Spectroscopic probe analysis for exploring probe-protein interaction: a mapping of native, unfolding and refolding of protein bovine serum albumin by extrinsic fluorescence probe

Steady state and dynamic fluorescence measurements have been used to investigate interaction between Bovine Serum Albumin (BSA) and fluorescence probe para-N,N-dimethylamino orthohydroxy benzaldehyde (PDOHBA), a structurally important molecule exhibiting excited state coupled proton transfer (PT) and charge transfer (CT) reaction. Fluorescence anisotropy, acrylamide quenching, and time resolved fluorescence measurements corroborate the binding nature of the probe with protein. The binding constant between BSA and PDOHBA has been determined by using Benesi-Hildebrand and Stern-Volmer equations. The negative value of ΔG indicates the spontaneity of this probe-protein complexation process. Observations from synchronous, three dimensional fluorescence spectra and circular dichroism spectra point toward the fact that the hydrophobicity as well as α-helix content of BSA are altered in presence of probe PDOHBA. The PT band of PDOHBA is found to be an excellent reporter for the mapping of destructive and protective behavior of SDS with variation of chaotrope concentration.     

Exploring structural change of protein bovine serum albumin by external perturbation using extrinsic fluorescence probe: spectroscopic measurement, molecular docking and molecular dynamics simulation

Structural modification through binding interaction of plasma protein bovine serum albumin (BSA) with an extrinsic charge transfer fluorophore 5-(4-dimethylamino-phenyl)-penta-2,4-dienoic acid (DMAPPDA) and its response to external perturbation due to interactions with surfactant sodium dodecyl sulphate (SDS) have been explored at physiological pH by steady state absorption, emission, fluorescence anisotropy, red edge excitation shift, far-UV circular dichroism and time resolved spectral measurements in combination with Molecular Docking and Molecular Dynamics (MD) simulation. Interaction of the probe with BSA is reflected by a small change in protein secondary structure with fluorescence enhancement and blue shift of probe emission. Molecular docking studies revealed that the probe binds to the hydrophobic cavity of sub-domain IIA of BSA. The distance for energy transfer from the tryptophan of BSA to the bound DMAPPDA measured by Fluorescence Resonance Energy Transfer is in good agreement with the molecular docking results. MD simulation predicts stabilization of the complex with respect to the bare molecule. Interaction of BSA and SDS with DMAPPDA supports the movement of the probe from hydrophilic free water region to a more restricted hydrophobic zone inside the protein.     

The binding of 4',6-diamidino-2-phenylindole to bovine serum albumin.

The binding of 4',6-diamidino-2-phenylindole (DAPI) to bovine serum albumin (BSA) has been investigated between pH 6 and 8, in 0.05 M phosphate buffer at 20 degrees C, by fluorescence titrations and the results analyzed according to a procedure previously reported (R. Favilla and A. Mazzini, Biochim. Biophys. Acta 788 (1984) 48). The dye binds to the protein with a blue shift of about 4 nm in its fluorescence emission maximum, but with an enhancement factor of 10 of its fluorescence quantum yield. The dissociation constant decreases from 100 microM to 54 microM as the pH is increased from 6 to 8, with a constant number of nearly three equivalent binding sites. The complete displacement of DAPI bound to BSA by Ca2+ suggests a possible specificity of this substantially electrostatic interaction. The fluorescence decay of DAPI bound to the protein shows a double exponential kinetics, with a tau 1 = 0.97 ns and tau 2 = 2.78 ns. These results, compared with those obtained for DAPI alone, tau 1 = 0.16 ns and tau 2 = 2.8 ns, are rationalized in terms of two different rotamers of DAPI. Both rotamers are able to bind to the protein, but only one of them undergoes an intramolecular proton transfer, from the 6-amidinium group to the indole aromatic ring, in the excited singlet state of DAPI alone. When DAPI interacts with BSA this transfer does not occur and consequently a large increase of fluorescence is observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of ionic strength on the binding of ascorbate to albumin

