Dendrimer Interactions with Hydrophobic Fluorescent Probes and Human Serum Albumin

The interactions between three types of polyamidoamine dendrimers (with anionic, cationic, and neutral charge on a surface) and fluorescent dye 1-anilinonaphthalene-8-sulfonate (ANS) were studied. Double fluorimetric titration method was employed to estimate a binding constant and the number of binding centers. As fluorescent probes can serve as models of toxin molecules, dendrimers, and human serum albumin (HSA) abilities to bind ANS were compared. In the presence of HSA and dendrimers, ANS located both in HSA and in dendrimers, but the interactions between ANS and HSA were stronger.     

荧光光谱法研究G3.0 PAMAM树状大分子与牛血清白蛋白的相互作用

聚酰胺-胺型(PAMAM)树状大分子是一类新型的纳米级、球型、高度分支、单分散性的聚合物,并具有安全、低毒、无免疫原性等许多独特的生物学性质。正是由于这些优势使其有望成为一种新型有效的生物材料,用于作为寡核苷酸的转运因子和药物转运载体。因此,深入了解树状大分子的生物学性质对进一步研究其在治疗方面的应用是至关重要的。文章应用荧光光谱法在生理条件下研究了具有表面氨基的3.0代聚酰胺-胺型(G3.0 PAMAM)树状大分子与牛血清白蛋白(BSA)间的相互作用。结果表明,加入G3.0 PAMAM树状大分子后,BSA内源性荧光发生猝灭,其猝灭机制属于静态猝灭,符合Stern-Volmer方程。通过计算得到该树状大分子与BSA间的结合常数为(1.067±0.025)L·mmol-1。通过同步荧光、红边激发荧光位移(REES)等方法的研究发现,树状大分子的存在会改变BSA的构象。此外还考查了体系的pH值和离子强度对该树状大分子与BSA相互作用的影响,由实验结果可推断静电作用是二者结合的主要作用机制。     

Estimation of PAMAM Dendrimers' Binding Capacity by Fluorescent Probe ANS

Dendrimers are globular, hyperbranched polymers which possess a high concentration of surface functional groups and internal cavities. These unique features make them very useful to many biomedical applications, especially as carrier molecules. This study presents results of estimation of polyamidoamine (PAMAM) dendrimers and human serum albumin (HSA) binding capacity of fluorescent probe 1-anilinonaphthalene-8-sulfonic acid (ANS). It has been shown that fluorescent probes can be used for quantitative analysis of dendrimers' binding capacity.     

Spectrophotometric Study on the Binding of Two Water Soluble Schiff Base Complexes of Mn (III) with ct-DNA

In this work, binding of two water soluble Schiff base complexes: Bis sodium (5-sulfosalicylaldehyde) o- phenylendiiminato) Manganese (III) acetate (Salophen complex) and Bis sodium (5-sulfosalicylaldehyde) 1, 2 ethylendiiminato) Manganese (III) acetate (Salen complex) with calf thymus (ct) DNA were investigated by using different spectroscopic and electrometric techniques including UV-vis, Circular dichroism (CD) and fluorescence spectroscopy, viscommetry and cyclic voltammetry (CV). Both complexes have shown a hyperchromic and a small bathochromic shift in the visible region spectra. A competitive binding study showed that the enhanced emission intensity of ethidium bromide (EB) in the presence of DNA was quenched by the addition of the two Schiff base complexes indicating that they displace EB from its binding site in DNA. Moreover structural changes in the CD spectra and an increase in the CV spectra with addition of DNA were observed. The results show that both complexes bind to DNA. The binding constants have been calculated using fluorescence data for two complexes also K_b was calculated with fluorescence Scatchard plot for Salophen. Ultimately, the experimental results show that the dominant interactions are electrostatic while binding mode is surface binding then followed by hydrophobic interactions in grooves in high concentration of complexes.     

