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。     

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.     

Spectroscopic Investigation of the Interactions of Cryptotanshinone and Icariin with Two Serum Albumins

The interactions of two drugs, cryptotanshinone (CTS) and icariin, with bovine serum albumin (BSA) and human serum albumin (HSA) have been investigated using multiple spectroscopic techniques under imitated physiological conditions. CTS and icariin can quench the fluorescence intensity of BSA/HSA by a static quenching mechanism with complex formation. The binding constants of CTS–BSA, CTS–HSA, icariin–BSA and icariin–HSA complexes were observed to be 1.67 × 10⁴, 4.04 × 10⁴, 4.52 × 10⁵ and 4.20 × 10⁵ L·mol^?1, respectively at 298.15 K. The displacement experiments suggested icariin/CTS are primarily bound to tryptophan residues of the proteins within site I and site II. The thermodynamic parameters calculated on the basis of the temperature dependence of the binding constants revealed that the binding of CTS–BSA/HSA mainly depends on van der Waals interaction and hydrogen bonds, and yet the binding of icariin–HSA/BSA strongly relies on the hydrophobic interactions. The binding distances between BSA/HSA and CTS/icariin were evaluated by the Föster non-radiative energy transfer theory. The results of synchronous fluorescence, 3D fluorescence, FT-IR and CD spectra indicates that the conformations of proteins were altered with the addition of CTS or icariin. In addition, the effects of some common ions on the binding constants of CTS/icariin to proteins are also discussed.     

Comparative studies on biophysical interactions between 4-dicyanomethylene-2,6-dimethyl-4H-pyran (DDP) with bovine serum albumin (BSA) and human serum albumin (HSA) via photophysical approaches and molecular docking techniques

Photophysical studies of 4-Dicyanomethylene-2,6-Dimethyl-4H-Pyran (DDP) dye with globular proteins, Human Serum Albumin (HSA) and Bovine Serum Albumin (BSA) were carried out in aqueous solution. An isosbestic point resulted on the addition of serum albumins, which signifies a complex or an equilibrium state of DDP dye with albumin. Addition of BSA to DDP dye results in a fluorescence enhancement accompanied with a significant hypsochromic shift, whereas with that of HSA, a fluorescence quenching with a considerable blue shift resulted. Excited state studies of DDP dye with serum albumins portray that the role of binding sites of dye with albumins vary considerably and the nature of interaction is presumably attributed to combined hydrogen-bonding and hydrophobic interactions. Molecular docking studies of DDP dye with albumins and two other derivatives 4-(Dicyanomethylene)-2-methyl-6-(4-dimethylaminostyryl)-4H-pyran (DCM) dye and 4-(Dicyanomethylene)-2-methyl-6-(4-t-buyl)-4H-pyran (DCT) dyes with BSA and HSA elucidates that the hydrogen-bonding interaction accompanied with several hydrophobic, pi–pi an pi–alkyl interactions coexist between dye and albumins. The binding energy, intermolecular energy and stability of the DDP, DCM and DCT dyes through docking techniques with albumins authenticate that the dye predominantly acts as hydrogen-bonding acceptor site and the protein molecule as the donor. DDP dye prefers to exist in four different binding sites of HSA, whereas, in the case of BSA, the most preferred site is found to be hydrophobic domain (site I). Interestingly, the most preferred site of DCT dye is III A subdomain of HSA, whereas DCM dye is oriented towards I B subdomain. DDP and DCT are smaller in size and reside in the domain preferred for smaller ligands (II A and IIIA) as resulted in several drugs-HSA interaction whereas DCM dye which is categorized as medium to larger ligand based on the extended structure resides in the most favoured site IB. Fluorescence techniques in combination with molecular docking methods elucidate binding characteristics and the domain in which the dye resides in a micro heterogeneous environment is established in this study.     

