Spectroscopic investigation of the interaction of the toxicant, 2-naphthylamine, with bovine serum albumin

The mechanism of interaction between bovine serum albumin (BSA) and 2-naphthylamine (2-NA) in aqueous solution was investigated by fluorescence spectroscopy, circular dichroism (CD) spectra, and UV-vis spectroscopy. It was proved from fluorescence spectra that the fluorescence quenching of BSA by 2-NA was a result of the formation of complex between 2-NA and BSA, and the binding constants (K(a) ) as well as the numbers of binding sites for 2-NA in BSA were determined according to the modified Stern-Volmer equation. The results of synchronous fluorescence and CD spectra demonstrated 2-NA could decrease the amount of α-helix of BSA, leading to the loosening of protein skeleton. UV-vis spectroscopy and resonance light scattering spectra (RLS) results also suggested the conformation of BSA were changed and the BSA aggregation occured, which could induce toxic effects on the organism.     

Nonenzymatic glycation of bovine serum albumin by fructose (fructation). Comparison with the Maillard reaction initiated by glucose

Nonenzymatic glycation by glucose (glucation) was compared with glycation by fructose (fructation). The rate and extent of protein-bound fluorescence generation upon fructation was about 10 times that upon glucation. In contrast, nonenzymatically glucated bovine serum albumin (BSA) released about twice as much formaldehyde upon periodate oxidation as did nonenzymatically fructated BSA. However, the rate of blocking of amino groups was similar in both proteins. Periodate oxidation of borohydride-reduced glycated BSA led to regeneration of amino groups with preservation of fluorescence. From the ratio between the decrease in formaldehyde-releasing ability and the regenerated amino groups, formaldehyde molar yields of 0.47 and 0.8 were computed for fructose- and glucose-derived Amadori groups, respectively. This is consistent with participation of both carbon 1 and carbon 3 in the Amadori rearrangement from fructose. The formaldehyde releasing ability of nonenzymatically fructated BSA attains asymptotic maximum values earlier than that of nonenzymatically glucated BSA. Thus, the higher rate of fluorescence generation in nonenzymatically fructated BSA could be explained by a faster conversion of its Amadori groups. Since fluorescence generation through the Maillard reaction has been correlated with long term complications of diabetes mellitus, the participation of nonenzymatic fructation in this pathological state deserves further exploration. This is especially relevant in tissues where fructose levels increase in diabetes as a result of the operation of the sorbitol pathway.     

Preparation and in vitro photodynamic activity of novel silicon(IV) phthalocyanines conjugated to serum albumins

The interactions of four novel silicon(IV) phthalocyanines (SiPc), namely SiPc[OC(3)H(5)(NMe(2))(2)](2) (1), SiPc[OC(3)H(5)(NMe(2))(2)](OMe) (2), {SiPc[OC(3)H(5)(NMe(3))(2)](2)}I(4) (3), and {SiPc[OC(3)H(5)(NMe(3))(2)](OMe)}I(2) (4) with human serum albumin (HSA), bovine serum albumin (BSA), and maleylated bovine serum albumin (mBSA) were studied by fluorescence spectroscopy. The fluorescence emission of the serum albumins was effectively quenched by these phthalocyanines mainly through a static quenching mechanism. The higher Stern-Volmer quenching constants for the unsymmetrically substituted phthalocyanines 2 and 4 suggested that they have a stronger interaction with these proteins than the symmetrically substituted analogues 1 and 3. A series of non-covalent BSA or mBSA conjugates of these phthalocyanines were also prepared and evaluated for their in vitro photodynamic activity against HepG2 human hepatocarcinoma cells. The bioconjugation could enhance the photocytotoxicity of 1 and 4 by up to eight folds, but the effects on 2 and 3 were negligible. The results could be partly explained by two counter-balancing effects, namely the enhanced uptake and increased aggregation tendency of phthalocyanine due to BSA conjugation. As shown by absorption spectroscopy, the tetracationic phthalocyanine 3 was significantly aggregated in the protein cavity and its photocytotoxicity remained the lowest among the four photosensitizers.     

