Binding equilibrium of I- to serum albumin with resonance Rayleigh scattering

The binding equilibrium between l- and human serum albumin (HSA) or bovine serum albumin (BSA) has been studied by means of the resonance Rayleigh scattering (RRS) and equilibrium dialysis. It has been found for the first time that RRS and multiple frequency scattering (MFS) are enhanced as the l- binding to the HSA and BSA, but fluorescence quenches. The equilibrium dialysis results suggest that the binding of l- to HSA and BSA fits a phase-distribution model other than Scsitchard model, and that the order of magnitude of its phase-distribution constant was found to be 104. It is most probable that Cl~ or other anion ions influence the binding of P by changing the ionic strength in the solution. The dialysis at different pH indicates that the binding mechanism is due to the electrostatic forces between the T-and protonated basic amino-acid residues.     

Photophysics in motionally constrained bioenvironment: interaction of norharmane with bovine serum albumin

Steady-state photophysics of norharmane (NHM), a bioactive alkaloid, has been studied in the presence of a model transport protein, bovine serum albumin (BSA). The emission spectrum undergoes a remarkable change upon addition of BSA to the aqueous solution of NHM in buffer. Addition of BSA leads to a marked increase in the fluorescence anisotropy of the neutral species of NHM, although the fluorescence anisotropy for the cationic species is almost invariant to BSA addition, suggesting that the neutral species is located in a motionally restricted environment of BSA, whereas the cationic species remains in the bulk aqueous phase. The binding constant (K) and free energy change (DeltaG) for the probe-protein binding have been calculated from the fluorescence data. Light has been thrown on the action of urea on protein-bound NHM. The denaturation study suggests that the protein, in its native form, binds with NHM. Polarity of the microenvironment around the probe has been determined from a comparison of the fluorescence properties of the two prototropic species of NHM in water-dioxane mixture with varying composition.     

Fluorescence of 1,3-Di(1-pyrenyl)propane probe incorporated into human serum albumin protein enforced conformations of the probe

 Steady-state and time-resolved fluorescence spectra of 1,3-di(1-pyrenyl)propane (1Py-(3)-1Py) incorporated into macromolecules of human serum albumin (HSA), into micelles of dodecyltrimethylammo-nium chloride (DTAC), and dissolved in 1,4-dioxane were compared. The steady-state fluorescence spectra indicated that in all the mentioned environments, upon excitation of 1Py-(3)-1Py, light was emitted from the single pyrene chromophores (1Py^*) and from the 1Py, 1Py^* excimers. The time-resolved fluo-rescence emission registered at 480 nm (excimer emission) for 1Py-(3)-1Py in the DTAC micelles and dissolved in 1,4-dioxane allowed to monitor formation of excimer with time constant τ₁=40.0 ns and 9.6 ns, for 1Py-(3)-1Py in the DTAC micelles and in 1,4-dioxane, respectively. However, when the 1Py-(3)-1Py probe was located inside of the macromolecules of HSA, only the decay of emission was observed for excimer with our set-up (t>2 ns after excitation). The instantaneous formation of excimer, unrelated to the decay of monomer excitation, indicates that the considerable fraction of 1Py-(3)-1Py in the hydrophobic pockets of HSA is present as the ground state dimer. The red shift (Δλ=8 nm) and broadening of UV absorption for 1Py-(3)-1Py in HSA (when compared with absorption 1Py-(3)-1Py in 1,4-dioxane) and comparison of exci-tation spectra of 1Py-(3)-1Py in HSA and in 1,4-dioxane also indicate that label molecules bound to some sites of HSA are in the ground state in the dimer conformation. Moreover, the close values of the ratios of intensities of monomer emission to excimer emission, registered 2 ns (5 ns gate) after excitation pulse with duration 300 ps and at the steady-state conditions, indicate that the interconversion between conformers of 1Py-(3)-1Py inside of the macro-molecules of HSA is slow in comparison with the decay time of Py chromophore in the excited state in HSA (two-exponential decay with decay times τ₁=2.41 ns, τ₂=69.0 ns). Thus, ratios of the intensities of monomer and excimer emissions of 1Py-(3)-1Py in HSA do not allow to obtain any information on the local microfluidity inside of the protein macromolecules but could be used for discrimination between different conformations of the probe, possibly located in different protein pockets. Received: 29 April 1996 Accepted: 15 August 1996     