新型含芘荧光探针分子与蛋白质相互作用的光谱研究

利用荧光发射光谱和紫外-可见吸收光谱,研究了新型含芘荧光探针分子芘丁酰谷氨酸(PLE)和芘甲酰谷氨酸(PYE)与溶菌酶(Lyso)和牛血清白蛋白(BSA)的相互作用,分析了结合过程和机理。结果显示:不同间隔链长度的探针分子在与Lyso作用时,表现的光谱性质差别不大,这主要是由于探针分子结合到了Lyso的表面;而不同间隔链长度的探针分子在与BSA作用过程中,却表现不同的光物理性质,这可能是探针分子作用于BSA空腔时其结合位点或者结合方式的不同引起的。目前的研究对揭示蛋白质分子的识别位点和定位切割具有重要的意义。     

A Fluorescence Analysis of ANS Bound to Bovine Serum Albumin: Binding Properties Revisited by Using Energy Transfer

Determination of binding parameters such as the number of ligands and the respective binding constants require a considerable number of experiments to be performed. These involve accurate determination of either free and/or bound ligand concentration irrespective of the measurement technique applied. Then, an appropriate theoretical model is used to fit the experimental data, and to extract the binding parameters. In this work, the interaction between bovine serum albumin (BSA) and 1-anilino-8-naphthalene sulphonate (ANS) is revisited. Using steady state fluorescence spectroscopy, the binding isotherm of BSA/ANS was obtained applying the Halfman-Nishida approach. The binding parameters, site number, and binding site association constants, were determined from the stoichiometric Adair model and Job's plot. The binding parameters obtained were then correlated to the distance of the respective binding site to the tryptophan residues using the energy transfer technique. This approach, that uses both tryptophans independently from each other, is presented as a tool to help understand the binding mechanism of the albumin fluorescent complex. The results show that ANS molecules bind to BSA in up to five different binding sites. Energy transfer from the tryptophan residues to the BSA/ANS complex shows that the four highest affinity binding sites (>10(4) M(-1)) are located at a reasonably close distance (18-27 A) to at least one of two tryptophan residues, while the lowest affinity binding site (approximately 10(4) M(-1)) is located over 34 A away from the both tryptophans.     

伊文思蓝与牛血清白蛋白相互作用的研究

利用荧光光谱和紫外吸收光谱研究了伊文思蓝（Evans blue,EB）与牛血清白蛋白（Bovine serum albumin,BSA）的相互作用。伊文思蓝与牛血清白蛋白作用,使牛血清白蛋白的荧光发生猝灭,利用Stern-Volmer方程和荧光寿命的测定,确定了伊文思蓝对牛血清白蛋白的荧光猝灭为静态猝灭和非辐射能量转移。实验测得伊文思蓝与牛血清白蛋白的结合常数KBSA-EB为1.122×106L·mol-1,结合点数n为0.994;根据Foerster非辐射能量转移理论,得到伊文思蓝与牛血清白蛋白之间的能量转移效率E为0.276,作用距离r为3.14nm。同时,利用同步荧光光谱研究了牛血清白蛋白的构象变化。     

Time-Resolved Fluorescence Studies on Bovine Serum Albumin Denaturation Process

The denaturation of Bovine Serum Albumin (BSA) by a chaotropic agent, guanidinium hydrochloride (GuH+Cl-) was studied by fluorescence lifetime analysis. The BSA was labelled with 1-anilino-8-naphthalene sulfonate (ANS) at two different molar ratios (1:1) and (1:10). The non-exponential fluorescence kinetics of the BSA-ANS complex at different stages of denaturation is analysed using three different models: a discrete tri-exponential sum, stretched exponential, and Gaussian lifetime distribution. In all cases, the fluorescence decay times decreased with protein denaturation. The results from the models show that there are at least two different binding sites located in the BSA protein with different water accessibility.     

Studies on the interactions of fluorescent-magnetic nanocomposites with biomolecules and cells labeling

In this study, the fluorescent-magnetic nanocomposites were synthesized. The interactions between nanocomposites and bovine serum albumin (BSA) were studied by absorption and FL titration experiments. The experiments show that binding of nanocomposites and BSA may be caused by the formation of QDs-BSA complex. A magnetic separation method was designed to directly demonstrate the interactions between the surface of bifunctional nanocomposites with CdSe/CdS quantum dots (QDs) and biomolecules (BSA and DNA). The fluorescence (FL) labeling on the Escherchia coli (E. coli) cells was also successfully developed for studying the biolabeling of the bifunctional nanocomposites. The results directly reveal that the bifunctional nanocomposites can separate biomolecules and label cells. The studies we have performed showed that the fluorescent-magnetic nanocomposites are proved to be a kind of novel biofuctional materials, which can be used in bioseparation and biolabeling applications.     