Spectroscopic investigation on the interaction of ICT probe 3-acetyl-4-oxo-6,7-dihydro-12H Indolo-[2,3-a] quinolizine with serum albumins

Interaction of 3-acetyl-4-oxo-6,7-dihydro-12H indolo-[2,3-a] quinolizine (AODIQ), a biologically active molecule, with model transport proteins, bovine serum albumin (BSA) and human serum albumin (HSA) have been studied using steady state and picosecond time-resolved fluorescence and fluorescence anisotropy. The polarity dependent intramolecular charge transfer (ICT) process is responsible for the remarkable sensitivity of this biological fluorophore to the protein environments. The CT fluorescence exhibits appreciable hypsochromic shift along with an enhancement in the fluorescence yield, fluorescence anisotropy (r) and fluorescence lifetime upon binding with the proteins. The reduction in the rate of ICT within the hydrophobic interior of albumins leads to an increase in the fluorescence yield and lifetime. Marked increase in the fluorescence anisotropy indicates that the probe molecule is located in a motionally constrained environment within the proteins. Micropolarities in the two proteinous environments have been determined following the polarity sensitivity of the CT emission. Addition of urea to the protein-bound systems leads to a reduction in the fluorescence anisotropy indicating the denaturation of the proteins. Polarity measurements and fluorescence resonance energy transfer (FRET) studies throw light in assessing the location of the fluorophore within the two proteinous media.     

Fluorescence Spectroscopic Studies on the Interaction of Oleanolic Acid and its Triterpenoid Saponins Derivatives with Two Serum Albumins

The interaction of oleanolic acid (OA) and its glycosylated derivatives (LL-2 and LL-4) with human and bovine serum albumins were investigated using the methods of fluorescence spectroscopy. The spectroscopic analysis of the fluorescence quenching that occurs when OA and its derivatives interact with serum albumin indicates that these quenching constants are inversely correlated with temperature and the quenching process involves static interactions. The binding affinity of OA and OA-derived compounds to bovine serum albumin (BSA) and human serum albumin (HSA) follow the trend LL-4 > LL-2 > OA, suggesting that glycosylation of OA can facilitate its binding to serum albumins. Additionally, the binding affinity of these compounds to HSA is stronger than it is to BSA. The calculated thermodynamic parameters suggest that hydrophobic interactions dominate these interaction processes. We also found that only a single type of binding site exists for OA and its derivatives to HSA and BSA. Synchronous fluorescence results indicate that the binding of OA, LL-2 and LL-4 to BSA and HSA can lead to the conformational changes around the tryptophan residues of the two serum albumins. These results provided valuable clues to the pharmacokinetics and the pharmacologic activities of OA and its types of triterpenoid saponins derivatives.     

光谱法测定伊曲康唑与牛血清和人血清白蛋白相互作用

用荧光光谱和紫外吸收光谱法, 在pH=7.4±0.1的0.1 mol·L-1磷酸缓冲溶液中, 研究了伊曲康唑与牛血清白蛋白(BSA)和人血清白蛋白(HSA)的相互作用. 实验结果表明, 伊曲康唑与牛血清白蛋白和人血清白蛋白作用的猝灭常数均随着温度的升高而降低, 伊曲康唑可以有规律地使血清白蛋白内源荧光猝灭, 其猝灭机理可认为是伊曲康唑与白蛋白形成复合物的静态猝灭. 获得了在不同温度下, 伊曲康唑与血清白蛋白作用的结合常数以及△G、△H和△S等热力学参数. 根据所得结果可推断伊曲康唑与白蛋白的作用力主要为疏水作用力, 同时, 利用荧光共振能量转移理论(FRET)计算得出了伊曲康唑与白蛋白结合位置的距离d. 而且, 利用同步荧光光谱和紫外光谱揭示了该反应中蛋白的结构和其微环境的变化.     

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.     

