Quenching of tryptophan fluorescence in various proteins by a series of small nickel complexes

A series of twelve anionic, cationic, and neutral nickel(II) complexes have been synthesized and characterized. The interaction of these complexes with bovine serum albumin (BSA), human serum albumin (HSA), lysozyme (Lyso), and tryptophan (Trp) has been studied using steady-state fluorescence spectroscopy. Dynamic and static quenching constants have been calculated, and the role played in quenching by the ligand and complex charge investigated. The nickel complexes showed selectivity towards the different proteins based on the environment surrounding the Trp residue(s). Only small neutral complexes with hydrophobic ligands effectively quenched protein fluorescence via static quenching, with association constants ranging from 10(2) M(-1) (free Trp) to 10(10) M(-1) (lysozyme), indicating a spontaneous and thermodynamically favorable interaction. The number of binding sites, on average, was determined to be one in BSA, HSA and free Trp, and two in lysozyme.     

Fluorescence spectroscopic studies on binding of a flavonoid antioxidant quercetin to serum albumins

Binding of quercetin to human serum albumin (HSA) was studied and the binding constant measured by following the red-shifted absorption spectrum of quercetin in the presence of HSA and the quenching of the intrinsic protein fluorescence in the presence of different concentrations of quercetin. Fluorescence lifetime measurements of HSA showed decrease in the average lifetimes indicating binding at a location, near the tryptophan moiety, and the possibility of fluorescence energy transfer between excited tryptophan and quercetin. Critical transfer distance (R _o ) was determined, from which the mean distance between tryptophan-214 in HSA and quercetin was calculated. The above studies were also carried out with bovine serum albumin (BSA).     

Femtosecond studies of tryptophan fluorescence dynamics in proteins: local solvation and electronic quenching

We report our systematic examination of tryptophan fluorescence dynamics in proteins with femtosecond resolution. Distinct patterns of femtosecond-resolved fluorescence transients from the blue to the red side of emission have been characterized to distinguish local ultrafast solvation and electronic quenching. It is shown that tryptophan is an ideal local optical probe for hydration dynamics and protein-water interactions as well as an excellent local molecular reporter for ultrafast electron transfer in proteins, as demonstrated by a series of biological systems, here in melittin, human serum albumin, and human thioredoxin, and at lipid interfaces. These studies clarify the assignments in the literature about the ultrafast solvation or quenching dynamics of tryptophan in proteins. We also report a new observation of solvation dynamics at far red-side emission when the relaxation of the local environment is slower than 1 ps. These results provide a molecular basis for using tryptophan as a local molecular probe for ultrafast protein dynamics in general.     

Photoprocesses of Xanthene Dyes Bound to Lysozyme or Serum Albumin

The ground and excited state processes of eosin, erythrosin and rose bengal in aqueous solution were studied in the presence of lysozyme or bovine serum albumin (BSA). Noncovalent protein-dye binding was analyzed by circular dichroism (CD), fluorescence and UV–Vis absorption spectroscopy. The effects of protein concentrations and pH were studied. Fluorescence quenching of the dye takes place due to binding to lysozyme and fluorescence enhancement due to low loading to BSA. The effects of proteins on the xanthene triplet state and its precursor were observed by time-resolved 530 nm photolysis. The triplet lifetime is quenched by lysozyme and prolonged by loading to BSA. Light-induced damages on both the dyes and proteins were observed under exclusion of oxygen. Photo-oxidation is efficient for lysozyme and lower for BSA. The CD signal of the eosin/BSA system is maximum at pH 4, where the photo-oxidation is minor.     