丝裂霉素C与牛血清蛋白结合作用的研究

结合光谱法与微量热法研究了水溶液中丝裂霉素C与牛血清白蛋白分子间的结合反应,测定了反应的结合常数K~A,结合位点数n及热力学函数△~rG~m,△~rH~m和△~rS~m,并确定了分子间作用力性质;依据Forster非辐射能量转移机制,确定了授体-受体间的结合距离和能量转移效率;采用同步荧光技术考察了丝裂霉素C对牛血清白蛋白构象的影响。     

The preparation of bovine serum albumin surface-imprinted superparamagnetic polymer with the assistance of basic functional monomer and its application for protein separation

Currently, small proteins imprinting are more reported since large proteins molecular imprinting faces challenge due to their bulk size and complex structure. In this work, bovine serum albumin (BSA) surface-imprinted magnetic polymer was successfully synthesized based on atomic transfer radical polymerization (ATRP) method in the presence of common monomer (N-isopropylacrylamide) with the assistant of basic functional monomer (N-[3-(dimethylamino)propyl]-methacrylamide), which provides a achievable attempt for imprinting larger target proteins based on the ATPR with the mild reaction conditions. The BSA-imprinted polymer exhibited higher adsorption capacity and selectivity to BSA over the non-imprinted polymer. Competitive adsorption tests indicated the BSA-imprinted polymer had better selective adsorption and recognition properties to BSA in the mixture. The obtained BSA-imprinted polymer was applied to bovine serum, which also showed selectivity to BSA. In addition, a conventional aqueous two-phase solution of PEG/sulphate was used as elution for adsorbed BSA, which was compared with common NaCl elution.     

光谱法研究噻菁染料与血清蛋白的相互作用

本文通过吸收和荧光光谱法研究了一种噻菁染料与人血清蛋白及牛血清蛋白的相互作用。吸收光谱数据表明,与血清蛋白结合后,噻菁染料单体的吸收峰发生红移,同时强度也有很大变化;还通过吸收光谱计算确定了噻菁染料与血清蛋白的结合位点数（ n ）。与人血清蛋白或牛血清蛋白结合后,噻菁染料的荧光量子产率增加。分析噻菁染料的荧光强度随溶液中血清蛋白浓度的变化得到了二者反应的表观结合常数（ K _a）和自由能变化（ ΔG ）。根据表观结合常数（ K _a）可以判断,人血清蛋白比牛血清蛋白与噻菁染料的结合更强。     

Interactions between protein and porphyrin-containing cyclodextrin supramolecular system: a fluorescent sensing approach for albumin

The interactions of meso-tetraphenylporphyrin (TPP), meso-tetraphenylporphyrin cobalt(ii) (CoTPP) and protein in the presence of a cyclodextrin derivative, heptakis(2,6-di-O-n-octyl)-beta-cyclodextrin (Oc-beta-CD), have been investigated. In the presence of Oc-beta-CD, significant increase of TPP fluorescence was realized, but the increased fluorescence was quenched by CoTPP. To further investigate the fluorescence-quenched system and explore its potential application in bioanalysis, a strategy has been devised to restore the quenching fluorescence of TPP upon interacting with protein. The restoration of TPP fluorescence in the present system is fast and accomplished upon interaction with bovine serum albumin (BSA) or human serum albumin (HSA). On the basis of the spectroscopic measurement and excited state fluorescence lifetime, the mechanism of TPP fluorescence quenching is attributed to formation of a ground-state complex of TPP and CoTPP, and the fluorescence restoration is attributed to the binding of CoTPP with the protein molecule which destroys the aggregate, releasing the free base porphyrin. With optimized conditions, the calibration equations are linear from 0.80 to 75.4 microg mL(-1) BSA and from 3.20 to 93.2 microg mL(-1) HSA. The corresponding detection limits are 0.32 microg mL(-1) for BSA and 1.06 microg mL(-1) for HSA, respectively. The method was used for the direct assay of HSA content in human serum. The result is comparable to that obtained by another method. The recovery from BSA in synthetic sample is also satisfactory.     