Dual temperature/pH-sensitive drug delivery of poly(N-isopropylacrylamide-co-acrylic acid) nanogels conjugated with doxorubicin for potential application in tumor hyperthermia therapy

The interaction of the amphiphilic drugs, i.e., amitriptyline hydrochloride (AMT) and promethazine hydrochloride (PMT), with serum albumins (i.e., human serum albumin (HSA) and bovine serum albumin (BSA)), has been examined by the various spectroscopic techniques, like fluorescence, UV-vis, and circular dichroism (CD). Fluorescence results indicate that in case of HSA-drug complexes the quenching of fluorescence intensity at 280 nm is less effective as compared to at 295 nm while in case of BSA-drug complexes both have almost same effect and for most of drug-serum albumin complexes there is only one independent class of binding. For all drug-serum albumin complexes the quenching rate constant (K(q)) values suggest the static quenching procedure. The UV-vis results show that the change in protein conformation of PMT-serum albumin complexes was more prominent as compared to AMT-serum albumin complexes. The CD results also explain the conformational changes in the serum albumins on binding with drugs. The increase in α-helical structure for AMT-serum albumin complexes is found to be more as compared to PMT-serum albumin complexes. Hence, the various spectroscopic techniques provide a quantitative understanding of the binding of amphiphilic drugs with serum albumins.     

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.     

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.     

Complexation of serum albumins and triton X-100: Quenching of tryptophan fluorescence and analysis of the rotational diffusion of complexes

The polarized and nonpolarized fluorescence of bovine serum albumin and human serum albumin in Triton X-100 solutions is studied at different pH values. Analysis of the constants of fluorescence quenching for BSA and HSA after adding Triton X-100 and the hydrodynamic radii of BSA/HSA–detergent complexes show that the most effective complexation between both serum albumins and Triton X-100 occurs at pH 5.0, which lies near the isoelectric points of the proteins. Complexation between albumin and Triton X-100 affects the fluorescence of the Trp-214 residing in the hydrophobic pockets of both BSA and HSA.     

VISIBLE LIGHT IRRADIATION OF HUMAN AND BOVINE SERUM ALBUMIN-BILIRUBIN COMPLEX*

Abstract— The UV absorption and the fluorescence emission spectra of both bovine (BSA) and human (HSA) serum albumin underwent noticeable changes upon irradiation of their 1:1 complexes with bilirubin; both these phenomena are suggestive of the photosensitized modification of aromatic amino acid residues. Amino acid analysis showed that after relatively short irradiation times of both albumins, only histidyl and tryptophyl residues appeared to be affected to a significant extent. After 60min of irradiation, some decrease in the tyrosine content was also observed, especially for HSA.
Conformational studies, obtained by exposing unirradiated and irradiated BSA and HSA to denaturing agents, showed that the three-dimensional organization of the 15 min irradiated samples was slightly different from that of the native proteins. On the other hand, after 15 min of irradiation, the association constant of the bilirubin-albumin complexes decreased from 2.07 to 0.54×10⁸ M ^-1 for HSA and from 2.16 to 0.87×10⁷ M ^-1 for BSA.
These data indicate that the histidyl residues are relatively unimportant for maintaining the native tertiary structure of BSA and HSA, but they are critical for determining the binding capacity of the albumins. Our data also imply that the tertiary structure of the BSA molecule is more labile than that of HSA.     

Interaction of pyrene-end-capped poly(ethylene oxide) with bovine serum albumin and human serum albumin in aqueous buffer medium: a fluorometric study

The photophysical behavior of a hydrophobically tailored water-soluble polymer, pyrene-end-capped poly(ethylene oxide) (PYPY), has been studied in aqueous buffered bovine serum albumin (BSA) and human serum albumin (HSA) media. In buffered aqueous solution the polymer shows dual emission corresponding to the monomer and the excimer of pyrene moiety. The relative intensity of the monomer to the excimer emission shows interesting variation with the addition of BSA and HSA and is indicative of significant interaction of these albumin proteins with the polymer. The binding interaction has been shown to have a prominent role on the steady state fluorescence anisotropy of the two emission bands. Attempt has been made to determine the micropolarities of the protein microenvironments from a comparison of the variation of the monomer to excimer relative fluorescence intensities of the probe in water–dioxane mixtures with varying composition.     

