Host-guest interaction between swelling clay minerals and poorly water-soluble drugs. 1: Complex formation between a swelling clay mineral and griseofulvin

The formation of a complex between a swelling clay mineral and griseofulvin (GF), a poorly water-soluble drug, was examined. A strong host-guest interaction between the neutral drug molecules and the clay mineral was observed not only in the solid state but also in aqueous dispersion. The powder X-ray diffraction patterns revealed the disappearance of a crystalline phase of GF through host-guest interaction for samples having low GF contents. The complex formation was confirmed to be due to monolayer adsorption on the basis of quantitative thermochemical analyses. The strong interaction between GF and the clay was also detected when the complex powder was dispersed in an aqueous medium on the basis of the intensity changes of from free GF solution in CD and fluorescence spectra as compared with those observed for the free GF solution.     

ENERGY TRANSFER AND ENERGY LOSSES IN BILAYER MEMBRANE VESICLES (LIPOSOMES)

Abstract. The efficiency of singlet-singlet energy transfer was studied in bilayer lipid membrane vesicles (liposomes) for the following donor-acceptor systems: (1) p -terphenyl (TP) and diphenyloctatetraene (DPO); (2) DPO and chlorophyll a (Chl a ); and (3) β-carotene and Chl a. The energy transfer efficiency φ_DA was measured by sensitized fluorescence of the acceptor. Fractional quenching of the donor φ_Q was found from the donor fluorescence in absence and presence of the acceptor. For TP-DPO and for DPO-Chl a , the transfer efficiency increased with increasing acceptor concentration but was essentially independent of the donor concentration. No energy transfer from β-carotene to Chl a could be detected. In liposomes, φ_DA differed only slightly from φ_Q at all donor and acceptor concentrations, thus demonstrating the absence of any appreciable energy losses. For solutions of the same donor-acceptor pairs in cyclohexane φ_Q was considerably larger than φ_DA. The difference represents energy lost, principally by internal conversion, due to collisional quenching. The principal function of the lipid membrane appears to be the suppression of such losses. In addition, the rate of energy transfer in lipid membranes is about double that in solutions (at the same intermolecular distance) due to more favorable orientation.     

EFFECT OF METAL IONS ON THE LUMINESCENCE OF ACRIDINE DYES BOUND TO DNA

Abstract— The luminescence of acridine dyes intercalated in DNA was studied as a function of the concurrent binding of metal ions to DNA, in an effort to deduce specific site interactions of the dyes. Two dyes, proflavine (PF) and acridine orange (AO), and two metal ions, silver and mercuric, were used. Both ions quench the fluorescence of the dyes in aqueous solution at room temperature. The metal ions have a different effect on the fluorescence of these dyes when they are intercalated between the base pairs of DNA. The fluorescence of AO is decreased when silver is bound, while the fluorescence of PF is enhanced. Since Ag⁺ initially binds to GC sites in DNA, which quench the PF fluorescence, it ostensibly 'turns off' the quenching by DNA at these sites, and this effect is greater than the quenching effect of the silver ion itself. Hg²⁺ ion initially binds to AT sites in DNA. Since both dyes fluoresce from AT sites, Hg²⁺ is expected to quench their fluorescence. This behavior is observed at low r (metal ion/base). At higher r values, however, where Hg²⁺ is expected to begin binding to GC sites, the fluorescence of PF is enhanced. These quenching turn-off effects are tentatively interpreted in terms of a change in the structure of the dye/DNA complex which occurs when a metal ion binds at the intercalation site. At 77 K. no fluorescence enhancement is observed when metal ions bind; Ag⁺ quenches the fluorescence and enhances the phosphorescence of both dyes. Qualitatively similar results are obtained with Hg²⁺.     

THE FORMATION OF HYDRATED ELECTRONS FROM THE EXCITED STATE OF INDOLE DERIVATIVES

Abstract— –Hydrogen atoms can be observed in u.v. irradiated aqueous solutions of indole derivatives. These H' atoms are produced in a reaction between H⁺ and solvated electrons which are formed in the excited state of indole. Protons are also known to be good quenching agents for the fluorescence of indole. However the pH dependence and effect of oxygen on the yield of hydrogen atoms indicates clearly that although both fluorescence and electron ejection originate in the excited singlet state the fluorescence quenching by protons is not caused by a transfer of electronic charge from the excited ring to H⁺. The temperature dependencies of both fluorescence and electron ejection yield an abnormally large "activation energy". It is proposed that this temperature dependence is to a large extent determined by a process characteristic of water as a solvent.     

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.     

ON THE NATURE OF INTERACTION BETWEEN PROFLAVINE and DNA

Abstract— The fluorescence decay of the mutagenic drug proflavine (PF) bound to DNA at a phosphate-to-drug ratio of 420 is found to be a non-exponential function of time. The deviation from exponentiality is shown to increase with increasing GC content of DNA. Thus, heterogeneity in the high affinity binding sites is clearly demonstrated. The nucleotides guanosine-5'-monophosphate (GMP) and cyti-dine-5'-monophosphate (CMP) are shown to form complexes with PF in 10^-3M sodium cacodylate buffer at pH 6.6. In addition, GMP quenches substantially the fluorescence of PF while CMP enhances it slightly. The fluorescence decay curves for 5 × I0^-6 M PF in the presence of GMP concentrations greater than 10^-2 M exhibit a deviation from exponentiality which parallels that exhibited by the fluorescence decay curves for PF bound to DNA of increasing GC content. It is inferred that (a) guanine is responsible for the quenching of the fluorescence of PF on binding to DNA; and (b) the forces involved in the interaction between PF and DNA are specific in nature. The implications of these findings concerning the mutagenic properties of acridine derivatives are discussed. Nanosecond depolarization studies reveal a quite fast depolarization of the fluorescence of bound PF; for the PF- Cl. perfringens DNA complex the rotational correlation time is about 26 ns. Time-resolved emission spectroscopy in the nanosecond scale demonstrates that, despite the change in the orientation of the drug, the electronic structure of the PF-DNA complex is not substantially altered during the lifetime of the excited singlet electronic state of the drug.     