Fluorescence investigation of affinity interaction between bovine serum albumin and triazine dye in reversed micelles

The effect of Cibacron Blue 3GA (CB) on the fluorescence emission spectra of bovine serum albumin (BSA) was investigated in cationic reversed micelles formed with cetyltrimethylammonium bromide (CTAB) compared with that in aqueous phase. The anionic CB had electrostatic interactions with cationic CTAB and affinity interactions with BSA in the reversed micelles. The addition of CB in the reversed micellar phase led to a great decrease in the fluorescence intensity of BSA and a remarkable red shift of the wavelength of emission maximum (λ_max). The fluorescence intensity of BSA decreased and the λ_max decreased 5 nm (blue shift) without the addition of CB in the reversed micellar phase. The fluorescence intensity of BSA with the addition of CB had the strongest value in the aqueous phase with the presence of CTAB, a less strong value in the reversed micellar phase, and a weak value in the aqueous phase without the presence of CTAB. The increase in λ_max of BSA with the addition of CB in the reversed micellar phase might indicate the decrease in the hydrophobic microenvironment of the Trp residue of BSA, contrary to those microenvironments in the absence of CB.     

Bromocresol green as a new spectrophotometric probe for serum albumins

Bromocresol green (BCG) has been employed as a new spectrophotometric probe to characterise the binding regions of human serum albumin (HSA) and bovine serum albumin (BSA). BCG binds with greater affinity onto BSA than onto HSA. Based on the abilities of ligands Naproxen and l-anilino-8-naphthalenesulphonic acid (ANS) to displace BCG from the serum albumins by competitive or non-competitive mechanism, binding regions were identified for these ligands. It has been found that both Naproxen and ANS share common binding sites with BCG in HSA with the relative ability of Naproxen > ANS on binding to HSA. In the case of BSA, ANS competes with BCG for the same binding sites, whereas Naproxen exhibits non-competitive binding. The highaffinity sites of Naproxen coincide with BCG binding sites while the low-affinity sites occur at sites distinct from the BCG binding region.     

Probing three-dimensional structure of bovine serum albumin by chemical cross-linking and mass spectrometry

Serum albumin is the principal transporter of fatty acids that are otherwise insoluble in circulating plasma. While the crystal structure of human serum albumin (HSA) as well as its binding with fatty acids has been characterized, the three dimensional structure of bovine serum albumin (BSA) has not been determined although both albumins share 76% sequence homology. In this study we used mass spectrometry coupled with chemical cross-linking, to probe the tertiary structure of BSA. BSA was modified with lysine specific cross-linkers, bis(sulfosuccinimidyl) suberate (BS(3)), disuccinimidyl suberate (DSS) or disuccinimidyl glutarate (DSG), digested with trypsin and analyzed by tandem mass spectrometry. With O-18 labeling during the digestion, through-space cross-linked peptides were readily identified in mass spectra by a characteristic 8 Da shift. From the cross-linked peptides identified in this study, we found that 12 pairs of lysine residues were separated within 20 A, while 5 pairs were spaced between 20 and 24 A. The spatial distance constraints generated from five K-K pairs in BSA were consistent with the corresponding distance obtained from the crystal structure of HSA, although only six equivalent K-K pairs could be compared. According to our data, the distance between K235 of IIA and K374 of IIB domain in BSA was farther by 7-11 A than that expected from the crystal structure of HSA, suggesting structural differences between BSA and HSA in this region. The distance constraints obtained for lysine residues using various cross-linkers should be valuable in assisting the determination of the 3-D structure of BSA.     