Fluorimetric studies and noncovalent labeling of protein with the near-infrared dye HITCI for analysis by CE-LIF

1,1',3,3,3',3'-Hexamethylindotricarbocyanine iodide (HITCI) is a commercially available, positively charged, indocarbocyanine dye used typically as a laser dye in the near infrared (NIR). The absorbance and fluorescence properties of HITCI in a variety of solvent systems were determined. Results indicate that the fluorescence of HITCI is not significantly affected by the pH. Titration of HITCI with human serum albumin (HSA) and trypsinogen was carried out to investigate the interactions between this dye and proteins. These studies revealed that the absorbance and fluorescence properties of the dye change upon binding to protein in a wide range of solution pH's. The potential use of HITCI as a noncovalent protein labeling probe, therefore, was explored. Determination and separation of HITCI and HITCI-protein complexes was performed by capillary electrophoresis with diode-laser induced fluorescence detection (CE-LIF). Both pre-column and on-column noncovalent labeling methods are demonstrated.     

Gaining an insight into the photoreactivity of a drug in a protein environment: a case study on nalidixic acid and serum albumin

The binding of nalidixic acid (NA) with human and bovine serum albumin (HSA and BSA) in buffer solution at pH 7.4 was investigated using circular dichroism (CD), UV absorption and fluorescence spectroscopy. Global analysis of multiwavelength spectroscopic data afforded the equilibrium constants of the most stable noncovalent drug/protein adducts of 1:1 and 2:1 stoichiometry and their individual CD, UV absorption, and fluorescence spectra. The primary binding site of the drug was located in subdomain IIIA (Sudlow Site II), whereas the secondary one was assigned to subdomain IIA. Conformational and CD calculations afforded the binding geometries. In the complexes, the fluorescence of the protein was strongly quenched by energy transfer and that of the drug was suppressed by electron transfer. Laser flash photolysis at 355 nm evidenced the formation of a radical pair consisting of a tyroxyl radical (lambdamax = 410 nm) and a reduced nalidixate anion radical NA(2-)* (lambdamax = 640 nm) with quantum yield of 0.4-0.5. Strong evidence was obtained that the process that involves Tyr411 in HSA (Tyr409 in BSA). A further transient with lambdamax approximately 780 nm observed in HSA was attributed to oxidation of the -(S200-S246)- bridge upon electron transfer to NA(-)*. Decay of the confined radical pairs occurred with rates approximately 10(7) s(-1). Formation of covalent drug-protein adducts in mixtures irradiated at lambdairr> 324 nm was proved using HPLC with fluorescence detection.     

Spectroscopic studies on the interaction of bovine (BSA) and human (HSA) serum albumins with ionic surfactants

Bovine (BSA) and human (HSA) serum albumins are frequently used in biophysical and biochemical studies since they have a similar folding, a well known primary structure, and they have been associated with the binding of many different categories of small molecules. One important difference of BSA and HSA is the fact that bovine albumin has two tryptophan residues while human albumin has a unique tryptophan. In this work results are presented for the interaction of BSA and HSA with several ionic surfactants, namely, anionic sodium dodecyl sulfate (SDS), cationic cethyltrimethylammonium chloride (CTAC) and zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate (HPS), as monitored by fluorescence spectroscopy of intrinsic tryptophans and circular dichroism spectroscopy. On the interaction of all three surfactants with BSA, at low concentrations, a quenching of fluorescence takes place and Stern-Volmer analysis allowed to estimate their 'effective' association constants to the protein: for SDS, CTAC and HPS at pH 7.0 these constants are, respectively, (1.4+/-0.1) x 10(5) M(-1), (8.9+/-0.1) x 10(3) M(-1) and (1.4+/-0.1) x 10(4) M(-1). A blue shift of maximum emission is observed from 345 to 330 nm upon surfactant binding. Analysis of fluorescence emission spectra allowed to separate three species in solution which were associated to native protein, a surfactant protein complex and partially denatured protein. The binding at low surfactant concentrations follows a Hill plot model displaying positive cooperativity and a number of surfactant binding sites very close to the number of cationic or anionic residues present in the protein. Circular dichroism data corroborated the partial loss of secondary structure upon surfactant addition showing the high stability of serum albumin. The interaction of the surfactants with HSA showed an enhancement of fluorescence at low concentrations, opposite to the effect on BSA, consistent with the existence of a unique buried tryptophan residue in this protein with considerable static quenching in the native state. The effects of surfactants at low concentrations were very similar to those of myristic acid suggesting a non specific binding through hydrophobic interaction modulated by eletrostatic interactions. The changes in the vicinity of the tryptophan residues are discussed based on the recently published crystallographic structure of HSA myristate complex (S. Curry et al., Nat. Struct. Biol. 5 (1998) 827).     