A fluorophore-nitroxide free radical dual-functional probe (FN) was utilized to study the kinetics of ascorbate (AH(-)) binding to Bovine Serum Albumin (BSA). Since the free radical fragment in the FN probe intramolecularly quenches fluorescence, ascorbate reduction of the nitroxide function is accompanied by a concomitant fluorescence intensity increase from the fluorophore. Thus, both fluorescence and the EPR techniques could be utilized to measure the reaction rate. In the presence of BSA protein, the observed rate of the overall process is the sum of that from at least two reactions: the reaction between free ascorbate and free probe, and the reaction between bound ascorbate and bound probe. Our findings show that the observed rate is strongly dependent on the ionic strength of the medium. A corollary of this observation is the indication of a purely electrostatic interaction between ascorbate and the BSA protein. This conclusion was further corroborated by 1H NMR measurement of the transverse relaxation time, T(2), of ascorbate protons in BSA solutions. Ascorbate ion was released from the ascorbate/BSA ensemble in the presence of increasing concentrations of NaCl. Binding constants of AH(-) to BSA were calculated at different ionic strengths at pH 7.4. Furthermore, an increase in ionic strength did not affect the ability of albumin to protect ascorbate against autoxidation. This suggests that the protein's protective antioxidant effect may be attributed to BSA binding of trace quantities of transition-metal cations (rather than ascorbate binding to BSA). This conclusion is supported by ascorbate UV-absorption measurements in the presence of albumin and Cu(2+) ions as a function of ionic strength.     

Protein-flavonol interaction: fluorescence spectroscopic study

Recent studies have shown that various synthetic as well as therapeutically active naturally occurring flavonols possess novel luminescence properties that can potentially serve as highly sensitive monitors of their microenvironments in biologically relevant systems. We report a study on the interactions of bovine serum albumin (BSA) with the model flavonol 3-hydroxyflavone (3HF), using the excited-state proton-transfer (ESPT) luminescence of 3HF as a probe. Upon addition of BSA to the flavonoid solutions, we observe remarkable changes in the absorption, ESPT fluorescence emission and excitation profiles as well as anisotropy (r) values. Complexation of 3HF with protein results in a pronounced shift (20 nm) of the ESPT emission maximum of the probe (from lambda(max)(em) = 513 nm to lambda(max)(em) = 533 nm) accompanied by a significant increase in fluorescence intensity. The spectral data also suggest that, in addition to ESPT, the protein environment induces proton abstraction from 3HF leading to formation of anionic species in the ground state. Fairly high values of anisotropy are observed in the presence of BSA for the tautomer (r = 0.25) as well as anion (r = 0.35) species of 3HF, implying that both the species are located in motion-restricted environments of BSA molecules. Analysis of relevant spectroscopic data leads to the conclusions that two binding sites are involved in BSA-3HF interaction, and the interaction is slightly positively cooperative in nature with a similar binding constant of 1.1 - 1.3 x 10(5) M(-1) for both these sites. Proteins 2001;43:75-81.     

A chlorotetracycline fluorescent probe--an indicator of calcium ion binding with calcium-binding proteins

It has been found in in vitro experiments that fluorescence intensity of deionized solution containing a chlorotetracycline fluorescent probe increases insignificantly at the addition of calmodulin of S-100 proteins. Subsequent introduction of Ca2+ into the medium results in the pronounced fluorescence increase depending on Ca2+ concentration. Addition of specific protein blockers--W7 (calmodulin inhibitor) and antibodies to S-100 brought about a decrease of fluorescence. In in vivo experiments on chlorotetracycline-stained neurons of Helix Pomatia ganglia subesophageal complex it has been shown that bringing of antibodies to S-100 and calmodulin significantly decreases the fluorescence intensity of these cells. These data suggest that the chlorotetracycline probe is an indicator of calcium ions binding with calcium-binding proteins both in in vitro and in vivo systems.     

Design and synthesis of ICT-based fluorescent probe for high-sensitivity protein detection and application to rapid protein staining for SDS-PAGE

A novel fluorescent molecular probe possessing styryl, sulfonyl, and cyanopyranyl moieties that was termed compound 1 was designed and synthesized to detect proteins through noncovalent bonding. Compound 1 did not produce fluorescence emission in the absence of proteins. However, its fluorescence spectrum showed a dramatic increase in the fluorescence intensity and strong orange emission after the addition of BSA. These changes were caused by intramolecular charge transfer (ICT). The fluorescence intensities of compound 1 were plotted as a function of the protein concentrations. A good linear relationship was observed up to a protein concentration of 325 mug/mL, and the detection limit was 70 ng/mL under the given assay conditions; this detection limit was higher than that of previously reported compounds. To demonstrate the application of compound 1, proteins in an SDS-PAGE gel were stained with compound 1 and were successfully imaged with a higher sensitivity and shorter staining operation time as compared to those of the silver staining method and SYPRO Ruby staining method. Thus, easy and high-sensitivity protein detection can be performed with the fluorescent probe, and this probe is ideally suited to proteomic applications.     

