Probing the cysteine 34 residue in human serum albumin using fluorescence techniques (original) (raw)
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Journal of Fluorescence, 2007
Determination of binding parameters such as the number of ligands and the respective binding constants require a considerable number of experiments to be performed. These involve accurate determination of either free and/or bound ligand concentration irrespective of the measurement technique applied. Then, an appropriate theoretical model is used to fit the experimental data, and to extract the binding parameters. In this work, the interaction between bovine serum albumin (BSA) and 1anilino-8-naphthalene sulphonate (ANS) is revisited. Using steady state fluorescence spectroscopy, the binding isotherm of BSA/ANS was obtained applying the Halfman-Nishida approach. The binding parameters, site number, and binding site association constants, were determined from the stoichiometric Adair model and Job's plot. The binding parameters obtained were then correlated to the distance of the respective binding site to the tryptophan residues using the energy transfer technique. This approach, that uses both tryptophans independently from each other, is presented as a tool to help understand the binding mechanism of the albumin fluorescent complex. The results show that ANS molecules bind to BSA in up to five different binding sites. Energy transfer from the tryptophan residues to the BSA/ANS complex shows that the four highest affinity binding sites (>10 4 M −1) are located at a reasonably close distance (18-27 Å) to at least one of two tryptophan residues, while the lowest affinity binding site (~10 4 M −1) is located over 34 Å away from the both tryptophans.
The Journal of Physical Chemistry B, 2009
Interaction of bovine serum albumin (BSA) with two series of dipolar molecules having both rigid and flexible structures has been studied by monitoring the spectral and temporal behavior of the intramolecular charge transfer fluorescence of the systems. The binding sites of the molecular systems in BSA have been located with the help of docking studies. Three different sites of varying hydrophobicity have been identified where these molecules are located. Binding in the hydrophobic domains of BSA leads to a blue shift of the fluorescence spectra and an enhancement of fluorescence intensity and lifetime. This enhancement is found to be the largest for flexible systems in which internal motion serves as a nonradiative decay route. In the BSA-bound condition, some of the dipolar molecules exhibit not-so-common "dip-rise-dip" time-resolved fluorescence anisotropy profiles. It is shown that a large difference of the fluorescence lifetimes of the protein-bound and unbound molecules is one of the factors that contributes to this kind of anisotropy profiles. As internal motion is often responsible for the short fluorescence lifetime of the flexible dipolar molecules, a large increase in the fluorescence lifetime of these systems occurs if binding to BSA leads to disruption/prevention of this motion. It thus appears that it might be possible to obtain information on the prevention/disruption of nonradiative pathway on protein binding from the anisotropy profiles of the kind discussed above. However, since the present study reveals cases where a large change in fluorescence lifetime also occurs due to other reasons, one needs to be careful prior to making any conclusion.
Journal of Photochemistry and Photobiology B: Biology, 2011
In this paper, the nature of the interactions between bovine serum albumin (BSA) and aurintricarboxylic acid (ATA) has been investigated by measuring steady state and time-resolved fluorescence, circular dichroism (CD), FT-IR and fluorescence anisotropy in protein environment under physiological conditions. From the analysis of the steady state and time-resolved fluorescence quenching of BSA in aqueous solution in presence of ATA it has been inferred that the nature of the quenching originates from the combined effect of static and dynamic modes. From the determination of the thermodynamic parameters obtained from temperature-dependent changes in K b (binding constant) it was apparent that the combined effect of hydrophobic association and electrostatic attraction is responsible for the interaction of ATA with BSA. The effect of ATA on the conformation of BSA has been examined by analyzing CD spectrum. Though the observed results demonstrate some conformational changes in BSA in presence of ATA but the secondary structure of BSA, predominantly of a-helix, is found to retain its identity. Molecular docking of ATA with BSA also indicates that ATA docks through hydrophobic interaction.