18β-Glycyrrhetinic acid interaction with bovine serum albumin

The interaction of 18β-glycyrrhetinic acid (GA): The metabolite of glycyrrhizic acid which is the main active component of a commonly used traditional Chinese medicine (TCM) Glycyrrhiza Uralensis Fisch with bovine serum albumin (BSA) has been investigated. Fluorescence emission spectra of serum albumin in the presence of GA, recorded at the excitation wavelength 280 nm, clearly show that GA act as quencher and have different quenching mechanism at a pH below or above the isoelectric point (pI). The binding sites number n and apparent binding constant K were measured. The thermodynamic parameters ΔH°, ΔG°, ΔS° at different temperatures were calculated. The effects of some common metal ions on binding are considered. Synchronous fluorescence and UV–vis spectra were used to study protein conformation. Energy transfer between GA and HSA was calculated by Förster's theory and the binding site was suggested to be site II. The binding of monoammonium glycyrrhizinate (GL) to BSA is also compared.     

Binding interaction of gatifloxacin with bovine serum albumin.

The binding of gatifloxacin to bovine serum albumin (BSA) in aqueous solution was studied using fluorescence spectroscopy and absorbance spectra, Further, the interactions influenced by Fe3+ and Cu2+ were also explored in this work. Based on Scatchard's site-binding model and florescence quenching, practical formulas for small molecule ligands to bio-macromolecules have been proposed. The binding parameters were measured according to suggested models, and the binding distance and the transfer efficiency of energy between gatifloxacin and BSA were also obtained in view of the F?rster theory of non-radiation energy transfer. The effect of gatifloxacin on the conformation of BSA has also been analyzed using synchronous fluorescence spectroscopy.     

A novel lophine-based fluorescence probe and its binding to human serum albumin

The binding of a lophine-based fluorescence probe, 4-[4-(4-dimethylaminophenyl)-5-phenyl-1H-imidazol-2-yl]benzoic acid methyl ester (DAPIM) with human serum albumin (HSA) was investigated by fluorescence spectroscopy under physiological conditions. While DAPIM shows extreme low fluorescence in aqueous solution, DAPIM binding with HSA emits strong fluorescence at 510 nm. The binding constant and binding number determined by Scatchard plot was 3.65 × 10⁶ M⁻¹ and 1.07, respectively. Competitive binding between DAPIM and other ligands such as warfarin, valproic acid, diazepam and oleic acid, were also studied fluorometrically. The results indicated that the primary binding site of DAPIM to HSA is site II at subdomain IIIA. DAPIM can be a useful fluorescence probe for the characterization of drug-binding sites. In addition to the interaction study, because the fluorescence intensity of DAPIM increased in proportion to HSA concentration, its potential in HSA assay for serum sample was also evaluated.     

Fluorescence quenching of bovine serum albumin.

The fluorescence emission spectra and 3D fluorescence spectra of bovine serum albumin (BSA) in cetyltrimethylammonium bromide (CTAB) reversed micelles were affected by the microenvironment. Blue shifts of the fluorescence emission peaks were found when BSA was present in CTAB reversed micelles. The fluorescence intensity changed with the water content. Similar changes in the peak regions of the 3D fluorescence spectra were also observed. CdS nanoparticles prepared in CTAB reversed micelles quenched the fluorescence of BSA significantly. The fluorescence of BSA was more effectively quenched by negative CdS nanoparticles than by positive or neutral CdS ones. The quenching degree increased linearly with increasing the concentration of negative CdS nanoparticles over the range of 5.0 x 10(-6) - 3.0 x 10(-5) mol L(-1). The quenching mechanism is discussed and the quenching constant is 1.32 x 10(4) L mol(-1).     

Spectroscopic studies on the interaction between riboflavin and albumins

The interactions between riboflavin (RF) and human and bovine serum albumin (HSA and BSA) were studied by using absorption and fluorescence spectroscopic methods. Intrinsic fluorescence emission spectra of serum albumin in the presence of RF show that the endogenous photosensitizer acts as a quencher. The decrease of fluorescence intensity at about 350 nm is attributed to changes in the environment of the protein fluorophores caused by the ligand. The quenching mechanisms of albumins by RF were discussed. The binding constants and binding site number were obtained at various temperatures. The distance between albumins and RF in the complexes suggests that the primary binding site for RF is close to tryptophan residue (Trp214) of HSA and Trp212 of BSA. The hydration process of albumins has also been discussed.     