Fluorometric investigation of the interaction of bovine serum albumin with surfactants and 6-mercaptopurine

Fluorescence quenching in solutions of bovine serum albumin has been investigated in the presence of 6-mercaptopurine and ionic surfactants. Spectroscopic analysis of the emission quenching at different temperatures revealed that the quenching mechanism of bovine serum albumin by 6-mercaptopurine was dynamic quenching mechanism. The Stern–Volmer quenching model has been successfully applied, and the activation energy of the interaction between 6-mercaptopurine and bovine serum albumin as much as 4.26 kJ mol⁻¹ was calculated. The distance r between donor (bovine serum albumin) and acceptor (6-mercaptopurine) was obtained according to fluorescence resonance energy transfer (FRET). The result of synchronous fluorescence spectra shows that the conformation of bovine serum albumin has been changed at the present of 6-mercaptopurine.     

Fluorescent Probing of Urea-induced Chemical Unfolding of Bovine Serum Albumin by Intramolecular Charge Transfer Fluorescence Probe E-3-(4-Dimethylamino-Naphthalen-1-yl)-Acrylic Acid

Changes in polarity at the immediate binding site in protein bovine serum albumin (BSA) produces distinct changes in the solvent polarity-dependent emission band of fluorescence probe E-3-(4-dimethylamino-naphthalen-1-yl)-acrylic acid. Steady-state spectroscopy and time-resolved spectroscopy have been used to investigate this binding process. Attaching the probe to BSA and then monitoring its spectral changes with increasing urea concentration and raising temperature has also tracked the denaturation of BSA chemically and thermally. The polarity of the microenvironment was investigated employing the Reichardt E_T(30) scale. Fluorescence anisotropy, red edge excitation shifts and acrylamide-induced quenching of fluorescence have been exploited to gain better insight into this binding process.     

Flavin-sensitized Photo-oxidation of Lysozyme and Serum Albumin

The excited state processes of riboflavin, flavin mononucleotide and flavin adenine dinucleotide in argon-saturated aqueous solution were studied in the presence of lysozyme or bovine serum albumin (BSA). UV–Vis absorption and fluorescence spectroscopy indicates that the noncovalent flavin-protein binding is relatively weak. Quenching of the flavin triplet state by BSA, observed by time-resolved photolysis, is less efficient than by lysozyme. Light-induced oxidation of the two proteins and reduction of the three flavins were studied. The quantum yields of the former and latter in the absence of oxygen are up to 0.1 and 0.04, respectively. The effects of pH and sensitizer and protein concentrations were examined in greater detail. The proposed reaction is electron transfer from the tryptophan moiety to the flavin triplet rather than excited singlet state.     

光谱法研究喜树碱与牛血清白蛋白的相互作用

采用多种光谱技术对喜树碱和牛血清白蛋白的相互作用进行了研究.结果表明喜树碱和牛血清白蛋白可形成基态复合物,引起牛血清白蛋白内源荧光猝灭.通过计算获得了二者在不同温度下的结合常数及结合位点数.根据喜树碱和牛血清白蛋白结合的热力学参数,确定了二者之间主要为疏水作用力.根据F(o)rster非辐射能量转移理论确定了喜树碱和牛血清白蛋白的作用距离.同步荧光光谱显示喜树碱主要与蛋白中色氨酸残基发生相互作用,改变其周围的局部构象.红外光谱提示喜树碱可引起蛋白的构象发生改变,α-螺旋二级结构减少.     

Fluorescence resonance energy transfer from tryptophan in human serum albumin to a bioactive indoloquinolizine system

The interaction between a bioactive molecule, 3-acetyl-4-oxo-6,7-dihydro-12H indolo-[2,3-a] quinolizine (AODIQ), with human serum albumin (HSA) has been studied using steady-state absorption and fluorescence techniques. A 1:1 complex formation has been established and the binding constant (K) and free energy change for the process have been reported. The AODIQ-HSA complex results in fluorescence resonance energy transfer (FRET) from the tryptophan moiety of HSA to the probe. The critical energy-transfer distance (R ₀) for FRET and the Stern-Volmer constant (K _sv) for the fluorescence quenching of the donor in the presence of the acceptor have been determined. Importantly, K _SV has been shown to be equal to the binding constant itself, implying that the fluorescence quenching arises only from the FRET process. The study suggests that the donor and the acceptor are bound to the same protein at different locations but within the quenching distance.     