Spectroscopic investigation on the interaction of Cr(VI) with bovine serum albumin

The interaction of potassium dichromate (Cr(VI)) with bovine serum albumin (BSA) was investigated by fluorescence, synchronous fluorescence, resonance light scattering (RLS), ultraviolet-visible absorption, and circular dichroism (CD) spectroscopies under simulated physiological conditions. The experimental results showed that Cr(VI) could quench the intrinsic fluorescence of BSA following a static quenching process, which indicates the formation of a Cr(VI)-BSA complex. The binding constant (KA) and binding site (n) were measured at different temperatures. The spectroscopic results also revealed that the binding of Cr(VI) to BSA can lead to the loosening of the protein conformation and can change the microenvironment and skeleton of BSA.     

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.     

Spectroscopic study of the interaction between lycopene and bovine serum albumin

The interaction of lycopene with bovine serum albumin (BSA) in aqueous solution was studied by fluorescence quenching, three‐dimensional fluorescence and circular dichroism spectroscopy. The data showed that the fluorescence of BSA was quenched by lycopene at different temperatures through a dynamic mechanism. The evaluation of three‐dimensional fluorescence spectra revealed a conformational modification of BSA induced by coupling with lycopene and an increase in protein diameter as a consequence of the ligand–protein interaction. Moreover, the information obtained from evaluation of the effect of lycopene on BSA conformation by circular dichroism strongly supported the existence of a slight unfolding of BSA induced by coupling to lycopene. Copyright © 2012 John Wiley & Sons, Ltd.     

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.     

Toxic effects of ethanol on bovine serum albumin

The toxic effects of ethanol on bovine serum albumin (BSA) were measured by resonance light scattering (RLS), fluorescence spectroscopy, ultraviolet spectrophotometry (UV), circular dichroism (CD), and transmission electron microscopy (TEM). The results indicated that ethanol had toxic effects on BSA, which led to protein denaturation and the effects increased with the ethanol dose. By means of RLS, BSA was found to aggregate in the presence of ethanol and particles smaller than 100 nm were observed from TEM. The fluorescence spectra showed that the intensity of the characteristic peak of BSA decreased and blue shifted, because of changes in the BSA skeleton structure, as well as alteration of the microenvironment of tryptophan (Trp) residues. The conformation changes of BSA were also shown by UV and CD spectrometry. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:66–71, 2010; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20314     

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.     

Fluorescence resonance energy transfer between bovine serum albumin and fluoresceinamine

Physical binding‐mediated organic dye direct‐labelling of proteins could be a promising technology for bio‐nanomedical applications. Upon binding, it was found that fluorescence resonance energy transfer (FRET) occurred between donor bovine serum albumin (BSA; an amphiphilic protein) and acceptor fluoresceinamine (FA; a hydrophobic fluorophore), which could explain fluorescence quenching found for BSA. FRET efficiency and the distance between FA and BSA tryptophan residues were determined to 17% and 2.29 nm, respectively. Using a spectroscopic superimposition method, the saturated number of FAs that bound to BSA was determined as eight to give a complex formula of FA₈–BSA. Finally, molecular docking between BSA and FA was conducted, and conformational change that occurred in BSA upon binding to FA molecules was also studied by three‐dimensional fluorescence microscopy. Copyright © 2015 John Wiley & Sons, Ltd.     

On the contemporaneous, reversible interaction of different ligands with serum albumins in dilute aqueous solutions: Fluorescein and phenylbutazone

The binding affinity of fluorescein and of phenylbutazone to human serum albumin (HSA) and to bovine serum albumin (BSA), respectively, as well as of the two drugs together to each protein in dilute aqueous solution has been studied by means of gel permeation chromatography, circular dichroism, U.V. absorption and fluorescence spectroscopy. Identity of and/or interdependence between primary binding sites for the two ligands considered on HSA and BSA are evidenced and correlated with a simple theoretical approach to mixed drugs binding.     