Deoxyribonucleic Acid and Bovine Serum Albumin Interaction with the Asymmetric Schiff Base Ligand and Its Molybdenum (VI) Complex: Multi Spectroscopic and Molecular Docking Studies

The interaction of an asymmetric Schiff base ligand derived from allylamine and 2,3-dihydroxybenzaldehyde and its molybdenum (VI) complex with deoxyribonucleic acid (DNA) and bovine serum albumin (BSA) were studied using spectroscopic and molecular docking methods. The spectroscopic results revealed that the DNA and BSA affinity for binding the Mo(VI) complex is greater than its ligand. Furthermore, the molecular docking calculations showed that H-bond, hydrophobic, π-π and π-cation interactions had the dominant roles in the stability of the compound-BSA complexes. The DNA interaction results suggested that the compounds interacted with DNA by the groove binding mechanism.     

The Interaction between Polycationic Poly-Lysine Dendrimers and Charged and Neutral Fluorescent Probes

The interactions between polycationic poly-lysine dendrimers and hydrophobic fluorescent probes (anionic ANS and neutral Prodan) were studied. R121 and R131 dendrimers were not able to interact with anionic and neutral hydrophobic groups. R124 was able to interact with neutral and anionic hydrophobic fluorescent probes, however mainly through hydrophobic forces. Dendrimers R155 and R169 showed the maximal effects. The strongest interactions observed for R169 can be explained by intramolecular folding (stacking) of its two L-proline residues. Using double fluorescence titration technique for ANS probe allowed to receive such constant of binding and the number of binding centers: for R121, 1.8·10³ (mol/l)⁻¹ and 1.07; for R124, 12.1·10³ (mol/l)⁻¹ and 0.48; for R131, 4.7·10³ (mol/l)⁻¹ and 0.48; for R155, 9.2·10³ (mol/l)⁻¹ and 1.36; for R169, 39.6·10³ (mol/l)⁻¹ and 0.97. Thus, neutral and anionic hydrophobic probes can be used for the fast preliminary screening of binding properties of newly synthesized polycationic dendrimers.     

Comprehensive Study on the Binding of Iron Schiff Base Complex with DNA and Determining the Binding Mode

The iron (III) [N, N′ Bis (5-(triphenyl phosphonium methyl) salicylidene)-1, 2 ethanediamine] chloride [Fe Salen]Cl, has been synthesized and characterized as described previously. The interaction of iron complex with calf thymus (CT) DNA has been studied extensively by experimental techniques. Absorption spectra showed both hypochromism and hyperchromism. Thermal denaturation study of DNA with complex revealed the ΔT_m of 5 °C. Competitive binding study shows that the enhanced emission intensity of ethidium bromide (EB) in the presence of DNA was quenched by adding of the iron complex indicating that it displaces EB from its binding site in DNA and the apparent binding constant has been estimated to be 5?×?10⁶?μM^?1. Fluorescence Scatchard plot revealed type B behavior for interaction of complex to DNA. Circular dichroism (CD) spectra measurements showed that the complex interacts with DNA via surface and groove bindings. Linear dichroism (LD) measurements confirmed the bending of DNA in the presence of complex. Furthermore, Isothermal titration calorimetry (ITC) experiments approved that the binding of complex is based on both electrostatic and hydrophobic interactions. More, ITC profile exhibits the existence of two binding phases for the complex.     

Optical bistability of the director field of the dendrimer-doped nematic liquid crystal

It was found that the second-generation carbosilane dendrimers with statistically distributed terminal aliphatic and azobenzene fragments induce negative orientational nonlinearity in nematic liquid crystals (NLC) (the NLC director turns perpendicular to the light field, decreasing the refractive index of an extraordinary wave). The light-induced Freedericksz transition in planar oriented NLCs is the first-order orientational phase transition characterized by a wide bistability region.     