Cyanylated Cysteine: A Covalently Attached Vibrational Probe of Protein−Lipid Contacts
The Journal of Physical Chemistry Letters, 2010
Cyanylated cysteine, or β-thiocyanatoalanine, is an artificial amino acid that can be introduced into peptides and proteins by post-translational chemical modification of solvent-exposed cysteine side chains, and thus it can be used in any protein with a suitable expression and mutagenesis system. In this study, cyanylated cysteine is introduced at selected sites in two model peptides that have been shown to bind to membrane interfaces: a membrane-binding sequence of the human myelin basic protein and the antimicrobial peptide CM15. Far-UV circular dichroism indicates that the secondary structures of the bound peptides are not influenced by introduction of the artificial side chain. Infrared spectra of both systems in buffer and exposed to dodecylphosphocholine micelles indicate that the CN stretching absorption band of cyanylated cysteine can clearly distinguish between membrane burial and solvent exposure of the artificial side chain. Since infrared spectroscopy can be applied in a wide variety of lipid systems, and since cyanylated cysteine can be introduced into proteins of arbitrary size via mutagenesis and post-translational modification, this new probe could see wide use in characterizing the protein-lipid interactions of membrane proteins.
Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications VII, 2015
Fluorescein (FL) and some of its precursors have proven to be effective fluorescent tracers in pharmaceutical and medical applications owing to their high quantum yield of fluorescence in physiological conditions and their high membrane permeability. In order to protect FL from metabolic effects during the process of its delivery, human serum albumin (HSA) has been used as a carrier because of its compatibility with the human body. In the present work, we used spectroscopic methods to characterize the binding mechanisms of FL and one of its derivatives, 5(6)carboxyfluorescein (CFL), in the HSA protein. The absorbance change of the two ligands (FL and CFL) was quantified as a function of the HSA concentration and the results indicate a moderate binding strength for the two ligands inside HSA (1.00 ± 0.12 x 10 4 M -1 ). The quenching effect of FL(CFL) on the fluorescence intensity of W214 (the sole tryptophan in HSA) indicates that FL and CFL occupy Site I in the protein which is known to bind several hydrophobic drugs. By performing site-competitive experiments, the location of the ligands is determined to be similar to that of the anticoagulant drug warfarin. At higher ratios of [ligand]/[HSA], we observed an upward curvature in the Stern-Volmer plots which indicates that the ligands occupy more pockets in Site I, close to W214. Our results indicate that both ligands bind in HSA with a moderate strength that should not affect their release when used as fluorescent reporters. The chemical and physical identities of the two ligands are also preserved inside the HSA binding sites. Downloaded From: http://proceedings.spiedigitallibrary.org/ on 03/14/2015 Terms of Use: http://spiedl.org/terms Proc. of SPIE Vol. 9339 933908-2 Downloaded From: http://proceedings.spiedigitallibrary.org/ on 03/14/2015 Terms of Use: http://spiedl.org/terms Proc. of SPIE Vol. 9339 933908-7 Downloaded From: http://proceedings.spiedigitallibrary.org/ on 03/14/2015 Terms of Use: http://spiedl.org/terms
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 2002
The binding of several different categories of small molecules to bovine (BSA) and human (HSA) serum albumins has been studied for many years through different spectroscopic techniques to elucidate details of the protein structure and binding mechanism. In this work we present the results of the study of the interactions of BSA and HSA with the anionic sodium dodecyl sulfate (SDS), cationic cethyltrimethylammonium chloride (CTAC) and zwitterionic N-hexadecyl-N,N-dimethyl-3ammonium-1-propanesulfonate (HPS) monitored by fluorescence spectroscopy of the intrinsic tryptophans at pH 5.0. Similarly to pH 7.0 and 9.0, at low concentrations, the interaction of BSA with these surfactants shows a quenching of fluorescence with Stern^Volmer quenching constants of (1.1 þ 0.1)U10 4 M 31 , (3.2 þ 0.1)U10 3 M 31 and (2.1 þ 0.1)U10 3 M 31 for SDS, HPS and CTAC, respectively, which are associated to the`effective' association constants to the protein. On the interaction of these surfactants with HSA, an opposite effect was observed as compared to BSA, i.e., an enhancement of fluorescence takes place. For both proteins, at low surfactant concentrations, a positive cooperativity was observed and the Hill plot model was used to estimate the number of surfactant binding sites, as