Magnetic core/shell Fe₃O₄/Au nanoparticles were used in the determination of drug binding to bovine serum albumin (BSA) using a fluorescence spectroscopic method. The binding constants and number of binding sites for protein with drugs were calculated using the Scatchard equation. Because of their superparamagnetic and biocompatible characteristics, magnetic core/shell Fe₃O₄/Au nanoparticles served as carrier proteins for fixing proteins. After binding of the protein to a drug, the magnetic core/shell Fe₃O₄/Au nanoparticles–protein–drug complex was separated from the free drug using an applied magnetic field. The free drug concentration was obtained directly by fluorescence spectrometry and the proteins did not influence the drug determination. So, the achieved number of binding sites should be reliable. The binding constant and site number for ciprofloxacin (CPFX) binding to BSA were 2.055 × 10⁵ L/mol and 31.7, and the corresponding values for norfloxacin (NOR) binding to BSA were 1.383 × 10⁵ L/mol and 38.8. Based on the achieved results, a suitable method was proposed for the determination of binding constants and the site number for molecular interactions. The method was especially suitable for studies on the interactions of serum albumin with the active ingredients of Chinese medicine. Copyright © 2015 John Wiley & Sons, Ltd.     

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.     

Hydration energy landscape of the active site cavity in cytochrome P450cam

Hydration of protein cavities influences protein stability, dynamics, and function. Protein active sites usually contain water molecules that, upon ligand binding, are either displaced into bulk solvent or retained to mediate protein–ligand interactions. The contribution of water molecules to ligand binding must be accounted for to compute accurate values of binding affinities. This requires estimation of the extent of hydration of the binding site. However, it is often difficult to identify the water molecules involved in the binding process when ligands bind on the surface of a protein. Cytochrome P450cam is, therefore, an ideal model system because its substrate binds in a buried active site, displacing partially disordered solvent, and the protein is well characterized experimentally. We calculated the free energy differences for having five to eight water molecules in the active site cavity of the unliganded enzyme from molecular dynamics simulations by thermodynamic integration employing a three-stage perturbation scheme. The computed free energy differences between the hydration states are small (within 12 kJ mol⁻¹) but distinct. Consistent with the crystallographic determination and studies employing hydrostatic pressure, we calculated that, although ten water molecules could in principle occupy the volume of the active site, occupation by five to six water molecules is thermodynamically most favorable. Proteins 32:381–396, 1998. © 1998 Wiley-Liss, Inc.     

Preferential binding of fisetin to the native state of bovine serum albumin: spectroscopic and docking studies

We have investigated the binding of the biologically important flavonoid fisetin with the carrier protein bovine serum albumin using multi-spectroscopic and molecular docking methods. The binding constants were found to be in the order of 10⁴ M^?1 and the number of binding sites was determined as one. MALDI-TOF analyses showed that one fisetin molecule binds to a single bovine serum albumin (BSA) molecule which is also supported by fluorescence quenching studies. The negative Gibbs free energy change (?G°) values point to a spontaneous binding process which occurs through the presence of electrostatic forces with hydrophobic association that results in a positive entropy change (+51.69 ± 1.18 J mol^?1 K^?1). The unfolding and refolding of BSA in urea have been studied in absence and presence of fisetin using steady-state fluorescence and lifetime measurements. Urea denaturation studies indicate that fisetin is gradually released from its binding site on the protein. In the absence of urea, an increase in temperature that causes denaturation of the protein results in the release of fisetin from its bound state indicating that fisetin binds only to the native state of the protein. The circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopic studies showed an increase in % α-helix content of BSA after binding with fisetin. Site marker displacement studies in accordance with the molecular docking results suggested that fisetin binds in close proximity of the hydrophobic cavity in site 1 (subdomain IIA) of the protein. The PEARLS (Program of Energetic Analysis of Receptor Ligand System) has been used to estimate the interaction energy of fisetin with BSA and the results are in good correlation with the experimental findings.     