荧光光谱法研究二溴羟基卟啉与蛋白质的结合作用机理

应用荧光光谱法研究了meso-四(3,5-二溴-4-羟基苯基)卟啉[T(DBHP)P]与牛血清白蛋白(BSA)之间的结合反应,基于T(DBHP)P对BSA内源荧光的猝灭机理,测定了两者之间在不同温度下的结合常数,温度为27 ℃时,荧光猝灭法测得反应的结合常数为K=1.30×106 L·mol-1,温度为48 ℃时,K=6.32×105 L·mol-1,结合常数随温度升高而减小,由此判定该猝灭类型为静态猝灭。根据Frster非辐射能量转移理论,确定了T(DBHP)P与BSA之间的能量转移效率E=0.91,能量给体(BSA)与受体[T(DBHP)P]之间的结合距离r=2.39 nm<7 nm,符合非辐射能量转移条件。依据热力学参数ΔG<0,ΔH<0,ΔS>0确定了T(DBHP)P与BSA之间的作用力主要是静电引力。同时,利用同步荧光光谱,考察了T(DBHP)P对BSA构象的影响,结果发现,T(DBHP)P的加入使BSA构象发生变化,BSA内部残基所处环境的疏水性降低。     

荧光光谱分析家蝇幼虫抗菌肽与大肠杆菌染色体DNA作用机理

利用荧光光谱方法研究了家蝇幼虫抗菌肽MDL-1与大肠杆菌染色体DNA的相互作用,并以溴化乙锭为荧光探针,考察了抗菌肽MDL-1结合大肠杆菌DNA的作用方式,探讨其作用模式,即：抗菌肽MDL-1与大肠杆菌DNA骨架的磷酸基团通过静电引力结合,使抗菌肽MDL-1和DNA的空间结构都发生变化,抗菌肽MDL-1进一步与DNA双螺旋的沟槽结合,然后抗菌肽MDL-1以嵌入或部分嵌入方式与DNA相互作用,计算出抗菌肽MDL-1与DNA的结合常数和成键位点数。本试验从分子水平上研究抗菌肽与细菌DNA的作用模式和结构特征,对深入了解抗菌肽的作用机理极为重要。     

Studying Complexes Between PPI Dendrimers and Mant-ATP

The aim of this study was to investigate the interactions between poly(propylene imine) (PPI) dendrimers and 2′-/3′-O-(N′-methylanthraniloyl)-ATP (Mant-ATP). Mant-ATP was used as a model molecule. Purine and pyrimidine nucleoside analogues are antimetabolites commonly used in therapy for cancer. Drug molecules can complex with dendrimers in two ways: therapeutic agents may be attached in dendrimer interior or bind to functional groups on the surface. Drugs attached to nanoparticles are characterized by improved solubility, pharmacokinetics and stability. Here, we have used poly(propylene imine) dendrimers of the 4th and 5th generations (PPI G4 and PPI G5) with primary amino surface groups partially modified with maltose (PPI-m) or without modification (PPI). We assessed the efficiency of complex formation in relation to dendrimer generation, pH of solution and the type of dendrimer used. A double fluorimetric titration method was used to estimate the binding constant (K _b ) and the number of binding centers per molecule of the binding agent (n).     

Modifying protein adsorption by layers of glutathione pre-adsorbed on Au(111)

Molecular interaction with metal surfaces raises fundamental questions regarding their binding tendency, their dispersion on the surface, as well as their conformation which may change their biological properties; addressing these questions, and being able to tune protein interactions, is of primary importance for the control of biointerfaces. In this study, one tripeptide, GSH (glu-cys-gly), was used to condition gold surfaces and thus influence the adsorption of bovine serum albumin (BSA). Depending on the pH value of the GSH solution, cationic, zwitterionic or anionic forms of the tripeptide could be stabilised on the surface, before interacting with BSA solutions. The amount of proteins was observed to depend both on the chemical state of the adsorbed underlying peptide and on the solvent of the protein solution, indicating an important role of electrostatic interactions upon protein adsorption. Moreover, atomic force microscopy (AFM), and synchrotron IR microscopy revealed a heterogeneous distribution of proteins on the GSH layer.     