Fluorescence lifetime and rotational correlation time of bovine serum albumin-sodium dodecyl sulfate complex labeled with 1-dimethylaminonaphthalene-5-sulfonyl chloride: Effect of disulfide bridges in the protein on these fluorescence parameters

A fluorescent dye, 1-dimethylaminonaphthalene-5-sulfonyl chloride, was used to label bovine serum albumin (BSA), intact and disulfide bridges-cleaved. The fluorescence lifetime and fluorescence anisotropy of the adducts in sodium dodecyl sulfate (SDS) solutions were studied by the nanosecond fluorescence depolarization method. The volume of equivalent sphere (V _e) was calculated to be 2.1×10^–19 cm³ for BSA with the intact disulfide bridges from the rotational correlation time. The value ofV _e was 4.4×10^–19 cm³ for the disulfide bridges-cleaved BSA. With an increase in SDS concentration, the rotational correlation time of the intact BSA became longer, while that of the disulfide bridges-cleaved BSA became shorter. This suggests that upon the binding of SDS, the total volume of the intact BSA increases while the expanded state of the protein, caused by the cleavage of the disulfide bridges, becomes compact.     

Role of hydrophobic and polar interactions for BSA-amphiphile composites

To evaluate the role of hydrophobic and electrostatic or other polar interactions for protein–ligand binding, we have studied the interactions of bovine serum albumin (BSA) with 2-alkylmalonic acid and 2-alkylbenzimidazole amphiphiles having different head group and alkyl chain length. The binding affinity for the protein–amphiphile interactions is found to depend predominantly on the length of hydrocarbon chain, suggesting the crucial role of hydrophobic forces, supported by polar interactions at the protein surface. The BSA fluorescence exhibits appreciable hypsochromic shift along with a reduction in fluorescence intensity and mean lifetime upon binding with 2-alkylmalonic acid. UV–visible, steady state and time-resolved fluorescence measurements were performed to compare the effects of amphiphiles on BSA as a function of the amphiphiles head group and alkyl chain length.     

Inhibition of amyloid aggregation of bovine serum albumin by sodium dodecyl sulfate at submicellar concentrations

Sodium dodecyl sulfate (SDS), as an anionic surfactant, can induce protein conformational changes. Recent investigations demonstrated different effects of SDS on protein amyloid aggregation. In the present study, the effect of SDS on amyloid aggregation of bovine serum albumin (BSA) was evaluated. BSA transformed to β-sheet-rich amyloid aggregates upon incubation at pH 7.4 and 65°C, as demonstrated by thioflavin T fluorescence, circular dichroism, and transmission electron microscopy. SDS at submicellar concentrations inhibited BSA amyloid aggregation with IC₅₀ of 47.5 μM. The inhibitory effects of structural analogs of SDS on amyloid aggregation of BSA were determined to explore the structure–activity relationship, with results suggesting that both anionic and alkyl moieties of SDS were critical, and that an alkyl moiety with chain length ≥10 carbon atoms was essential to amyloid inhibition. We attributed the inhibitory effect of SDS on BSA amyloid aggregation to interactions between the detergent molecule and the fatty acid binding sites on BSA. The bound SDS stabilized BSA, thereby inhibiting protein transformation to amyloid aggregates. This study reports for the first time that the inhibitory effect of SDS on albumin fibrillation is closely related to its alkyl structure. Moreover, the specific binding of SDS to albumin is the main driving force in amyloid inhibition. This study not only provides fresh insight into the role of SDS in amyloid aggregation of serum albumin, but also suggests rational design of novel antiamyloidogenic reagents based on specific-binding ligands.     