Surface-enhanced Raman spectroscopy study of the interaction of the antitumoral drug emodin with human serum albumin

Surface-enhanced Raman spectroscopy was employed in this work to study the interaction between the antitumoral drug emodin and human serum albumin (HSA), as well as the influence of fatty acids in this interaction. We demonstrated that the drug/protein interaction can take place through two different binding sites which are probably localized in the IIA and IIIA hydrophobic pockets of HSA and which correspond to Sudlow's I and II binding sites, respectively. The primary interaction site of this drug seems to be site II in the defatted albumin. Fatty acids seem to displace the drug from site II to site I in nondefatted HSA, due to the high affinity of fatty acids for site II. The drug interacts with the protein through its dianionic form in defatted HSA (when placed in the site II) and through its neutral form in the site I of nondefatted albumins.     

Binding interaction of a biological photosensitizer with serum albumins: a biophysical study

A photophysical study on the binding interaction of an efficient cancer cell photosensitizer, norharmane (NHM), with model transport proteins, bovine serum albumin (BSA) and human serum albumin (HSA), has been performed using a combination of steady-state and time-resolved fluorescence techniques. The emission profile undergoes a remarkable change upon addition of the proteins to the buffered aqueous solution of the photosensitizer. The polarity-dependent prototropic transformation is responsible for the remarkable sensitivity of this biological fluorophore to the protein environments. A marked increase in the fluorescence anisotropy in the proteinous environments indicates that the albumin proteins introduce motional restriction on the drug molecule. Light has been thrown on the denaturing action of urea on the probe-bound protein. The probable binding site of the drug in proteins has also been assessed from the combination of denaturation study, micropolarity measurement, and fluorescence resonance energy transfer (FRET) study. The present study suggests that the stability of serum albumins is enhanced upon binding with the drug.     

Luminescence,circular dichroism and in silico studies of binding interaction of synthesized naphthylchalcone derivatives with bovine serum albumin

Chalcones possess various biological properties, for example, antimicrobial, anti‐inflammatory, analgesic, antimalarial, anticancer, antiprotozoal and antitubercular activity. In this study, naphthylchalcone derivatives were synthesized and characterized using ¹H NMR ¹³C NMR, Fourier transform infrared and mass techniques. Yields for all derivatives were found to be >90%. Protein–drug interactions influence the absorption, distribution, metabolism and excretion (ADME) properties of a drug. Therefore, to establish whether the synthesized naphthylchalcone derivatives can be used as drugs, their binding interaction toward a serum protein (bovine serum albumin) was investigated using fluorescence, circular dichroism and molecular docking techniques under physiological conditions. Fluorescence quenching of the protein in the presence of naphthylchalcone derivatives, and other derived parameters such as association constants, number of binding sites and static quenching involving confirmed non‐covalent binding interactions in the protein–ligand complex were observed. Circular dichroism clearly showed changes in the secondary structure of the protein in the presence of naphthylchalcones, indicating binding between the derivatives and the serum protein. Molecular modelling further confirmed the binding mode of naphthylchalcone derivatives in bovine serum albumin. A site‐specific molecular docking study of naphthylchalcone derivatives with serum albumin showed that binding took place primarily in the aromatic low helix and then in subdomain II. The dominance of hydrophobic, hydrophilic and hydrogen bonding was clearly visible and was responsible for stabilization of the complex.     

Studies on the interactions between the cyclic nucleotide-binding sites of cGMP-dependent protein kinase

The rate and equilibrium kinetics of [3H]cGMP binding to the two rapidly exchanging and two slowly exchanging sites of dimeric cGMP-dependent protein kinase from bovine lung were studied. As observed by McCune and Gill (McCune, R. W., and Gill, G. N. (1979) J. Biol. Chem. 254, 5083-5091), unlabeled cGMP retarded the dissociation of [3H]cGMP bound to the "slow" site. This effect was due to interaction of unlabeled cGMP with the "rapid" rather than the slow site. First, the potencies of unlabeled cGMP and a number of cGMP analogs correlated nearly perfectly with their affinities for the rapid site. Second, the rate of dissociation in the absence of unlabeled ligand was independent of the degree of saturation of the slow sites. Third, unlabeled ligand inhibited the rate of dissociation more (about 10-fold) than theoretically predicted (maximum 2-fold) from interaction between two similar sites in one macromolecule. A favorable free energy coupling appeared to exist between the rapid and slow sites but not between the slow sites. cGMP associated faster to the slow site than the rapid site. Mg/ATP decreased the rate of association to either site by 50% and increased about ten-fold the rate of dissociation from the slow site. The dissociation of cGMP from the slow site could be described by a single activation energy (Ea = 71 kJ X mol-1) for the whole temperature range (0-37 degrees C) tested. These data indicated that the cyclic nucleotide-binding sites of the cGMP-kinase are kinetically more homologous to those in the cAMP-dependent protein kinases than previously recognized.     