well as the association constants of the surfactants to the proteins. It is worthy of notice that the binding constants for the surfactants at pH 5.0 are lower as compared to pH 7.0 and 9.0. This is probably due to fact that the protein at this acid pH is quite compact reducing the accessibility of the surfactants to the hydrophobic cavities in the binding sites. The interaction of myristic acid with both proteins shows a similar fluorescence behaviour, suggesting that the mechanism of the interaction is the same. Recently published crystallographic studies of HSA^myristate complex were used to perform a modelling study with the aim to explain the fluorescence results. The crystallographic structure reveals that a total of five myristic acid molecules are asymmetrically bound in the macromolecule. Three of these sites correspond to higher affinity ones and correlate with high association constants described in the literature. Our models for BSA and HSA with bound SDS suggest that the surfactant could be bound at the same sites as those reported in the crystal structure for the fatty acid. The differences in tryptophan vicinity upon surfactant binding are explored in the models in order to explain the observed spectroscopic changes. For BSA the quenching is due to a direct contact of a surfactant molecule with the indole of W 131 residue. It is clear that the binding site in BSA which is very close, in contact with tryptophan W 131 , corresponds to a lower affinity site, explaining the lower binding constants obtained from fluorescence studies. In the case of HSA the enhancement of fluorescence is due to the removal of static quenching of W 214 residue in the intact protein caused by nearby residues in the vicinity of this tryptophan. ß 2002 Elsevier Science B.V. All rights reserved. 0167-4838 / 02 / $^see front matter ß 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 -4 8 3 8 ( 0 1 ) 0 0 2 8 7 -
Colloids and Surfaces B-biointerfaces, 2007
Human serum albumin (HSA) is frequently used in biophysical and biochemical studies since it has a well-known primary structure and it has been associated with the binding of many different categories of small molecules. In the present study, results are presented for the binding of cetylpyridinium chloride (CPC) with HSA at various pH and 25 • C, as monitored using ion selective membrane electrodes and fluorescence spectroscopy of intrinsic tryptophan. The obtained binding isotherms were analyzed on basis of binding capacity concept and Hill plot in order to determine the Hill parameters of binding sets. The system behaved as a system with two sets of binding sites in all studied situations. The results represent a positive cooperative behavior and the essential role of hydrophobic interactions in both binding sets. The intrinsic binding affinity of second binding set have a similar values and trends at acidic and neutral pHs, that represents the similar unfolded structure at these pHs. CPC quenched the fluorescence arising from Trp group incorporated to HSA. A biphasic behavior was observed in quenching process that confirmed the results of binding study correspond to the existence of two binding sets. The similarity of unfolded structure in acidic and neutral pH was also confirmed by fluorescence study. The quenching of HSA fluorescence takes place with a Stern-Volmer constant of 0.643 × 10 4 , 1.23 × 10 4 and 7.40 × 10 4 at pH 3.5, 6.8 and 9.5, respectively. The Stern-Volmer behavior observed at low molar ratio of [CPC]/[HSA] (about 6), that represents the occurrence of conformational changes after this molar ratio. Comparing, the K SV values and binding parameters indicate that the binding is dominated by hydrophobic effects and, in minor degree, by electrostatic interactions.
Asian Journal of Chemistry
The interaction of 4′-hydroxychalcone (4′HC) with bovine serum albumin (BSA) and human serum albumin (HSA) was studied under physiological condition (pH=7.0). The fluorescence intensity of both serum albumins was quenched in presence of 4′HC at different temperatures. Stern-Volmer analysis and bimolecular quenching constants indicates the presence of static quenching in BSA. Whereas, fluorescence quenching of HSA is due to both the mechanism of static and dynamic quenching. The formation of ground state complex is further confirmed by absorption spectroscopy. The interaction of 4′HC with BSA is stronger than with HSA. FRET study shows the possible energy transfer between 4′HC with BSA and HSA. The binding site of the protein was identified by molecular docking study. The FTIR and CD analysis indicates conformational change in both the serum albumins. The thermodynamic study indicates that the association of BSA and HSA with 4′HC is spontaneous, enthalpy driver and involves electrost...