Sensitive determination of protein based on the fluorescence enhancement effect of terbium (III)–epinephrine–protein–sodium dodecylsulfate system

It was found that the fluorescence of Tb³⁺–epinephrine (E) complex can be enhanced by both bovine serum albumin (BSA) and sodium dodecylsulfate (SDS), and stabilized by ascorbic acid (AA). It is considered that the fluorescence enhancement of the Tb³⁺–E–BSA–AA–SDS system originates not only from the hydrophobic microenvironment provided by BSA–SDS, but also from the energy transfer from BSA to Tb³⁺ in this system. Therefore, a new fluorescence method for the determination of protein concentrations as low as 1.3 × 10^?9 g mL^?1 BSA is established using Tb³⁺–epinephrine complex as probe. The method has been applied for the determination of BSA and human serum albumin in actual samples, and the results obtained are satisfactory. Compared with other fluorescence methods, this method is simpler and more sensitive for the determination of protein. The mechanism of the fluorescence enhancement of the system is studied in detail. Copyright © 2009 John Wiley & Sons, Ltd.     

Interaction of bovine (BSA) and human (HSA) serum albumins with ionic surfactants: spectroscopy and modelling

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-3-ammonium-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+/-0.1)x10(4) M(-1), (3.2+/-0.1)x10(3) M(-1) and (2.1+/-0.1)x10(3) M(-1) 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 W131 residue. It is clear that the binding site in BSA which is very close, in contact with tryptophan W131, 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 W214 residue in the intact protein caused by nearby residues in the vicinity of this tryptophan.     

A novel amphiphilic thiosemicarbazone derivative for binding and selective sensing of human serum albumin

The interaction of ligands and drug molecules with protein is of major interest in drug pharmacokinetics and pharmacodynamics. In this study, we synthesized a novel thiosemicarbazone‐based amphiphilic molecule for selective binding and detection of human serum albumin (HSA) with significant increase in fluorescence intensity. The compound 5‐(octyloxy) naphthalene substituted salicylaldehyde thiosemicarbazone was designed to interact with site I of HSA. The weak fluorescence of the probes in aqueous solution showed a dramatic increase in fluorescence intensity upon binding with HSA, while the responses to various other proteins and enzymes were negligible under similar experimental conditions. Changes in fluorescence intensity and formation of a new emission maximum of the compound in the presence of HSA as well as an increase in steady‐state anisotropy values reflected well the nature of binding and location of the probe inside the protein environment. Copyright © 2012 John Wiley & Sons, Ltd.     

Fluorescence probe properties of intramolecular charge transfer diphenylbutadienes in micelles and vesicles

This paper reports absorption and fluorescence spectral studies of methyl 4-[(1E,3E)-4-phenylbuta-1,3-dienyl]benzoate (1), N,N-dimethyl-N-[4-[(1E,3E)-4-phenylbuta-1,3-dienyl]phenyl]amine (2), methyl 4-[(1E,3E)-4-[4-(dimethylamino)phenyl]buta-1,3-dienyl]benzoate (3) and 1-methyl-4-[(1E,3E)-4-[4-methoxyphenyl]buta-1,3-dienyl]benzoate (4) in homogeneous media of 1,4-dioxane and 1,4-dioxane-water binary mixtures, and in microheterogeneous media of cetyl trimethyl ammonium bromide (CTAB), sodium dodecyl sulfate (SDS) and Triton-X-100 micelles, and dipalmotoyl phosphatidylcholine (DPPC) vesicles. The binding site of the diene probes in micelles and vesicles has been determined and it has been found that while in micelles dienes occupy the polar interfacial regions, in vesicles the probes are located deep inside the hydrophobic bilayer. The binding of dienes to the vesicles is stronger than their binding to the micelles as indicated by the binding constant values. The fluorescence emission of the probe dienes in micelles is from a conformationally relaxed intramolecular charge transfer excited state. However, in vesicles, since the excited state conformational motions are restricted due to the rigidity of the alkyl chain, the dienes fluoresce from their planar locally excited states.     

Probing protein-surfactant interaction by steady state and time-resolved fluorescence spectroscopy

The microenvironment of the probe coumarin 153 (C-153) in 1% bovine serum albumin (BSA) is more hydrophobic in nature compared to that in pure micelles or protein-surfactant complexes. In the native state of protein, we have not observed any solvation using C-153 as a probe but we have observed a slow dynamics on protein surface using 8-anilino-1-naphthalenesulfonic acid (ANS) as a probe. This may be due to the location of the probe (C-153) in the hydrophobic, solvent-inaccessible pocket of the BSA. Solvation dynamics in the BSA-surfactant (SDS) complexes in the solution phase is markedly different from that in pure micelles. This is may be due to the formation of 'necklace and bead' structure in the complexes. The rotational motion is also severely hindered in the surface of the protein.     