Thermochemistry of the specific binding of C12 surfactants to bovine serum albumin

The specific binding to bovine serum albumin (BSA) of anionic and non-ionic surfactants with C12 acyl chains has been studied by high sensitivity isothermal titration calorimetry. This method proved particularly effective in resolving the binding of anionic surfactants into separate classes of sites with different affinity. For sodium dodecylsulfate (SDS) the measured binding curves could be rationalized as association to two classes (high affinity/low affinity) of sites comprising, respectively, three and six similar (i.e. thermodynamically equivalent), independent sites. Changes in the thermodynamic functions enthalpy, standard free energy, standard entropy and heat capacity could be discerned for each class of binding site, as well as for micelle formation. These data suggest that binding to low affinity sites (in analogy with micelle formation) exhibits energetic parameters; in particular, a large negative change in heat capacity, which is characteristic of hydrophobic interactions. The thermodynamics of high affinity binding, on the other hand, is indicative of other dominant forces; most likely electrostatic interactions. Other anionic ligands investigated (laurate and dodecyl benzylsulfonate) showed a behavior similar to SDS, the most significant difference being the high affinity binding of the alkylbenzyl sulfonate. For this ligand, the thermodynamic data is indicative of a more loosely associated complex than for SDS and laurate. BSA was found to bind one or two of the non-ionic surfactants (NIS) hepta- or penta(ethylene glycol) monododecyl ether (C12EO7 and C12EO5) with binding constants about three orders of magnitude lower than for SDS. Hence, the free energy of the surfactant in the weakly bound BSA-NIS complex is only slightly favored over the micellar state. The binding process is characterized by very large exothermic enthalpy changes (larger than for the charged surfactants) and a large, positive increment in heat capacity. These observations cannot be reconciled with a molecular picture based on simple hydrophobic condensation onto non-polar patches on the protein surface.     

Vitamin C-bovine serum albumin binding behaviour

The binding of ascorbic acid and dehydroascorbic acid to bovine serum albumin is greatly heterogeneous. The Hill plots, as evaluated from the fluorescence quenching measurements, clearly show a biphasic behaviour. Scatchard analysis moreover indicates that the potency and the pattern of the binding can change gradually in the process of occupation of various sites because of albumin structural modifications.     

Evaluation of drug-human serum albumin binding interactions with support vector machine aided online automated docking

MOTIVATION: Human serum albumin (HSA), the most abundant plasma protein is well known for its extraordinary binding capacity for both endogenous and exogenous substances, including a wide range of drugs. Interaction with the two principal binding sites of HSA in subdomain IIA (site 1) and in subdomain IIIA (site 2) controls the free, active concentration of a drug, provides a reservoir for a long duration of action and ultimately affects the ADME (absorption, distribution, metabolism, and excretion) profile. Due to the continuous demand to investigate HSA binding properties of novel drugs, drug candidates and drug-like compounds, a support vector machine (SVM) model was developed that efficiently predicts albumin binding. Our SVM model was integrated to a free, web-based prediction platform (http://albumin.althotas.com). Automated molecular docking calculations for prediction of complex geometry are also integrated into the web service. The platform enables the users (i) to predict if albumin binds the query ligand, (ii) to determine the probable ligand binding site (site 1 or site 2), (iii) to select the albumin X-ray structure which is complexed with the most similar ligand and (iv) to calculate complex geometry using molecular docking calculations. Our SVM model and the potential offered by the combined use of in silico calculation methods and experimental binding data is illustrated.