Cd（Ⅱ）与HSA或BSA的结合平衡研究

用平衡透析法详细研究了生理pH(7.43)条件下Cd(Ⅱ)与HSA或BSA的结合平衡.通过非线性最小二乘法拟合Bjerrum方程,首次报道了Cd(Ⅱ)-HSA和Cd(Ⅱ)-BSA体系的逐级稳定常数值,其K_1～K_3的数量级均为10~4;Hill系数和自由能偶合定量分析表明Cd(Ⅱ)与HSA或BSA的结合均产生在类似体系中少见的强的正协同效应,且Cd(Ⅱ)与HSA结合产生的正协同效应大于BSA;Scatchard图分析表明,Cd(Ⅱ)在HSA和BSA中均有3个强结合部位.通过Cd(Ⅱ)与Cu(Ⅱ),Zn(Ⅱ)或Ca(Ⅱ)等竞争结合HSA或BSA的结果,进一步讨论了Cd(Ⅱ)在HSA或BSA中强结合部位的可能位置和(或)配体.     

La(Ⅲ)与HSA或BSA的结合平衡研究

用平衡透析法详细研究了pH=6.3条件下La(Ⅲ)与HSA或BSA的结合平衡.Scatchard图分析表明,La(Ⅲ)在HSA中有2个强结合部位和8个弱结合部位;在BSA中有2个强结合部位和6个弱结合部位.从La(Ⅲ)与Cu(Ⅱ),Zn(Ⅱ)和Cd(Ⅱ)等的竞争结合HSA或BSA的结果推测:La(Ⅲ)在HSA或BSA中的一个强结合部位的配位原子可能全部是氧原子.通过非线性最小二乘法拟合Bjerrum方程,首次报道了La(Ⅲ)-HSA和La(Ⅲ)-BSA体系的逐级稳定常数值,其K₁的数量级为10⁴.Hill系数及自由能偶合分析表明La(Ⅲ)与HSA或BSA的结合均产生一定的负协同效应.     

Interaction of Globular Plasma Proteins with Water‐Soluble CdSe Quantum Dots

The interactions between water‐soluble semiconductor quantum dots [hydrophilic 3‐mercaptopropionic acid (MPA)‐coated CdSe] and three globular plasma proteins, namely, bovine serum albumin (BSA), β‐lactoglobulin (β‐Lg) and human serum albumin (HSA), are investigated. Acidic residues of protein molecules form electrostatic interactions with these quantum dots (QDs). To determine the stoichiometry of proteins bound to QDs, we used dynamic light scattering (DLS) and zeta potential techniques. Fluorescence resonance energy transfer (FRET) experiments revealed energy transfer from tryptophan residues in the proteins to the QD particles. Quenching of the intrinsic fluorescence of protein molecules was noticed during this binding process (hierarchy HSA<β‐Lg <BSA, lower binding affinity for hydrophobic protein molecules). Upon binding with QD particles, the protein molecules underwent substantial conformational changes at the secondary‐structure level (50 % helicity lost), due to loss in hydration.