Conformational alteration in serum albumin as a carrier for pyridoxal phosphate: a distinction from pyridoxal phosphate-dependent glutamate decarboxylase

The conformation of bovine serum albumin (BSA), a pyridoxal phosphate (pyridoxal-P) carrier, was investigated by using uv/visible spectrophotometry, fluorescence spectroscopy, circular dichroism, and differential scanning microcalorimetry. Upon interacting with pyridoxal-P, the uv/visible absorption spectrum of BSA exhibits peaks at 330 and 392 nm due to the formation of a Schiff base. Pyridoxal-P quenches the fluorescence emission intensity (excited at 295 or 280 nm) by 24% and enhances fluorescence steady-state polarization of BSA by 20%. These observations suggest a conformational change in BSA when it interacts with pyridoxal-P. However, this conformational change appears to be small since circular dichroism showed only a 2-4% decrease in the alpha-helical content of BSA and no change in the beta-sheet content, and differential scanning microcalorimetry yielded only a 10% change in the enthalpy of thermal unfolding of BSA. 2-Aminoethylisothiouronium bromide, an antioxidant, causes no effect on either uv/visible absorption spectrum or fluorescence emission intensity of BSA, suggesting that BSA lacks sensitive sulfhydryl groups. To help in understanding BSA as a carrier for pyridoxal-P, the results were compared with those for glutamate decarboxylase (GAD), a pyridoxal-P-dependent protein, which requires pyridoxal-P as the cofactor for activity. Although BSA and GAD exhibit comparable molecular weights (66430 versus 65300), numbers of amino acid residues (582 versus 585), and binding affinity (>10(6) M-1), distinct conformational alterations occur between the two proteins upon interacting with pyridoxal-P: a small conformational change for BSA versus a large conformational change for GAD. In contrast to the case of BSA, AET causes significant effects on both the uv/visible spectrum and fluorescence emission intensity of GAD, because GAD contains sensitive sulfhydryl groups. Factors such as disulfide bond and active site sequence were discussed to understand BAS as a carrier for pyridoxal-P and a pyridoxal-P-independent protein.     

Biochemical evaluation of a synthesized isoflavone-selenium complex by molecular spectra

A coordination compound of 5, 7-dihydrox-4'-methoxyisoflavone and selenium was synthesized and its structure was identified by IR, LC-MS and (1)H-NMR. Its biochemical effects were investigated using bovine serum albumin (BSA) as a target protein molecule, in which process three-dimensional (3D) fluorescence spectra, ultraviolet spectra, circular dichroism (CD) spectra and fluorescence probe techniques were employed. The interaction of SEIF and BSA was discussed by fluorescence quenching method and F?rster non-radiation energy transfer theory. The thermodynamic parameters ΔH (θ), ΔG (θ), ΔS (θ) at different temperatures were calculated according to Van't Hoff isobaric equation and the results indicated the interaction was an exothermic as well as a spontaneous process. The binding site was explored by fluorescence probe method using warfarin and ibuprofen as markers. Intramolecular forces which are responsible for maintaining the binding were mainly hydrogen bond and van der Waals power. The average distance from the tryptophan residue in domain II of BSA (donor) to SEIF (acceptor) is 3.57 nm at body temperature. The conformation changes of BSA were investigated by 3D fluorescence and CD spectra.     

Analysis of heterogeneous fluorescence decays in proteins. Using fluorescence lifetime of 8-anilino-1-naphthalenesulfonate to probe apomyoglobin unfolding at equilibrium