Interaction of loratadine with serum albumins studied by fluorescence quenching method

The interactions between loratadine and bovine serum albumin (BSA) and human serum albumin (HSA) were studied using tryptophan fluorescence quenching method. The fluorescence intensity of the two serum albumins could be quenched 70% at the molar ratio [loratadine]:[BSA (or HSA)]=10:1. In the linear range (0-50 micromol L(-1)) quenching constants were calculated using Stern-Volmer equation. Temperature in the range 298 K-310 K had a significant effect (p<0.05) on the two serum albumins through ANOVA analysis and t-test. Furthermore the conformation changes in the interactions were studied using FTIR spectroscopy.     

Interactions between 1-benzoyl-4-p-chlorophenyl thiosemicarbazide and serum albumin: investigation by fluorescence spectroscopy

The interactions between 1-benzoyl-4-p-chlorphenyl thiosemicarbazide (BCPT) and bovine serum albumin (BSA) or human serum albumin (HSA) have been studied by fluorescence spectroscopy. By the analysis of fluorescence spectrum and fluorescence intensity, it was showed that BCPT has a strong ability to quench the intrinsic fluorescence of both bovine serum albumin and human serum albumin through a static quenching procedure. The binding constants of BCPT with BSA or HSA were determined at different temperatures based on the fluorescence quenching results. The binding sites were obtained and the binding force were suggested to be mainly hydrophobic. The effect of common ions on the binding constants was also investigated. A new fluorescence spectroscopy assay of the proteins is presented. The linear range is 5.36-67.0 microg mL(-1) with recovery of 101.1% for BSA, and the linear range is 8.28-144.9 microg mL(-1) with recovery of 102.6% for HSA. Determination of the proteins in bovine serum or in human serum by this method gives results which are very close to those obtained by using Coomassie Brilliant Blue G-250 colorimetry. A practical method was proposed for the determination of BCPT in human serum samples.     

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.     

Combined multispectroscopic and molecular docking investigation on the interaction between delphinidin‐3‐O‐glucoside and bovine serum albumin

Anthocyanin is one of the flavonoid phytopigments with specific health benefits. The interaction between delphinidin‐3‐O‐glucoside (D3G) and bovine serum albumin (BSA) was investigated by fluorescence spectroscopy, synchronous fluorescence spectroscopy, three‐dimensional fluorescence spectroscopy, ultraviolet‐visible absorption spectroscopy, circular dichroism spectroscopy and molecular modeling. D3G effectively quenched the intrinsic fluorescence of BSA via static quenching. The number of binding sites and binding constant K_a were determined, and the hydrogen bonds and van der Waals forces played major roles in stabilizing the D3G–BSA complex. The distance r between donor and acceptor was obtained as 2.81 nm according to Förster's theory. In addition, the effects of pH and metal ions on the binding constants were discussed. The results studied by synchronous fluorescence, three‐dimensional fluorescence and circular dichroism experiments indicated that the secondary structures of the protein has been changed by the addition of D3G and the α‐helix content of BSA decreased (from 56.1% to 52.4%). Furthermore, the study of site marker competitive experiments and molecular modeling indicated that D3G could bind to site I of BSA, which was in the large hydrophobic cavity of subdomain IIA. Copyright © 2014 John Wiley & Sons, Ltd.     

The influence of several nutritional supplements on the rational use of cabozantinib

To promote the rational use of cabozantinib (CBZ), this paper studied the influence of several nutritional supplements on the interaction between CBZ and bovine serum albumin (BSA), an appropriate alternative model for human serum albumin (HSA) that is one of the important transporter proteins in plasma, by fluorescence spectroscopy and UV–vis spectroscopy. The results showed that CBZ could quench the fluorescence of BSA via a dynamic–static quenching process, and the six nutritional supplements did not change the quenching mode of BSA by CBZ. However, all of them could reduce the binding constant of the CBZ–BSA system at 293 K and increase the polarity around tryptophan residues. Among them, nicotinamide and vitamin B₁₂ (VB₁₂) had a greater effect on the binding constants of the CBZ–BSA system. In the meantime, the thermodynamic parameters of the CBZ–BSA system were examined, indicating that the interaction of CBZ with BSA was spontaneous and dominated by hydrophobic forces. Further research discovered that the combining of CBZ with BSA was primarily located within Site I of BSA, and the binding distance r was 2.48 nm. Consequently, while taking CBZ, patients should use VB₁₂ and nicotinamide carefully, which may interfere with the transport of drugs.     