Study of the interaction of indirubin with bovine serum albumin

This study examined the interaction of indirubin with bovine serum albumin (BSA) at three temperatures (286, 297, 308 K) at pH 7.40. In the presence of indirubin, the drug-BSA binding mode, binding constant and the protein structure changes in aqueous solution were determined by fluorescence quenching methods including Fourier transform infrared (FT-IR) spectroscopy and UV-Vis spectroscopy. The FT-IR change indicates that indirubin binds to BSA. The change in protein secondary structure accompanying ligand binding has been proved by fluorescence spectra data. The thermodynamic parameters, the enthalpy change (DeltaH), and the entropy change (DeltaS) calculated by the van't Hoff equation possess small negative (-2.744 kJ.mol(-1)) and positive values (112.756 J.mol(-1).K(-1)), respectively, which indicated that hydrophobic interactions play the main role in the binding of indirubin to BSA. Furthermore, the displacement experiment shows that indirubin can bind to the subdomain IIA and the distance between the tryptophan residues in BSA and indirubin bound to site I was estimated to be 2.24 nm according to F?ster's equation on the basis of fluorescence energy transfer.     

头孢美唑钠与BSA及纳米银-BSA体系相互作用的研究

纳米银与牛血清白蛋白(BSA)均匀混合形成纳米银-BSA体系,运用荧光光谱,紫外吸收光谱,同步荧光光谱研究了注射用头孢美唑钠(Cefmetazole Sodium for Injection,CS)与BSA及纳米银-BSA体系的相互作用.头孢美唑钠对BSA具有荧光猝灭作用,其猝灭方式为静态猝灭,求出了猝灭常数,结合常数...     

Binding equilibrium study of phosphotungstic acid and HSA or BSA with UV spectrum, fluorescence spectrum and equilibrium dialysis

The binding equilibrium between phosphotungstic acid (H7[P(W2O7)6] · XH2O;PTA) and human serum albumin (HSA) or bovine serum albumin (BSA) has been studied by UV-Vis, fluorescence spectroscopies and equilibrium dialysis. It has been observed that UV absorption enhanced and the fluorescence quenched as the PTA binding to HSA or BSA at physiological pH 7.43(?.02). The Scatchard analysis indicated that there exists a strong binding site of PTA in both HSA and BSA, and the successive stability constants of these two systems are obtained by nonlinear least-squares methods fitting Bjerrum formula.     

硫唑嘌呤与牛血清白蛋白相互作用的热力学研究

用荧光光谱法和紫外-可见光谱法研究了在模拟人体生理条件下,硫唑嘌呤和牛血清白蛋白（BSA）结合反应的特征,发现硫唑嘌呤对BSA有较强的荧光猝灭作用,且硫唑嘌呤的紫外吸收光谱和BSA的荧光光谱有一定程度的重叠,由此可得出其作用距离和结合过程的基本热力学参数。     

Characterization of the interaction between a new merocyanine dye and bovine serum albumin

A new merocyanine dye was synthesized, and its acidity constant was determined by spectrophotometric and chemometrics methods. The interactions of the new cyanine dye with bovine serum albumin (BSA) have been studied by fluorescence and UV absorption spectroscopy at pH 7.40. A visual color change from red to blue was observed by addition of BSA to aqueous solution of the dye. The quenching constants and binding parameters (binding constants and number of binding sites) were determined at different temperatures. The calculated thermodynamic parameters confirmed that the binding reaction is mainly entropy-driven, whereas electrostatic interaction plays major role in the reaction. The displacement experiment confirmed binding of the dye to the subdomain IIA (site 1) of albumin. Moreover, synchronous fluorescence spectroscopy studies revealed the dye induces some local conformational change in BSA. The binding distance, r, between donor (serum albumin) and acceptor (dye) was obtained according to Förster’s theory.