Chlorpromazine interactions to sera albumins. A study by the quenching of fluorescence

Binding of chlorpromazine (CPZ) and hemin (Hmn) to human (HSA) and bovine (BSA) serum albumin was studied by fluorescence quenching technique. Intrinsic fluorescences of BSA and HSA were measured by selectively exciting their tryptophan residues. Gradual quenching was observed by titration of both proteins with CPZ and Hmn. CPZ is a widely used anti-psychosis drug that causes severe side effects and strongly interacts with biomembranes, both in its lipidic and proteic regions. CPZ also interacts with blood components, influences bioavailability, and affects the function of several biomolecules. Albumin plays an important role in the transport and storage of hormones, ions, fatty acids and others substances, including CPZ, affecting the regulation of their plasmatic concentration. Hmn is an important ferric residue of hemoglobin that binds within the hydrophobic region of albumin with great specificity. Hmn added to HSA and BSA solutions at a molar ratio of 1:1 quenched about half of their fluorescence. Stern-Volmer plots obtained from experiments carried out at 25 and 35 degrees C showed the quenching of fluorescence of HSA and BSA by CPZ to be a collisional phenomenon. Hmn quenches fluorescence by a static process, which specifically indicates the formation of a complex. Our results suggest the prime binding site for CPZ and Hmn on both HSA and BSA to be near tryptophan residues.     

Probing the Binding Interaction of a Phenazinium Dye with Serum Transport Proteins: A Combined Fluorometric and Circular Dichroism Study

In the present investigation, an attempt has been made to study the interaction of phenosafranin (PSF), a cationic phenazinium dye with the transport proteins, bovine serum albumin (BSA) and human serum albumin (HSA), employing steady-state and time-resolved fluorometric and circular dichroism (CD) techniques. The photophysical properties of the dye are altered on binding with the serum proteins. An explicit study with respect to the modification of the fluorescence and fluorescence anisotropy upon binding, effect of denaturant, fluorescence lifetime and CD measurements reveal that the dye binds to both BSA and HSA with almost the same affinity. Far-UV CD spectra indicate a decrease in the percentage of α-helicity only for BSA upon binding with the probe. Near-UV CD responses indicate an alteration in the tertiary structure of both the transport proteins because of binding.     

辅酶NADH与色氨酸共振能量转移的荧光动力学研究

采用时间相关单光子计数技术, 结合紫外-可见吸收光谱和稳态荧光光谱, 对不同环境下的色氨酸和辅酶还原型烟酰胺腺嘌呤二核苷酸(NADH)之间的共振能量转移荧光动力学进行了研究. 单体色氨酸、 牛血清白蛋白以及乳酸脱氢酶蛋白与NADH之间相互作用的光谱数据表明, 只有存在NADH结合位点的乳酸脱氢酶和NADH之间发生了荧光共振能量转移. 进一步通过加入丙酮酸来阻断乳酸脱氢酶和NADH之间的荧光共振能量转移通道, 时间分辨荧光光谱和衰减相关光谱(DAS)证实, 蛋白结合位点的存在是NADH和色氨酸之间发生荧光共振能量转移的前提条件. DAS揭示了乳酸脱氢酶平均荧光寿命的减小主要是源于色氨酸中7.35 ns的荧光寿命成分与NADH之间的荧光共振能量转移, 同时给出了NADH和色氨酸之间的能量转移效率, 为研究NADH和蛋白之间的相互作用提供了新思路.     

A fluorescence method for quantitative measurements of specific protein at powder surfaces

A new method using fluorescence labelled proteins was developed to determine the quantitative amount of specific protein at the powder surface. The method is based on steady-state fluorescence measurements of pyrene labelled proteins with oxygen gas phase quenching at the powder surface. The surface load of protein was measured for spray-dried dextran powders containing bovine serum albumin (BSA). The results show a patchwise surface load of about 1.3 mg m⁻² at a concentration of 0.33% (dry weight) BSA in the powder. The patchwise surface load stays constant with increased BSA concentration in the powder.     