Improvement of the acridine orange-protein-surfactant system for protein estimation based on aromatic ring stacking effect of sodium dodecyl benzene sulphonate.

The fluorescence of acridine orange (AO) is greatly quenched by the anionic surfactant sodium dodecyl benzene sulphonate (SDBS), but when protein is added into the AO-SDBS system, the fluorescence intensity of the latter is enhanced again. Based on this, a new fluorimetric method of determination of protein was developed. Under optimum conditions, the enhanced intensity of fluorescence is in proportion to the concentration of protein, such as bovine serum albumin (BSA), human serum albumin (HSA) and egg albumin (EA), over a wide range with detection limits at the 10(-9) g/mL level. This method has been satisfactorily used for the determination of protein in samples. We compared results using 280 nm and 490 nm excitation wavelengths and the mechanism of the assay.     

Effects of alcohol on the solubility and structure of native and disulfide-modified bovine serum albumin

Differential precipitation of human plasma by ethanol is one of the most important processes for purifying therapeutic proteins, including human serum albumin. Better understanding of the effects of ethanol on the structure and stability of proteins is critical for effective and safe application of ethanol-induced protein precipitation. Here, we examined the effects of ethanol on the structure and solubility of bovine serum albumin (BSA) and SH-modified BSA. Ethanol caused BSA denaturation in a bimodal fashion, i.e., reduction of α-helix at low concentration and subsequent induction of the α-helical structure at higher concentration. In contrast, the solubility of BSA decreased monotonically. The secondary structure of SH-modified BSA was different from that of native BSA. Ethanol resulted in enhanced secondary structures of SH-modified BSA and decreased solubility monotonically. These results suggest the favorable interaction of ethanol with hydrophobic residues, leading to protein denaturation, but the unfavorable interaction with charged residues, leading to a reduction of protein solubility.     

Design and synthesis of the BODIPY–BSA complex for biological applications

A quinoxaline‐functionalized styryl–BODIPY derivative (S1) was synthesized by microwave‐assisted Knoevenagel condensation. It exhibited fluorescence enhancement upon micro‐encapsulation into the hydrophobic cavity of bovine serum albumin (BSA). The S1–BSA complex was characterized systematically using ultraviolet (UV)–visible absorption, fluorescence emission, kinetics, circular dichroism and time‐resolved lifetime measurements. The binding nature of BSA towards S1 was strong, and was found to be stable over a period of days. The studies showed that the S1–BSA complex could be used as a new biomaterial for fluorescence‐based high‐throughput assay for kinase enzymes.     

Functionalization of a protein surface with per-O-methylated β-cyclodextrin

Per-O-methylated β-cyclodextrin (CD) bearing an iodoacetamide group at the 6-position was synthesized to functionalize protein surfaces. Bovine serum albumin (BSA) was quantitatively modified with the CD derivative by the S(N) 2 reaction of iodoacetamide with a cysteine residue (Cys34) on the BSA surface. The resultant CD-functionalized BSA (BSA-CD) spontaneously dimerized upon addition of an anionic tetraarylporphyrin (TPPS) through the supramolecular 1:2 complexation between TPPS and CD on the protein surface. The BSA-CD/TPPS complex further complexed with ferric protoporphyrin IX (hemin) in the hydrophobic pockets of albumin to form a hemin/BSA-CD/TPPS ternary complex in which static fluorescence quenching occurred owing to intramolecular electron transfer from the photoexcited TPPS to hemin.