Sonochemical Synthesis and Characterization of the Copper(II) Nanocomplex: DNA- and BSA-Binding,Cell Imaging,and Cytotoxicity Against the Human Carcinoma Cell Lines

The focus of the present work is the preparation of new metal-based nanodrug to overcome limitations of chemotherapy such as poor water solubility of most common chemotherapeutic drugs. The copper(II) complex of 1,2,4-triazine derivatives, [Cu(dppt)₂(H₂O)₂]²⁺ (dppt is 5,6-diphenyl- 3- (2-pyridyl)-1,2,4-triazine), has been synthesized at nano-size by sonochemical method and characterized by FTIR, zetasizer, and scanning electron microscopy (SEM). The interaction of the complex and nanocomplex with fish sperm DNA (FS–DNA) and BSA have been investigated under physiological conditions by a series of experimental methods. The results have indicated that the complex binds to FS–DNA by two biding modes, viz., electrostatic and intercalates into the base pairs of DNA. The competitive study with ethidium bromide (EB) shows that the complex and nanocomplex competes for the DNA–binding sites with EB. Protein binding studies show that the complex and nanocomplex could bind with BSA. The results of synchronous fluorescence of BSA show that additions of the complex affect the microenvironment of both tyrosine and tryptophan residues during the binding process. The in vitro cytotoxicity of the complex (solution in DMSO) and nanocomplex (colloid in H₂O) against the human carcinoma cell lines (MCF-7 and A-549) was evaluated by MTT assay. The results of in vitro cytotoxicity indicate that the complex and nanocomplex have excellent cytotoxicity activity against MCF-7 and A-549. Results of the microscopic analyses of the cancer cells confirm the results of the cytotoxicity.     

Spectroscopic and docking studies of the binding of two stereoisomeric antioxidant catechins to serum albumins

The interactions of two stereoisomeric antioxidant flavonoids, catechin (C) and epicatechin (EC) with bovine serum albumin (BSA) and human serum albumin (HSA), have been investigated by steady state and time resolved fluorescence, phosphorescence, circular dichroism (CD), FTIR and protein–ligand docking studies. The steady-state fluorescence studies indicate a single binding site for both the ligands. FTIR spectra suggest that in both the albumins, C and EC stabilize the α-helix at the cost of a corresponding loss in the β-sheet structure. CD studies have been carried out using (±)C, and both the epimers (+)C and (?)C. The low temperature phosphorescence and protein–ligand [(+), (?) and (±) forms of C and EC] docking studies indicate that the ligands bind in the proximity of Trp 134 of BSA and Trp 214 of HSA, thereby changing their solvent accessible surface areas (ASA). Asn 158 and Glu 130 side chains are found to be within the hydrogen bonding distance from the phenolic –OH groups of C and EC in the case of BSA complex. C and EC are located within the binding pocket of sub-domain IIa of HSA.     

不同巯基试剂修饰的CdTe量子点与BSA相互作用研究

修饰试剂对量子点的合成、性质具有重要的影响,但目前有关修饰试剂对量子点与蛋白质间相互作用的影响尚不清楚。采用紫外-可见吸收光谱、荧光光谱及红外光谱研究了3种巯基化合物(巯基乙酸,TGA;L-半胱氨酸,L-Cys;还原型谷胱甘肽,GSH)修饰的CdTe量子点与牛血清白蛋白(BSA)的相互作用。通过Stern-Volmer方程对数据进行了分析,得到了不同CdTe量子点与BSA相互作用过程的ΔHθ,ΔGθ和ΔSθ,并比较了CdTe量子点的不同修饰剂对BSA荧光猝灭的影响。研究结果表明,3种修饰试剂包覆的CdTe量子点与BSA的相互作用均为静态猝灭过程,猝灭常数KSV(L-Cys)KSV(TGA)≈KSV(GSH);TGA和L-Cys修饰的CdTe量子点与BSA的结合力主要为疏水作用力,而GSH修饰的量子点与其结合力既有氢键作用力又有疏水作用力;这些结果说明量子点与BSA的作用过程与量子点表面修饰试剂的类型有关。