The solvatochromic fluorescent dye 8-anilino-1-naphthalenesulfonate (ANS) is one of the popular probes of protein folding. Folding kinetics is tracked with ANS fluorescence intensity, usually interpreted as a reflection of protein structure-the hydrophobicity of the binding environments. Such simplistic view overlooks the complicated nature of ANS-protein complexes: the fluorescence characteristics are convoluted results of the ground state populational distribution of the probe-protein complex, the structural changes in the protein and the excited state photophysics of the probe. Understanding of the interplay of these aspects is crucial in accurate interpretation of the protein dynamics. In this work, the fluorescence decay of ANS complexed with apomyoglobin at different conformations denatured by pH is modeled. The fluorescence decay of the ANS-apomyoglobin complex contains information on not only apomyoglobin structure but also molecular populational distributions. The challenge in modeling fluorescence decay profiles originates from the convolution of heterogeneous binding and excited-state relaxation of the fluorescent probe. We analyzed frequency-domain fluorescence lifetime data of ANS-apomyoglobin with both maximum entropy methods (MEM) and nonlinear least squares methods (NLLS). MEM recovers a model of two expanding-and-merging lifetime distributions for ANS-apomyoglobin in the equilibrium transition from the native (N) through an intermediate (I-1) to the acid-unfolded state U(A). At pH 6.5 and above, when apomyoglobin is mostly populated at the N-state, ANS-apomyoglobin emits a predominant long-lifetime fluorescence from a relaxed charge transfer state S(1,CT) of ANS, and a short-lifetime fluorescence that is mainly from a nascent excited-state S(1,np) of ANS stabilized by the strong ANS-apomyoglobin interaction. Lowering the pH diminishes the contribution from the S(1,np) state. Meanwhile, more protein molecules become populated at the U(A) state, which exhibits a short lifetime that is not distinguishable from the S(1,np) state. At pH 3.4, when the population of the U(A) becomes significant, the short-lifetime fluorescence comes predominantly from ANS binding to the U(A). Further lowering the pH leads to more exposure of the bound ANS. The long lifetime shifts toward and finally merges with the short lifetime and becomes one broad distribution that stands for ANS binding to the U(A) below pH 2.4. The above expanding-and-merging model is consistent with F-statistic analysis of NLLS models. The consistency of this model with the knowledge from the literature, as well as the continuity of the decay parameters changing upon experimental conditions are also crucial in drawing the conclusions.     

Molecular mechanism of polyethylene glycol mediated stabilization of protein

The effect of different molar ratios of polyethylene glycol (PEG) on the conformational stability of protein, bovine serum albumin (BSA), was studied. The binding of PEG with BSA was observed by fluorescence spectroscopy by measuring the fluorescence intensity after displacement of PEG with chromophore ANS and had further been confirmed by measuring the intrinsic fluorescence of tryptophan residues of BSA. Co-lyophilization of BSA with PEG at optimum BSA:PEG molar ratio led to the formation of the stable protein particles. Circular dichroism (CD) spectroscopy study suggested that a conformational change had occurred in the protein after PEG interaction and demonstrated the highest stability of protein at the optimum BSA:PEG molar ratio of 1:0.75. Additional differential scanning calorimetry (DSC) study suggested strong binding of PEG to protein leading to thermal stability at optimum molar ratio. Molecular mechanism operating behind the polyethylene glycol (PEG) mediated stabilization of the protein suggested that strong physical adsorption of PEG on the hydrophobic core of the protein (BSA) along with surface adsorption led to the stability of protein.     

Complexation of beta-lactam antibiotic drug carbenicillin to bovine serum albumin: energetics and conformational studies

Binding of the antibiotic drug carbenicillin to bovine serum albumin (BSA) has been studied using isothermal titration calorimetry (ITC) in combination with fluorescence and circular dichroism (CD) spectroscopies. The thermodynamic parameters of binding have been evaluated as a function of temperature, ionic strength, and in the presence of anionic, cationic and nonionic surfactants, tetrabutylammonium bromide, and sucrose. The values of van't Hoff enthalpy do not agree with the calorimetric enthalpy indicating conformational changes in the protein upon drug binding. These observations are supported by the intrinsic fluorescence and CD spectroscopic measurements. A reduction in the binding affinity of carbenicillin to BSA is observed with increase in ionic strength of the solution, thereby suggesting, prevailing of electrostatic interactions in the binding process. The involvement of hydrophobic interactions in the binding of the drug to the protein is also indicated by a slight reduction in binding constant in the presence of tetrabutylammonium bromide. The experiments in the presence of sucrose suggest that hydrogen bonding is perhaps not dominant in the binding. The anionic surfactant sodium dodecyl sulphate (SDS) is observed to completely interfere in the ionic interactions in addition to its partial denaturing capacity. However, the presence of cationic surfactant hexadecyl trimethylammonium bromide (HTAB) and nonionic surfactant Triton-X 100 induce a slight reduction in the values of binding affinity. These calorimetric and spectroscopic results, provide quantitative information on the binding of carbenicillin to BSA and suggests that the binding is dominated by electrostatic interactions with contribution from hydrophobic interactions. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 831-840, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.