甘草酸二铵与牛血清白蛋白相互作用的光谱

在模拟人体生理条件下,用常规荧光光谱和紫外－可见吸收光谱研究甘草酸二铵和牛血清白蛋白的相互作用机制,并利用同步荧光光谱和三维荧光光谱研究了药物对牛血清白蛋白构象的影响。研究表明,甘草酸二铵对牛血清白蛋白具有荧光猝灭作用,且为单一的动态猝灭过程。根据Stern-Volmer方程求出了两者相互作用的动态猝灭常数,并根据Förster非辐射能量转移理论确定了药物与蛋白的结合距离。     

光谱法研究牡荆素与蛋白质相互作用及共存金属离子的影响

利用荧光光谱法和紫外-可见光谱法研究了牡荆素(VT)与牛血清白蛋白(BSA)之间的相互作用.VT对BSA的荧光猝灭为动态猝灭过程,测定了不同温度下的猝灭常数;根据F(o)rster非辐射能量转移理论,计算出VT在BSA中的结合位置与色氨酸残基间的距离为2.675.nm;通过热力学参数推断出VT与BSA之间主要靠疏水作用...     

Nonspecific interactions of a carboxylate-substituted PPE with proteins. A cautionary tale for biosensor applications

Two carboxylate-substituted, fluorescent (Phi = 0.08), water-soluble poly(p-phenyleneethynylene)s (PPE) and a water-soluble model compound were exposed to a series of proteins and bovine serum. While the anionic PPEs do not have any specific binding sites, they form stable complexes with histone, lysozyme, myoglobin, and hemoglobin. The complex formation was evidenced by fluorescence quenching. Bovine serum albumin does not quench the fluorescence of the PPEs but enhances it, probably due to its surfactant character. These results imply that the use of charged conjugated polymers as biosensors, while an attractive proposition, has to take into account strong nonspecific interactions between conjugated polymers and the host of proteins that is found in cells and complex biological fluids.     

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.     

Preparation of phenylboronic acid functionalized cation-exchange monolithic columns for protein separation and refolding

In this study, we described a simple and effective modification procedure to prepare poly (methacrylate-co-ethylene glycol dimethacrylate) monolithic columns functionalized with 3-aminophenylboronic acid. The column morphology, pore size and specific surface area of the fabricated monolith were characterized by scanning electron microscopy, X-ray photoelectron spectroscopy, thermogravimetric analysis, and mercury intrusion porosimeter, respectively. The frontal analysis was carried out for dynamic loading capacity of the model protein on the modified column. The chromatographic performance of the cation-exchange monolith was evaluated through separating a mixture of five proteins such as lysozyme, cytochrome c, ribonuclease A, trypsin and bovine serum albumin and one-step purification of lysozyme from egg whites, and the expected results were obtained. In addition, the functionalized column was used to refold ribonuclease A and cytochrome c, and this procedure was monitored by circular dichroism and fluorescence spectroscopy. Compared with the conventional dilution refolding method, the ion-exchange chromatography refolding method developed here is more effective for specific bioactivity recovery.     

Effect of Native Disulfide Bonds on the Denaturation of Proteins in 6 mol/L Guanidinium Chloride——A Fourier Transformed Infrared Study

The unfolding of bovine serum albumin, lysozyme and ribonuclease A denatured in 6 mol/L GuHCl with their disulfide bridges intact and reduced have been compared by FTIR studies. The peak positions and heights in the deconvolved spectra of amide I bands of the above denatured proteins with native disulfide bonds show marked differences whereas those for the denatured proteins without disulfide linkages are closely similar. The above and other evidence suggest that denatured proteins with intact disulfides still have considerable ordered conformation even in 6 mol/L GuHCl.