EFFECTS OF METAL CATIONS, RETINAL, AND THE PHOTOCYCLE ON THE TRYPTOPHAN EMISSION IN BACTERIORHODOPSIN

Abstract— The picosecond fluorescence kinetics of tryptophan residues in bacteriorhodopsin and some perturbed analogs are measured to study the different tryptophan environments and their changes upon metal cation removal, retinal removal, and M₄₁₂ trapping. In bacteriorhodopsin, the emission shows four decay components designated Or, C2r, C3r, and C4r in order of increasing lifetimes. The emission wavelength of C3r and C4r is near that found in aqueous solution, while that of C1r is the shortest. The removal of retinal triples the total emission intensity and reduces the number of components to two, suggesting that the observed variation of the lifetimes in bacteriorhodopsin results from the variation of the energy transfer efficiency between different tryptophans and retinal. We conclude that the Or and C2r emission is from the closest tryptophans to the retinal. The quenching of the C3r emission by all metal cations, including those that cannot act as energy acceptors, e.g. Ca²⁺, is attributed to protein conformation changes caused by metal cation binding which leads to a stronger energy transfer coupling between tryptophans and retinal. The additional quenching of the C2r emission in Eu³⁺bound bacterioopsin is proposed to result from direct energy transfer between tryptophans and Eu³⁺.     

Simultaneous fluorescence light-up and selective multicolor nucleobase recognition based on sequence-dependent strong binding of berberine to DNA abasic site

Label-free DNA nucleobase recognition by fluorescent small molecules has received much attention due to its simplicity in mutation identification and drug screening. However, sequence-dependent fluorescence light-up nucleobase recognition and multicolor emission with individual emission energy for individual nucleobases have been seldom realized. Herein, an abasic site (AP site) in a DNA duplex was employed as a binding field for berberine, one of isoquinoline alkaloids. Unlike weak binding of berberine to the fully matched DNAs without the AP site, strong binding of berberine to the AP site occurs and the berberine's fluorescence light-up behaviors are highly dependent on the target nucleobases opposite the AP site in which the targets thymine and cytosine produce dual emission bands, while the targets guanine and adenine only give a single emission band. Furthermore, more intense emissions are observed for the target pyrimidines than purines. The flanking bases of the AP site also produce some modifications of the berberine's emission behavior. The binding selectivity of berberine at the AP site is also confirmed by measurements of fluorescence resonance energy transfer, excited-state lifetime, DNA melting and fluorescence quenching by ferrocyanide and sodium chloride. It is expected that the target pyrimidines cause berberine to be stacked well within DNA base pairs near the AP site, which results in a strong resonance coupling of the electronic transitions to the particular vibration mode to produce the dual emissions. The fluorescent signal-on and emission energy-modulated sensing for nucleobases based on this fluorophore is substantially advantageous over the previously used fluorophores. We expect that this approach will be developed as a practical device for differentiating pyrimidines from purines by positioning an AP site toward a target that is available for readout by this alkaloid probe.     

FLUORESCENCE STUDY OF THE PHYSICO-CHEMICAL PROPERTIES OF A BENZO(A)PYRENE7,8-DIHYDRODIOL9,10-OXIDE DERIVATIVE BOUND COVALENTLY TO DNA

Abstract— Covalent complexes between 7 ,8-dihydrodiol 9.10-oxide benzo(a)pyrene (BPDE) and DNA with a modification of one BPDE molecule per 1000 DNA bases were prepared in vitro . The same stereoselective and chemically homogeneous binding of BPDE to native DNA was observed, as reported earlier for human and bovine bronchial explants. The fluorescence of the pyrene-like aromatic moiety of BPDE bound to DNA in vitro was used as a probe of the microenvironment of the BPDE molecule in order to obtain information about the structure of the BPDE-DNA complex dissolved in aqueous solution. Fluorescence techniques, based on the quenching of the singlet excited states by metal ions such as Ag⁺, by iodide ions, and by molecular oxygen are described, which provide a method for differentiating between external and internal (intercalation) binding of polycyclic aromatic molecules to DNA. Silver ions, which bind specifically to DNA bases, exhibit a strong quenching effect on noncovalently bound, intercalated benzo(a)pyrene; on the other hand, there is no quenching effect on the fluorescence of BPDE in the covalent DNA adduct. Quenchers such as O₂ and iodide ions, which do not specifically bind to DNA and are dissolved in the solution external to the DNA molecule, exhibit a quenching effect on the BPDE chromophore. Furthermore, the fluorescence yield of the BPDE-DNA complex decreases with increasing DNA concentration, an effect which is not observed with non-covalently bound intercalated benzo(a)pyrene-DNA complexes, and which is attributed to intermolecular DNA-DNA interactions. The results of these studies indicate that the pyrene-like chromophore in the covalent BPDE-DNA complex is not intercalated between the base pairs, and that it is located in an accessible region external to the DNA helix. Possible structures are discussed.     

牛血清白蛋白与金霉素结合反应的机制研究

以光谱技术与微量热技术相结合的方法研究水溶液中金霉素与牛血清白蛋白分子间结合作用的热力学性质.荧光猝灭法测得该反应的结合常数K=2.09×105L/mol,结合位点数n=1.75,微量法测得反应的焓变△rHm= -17.50 kJ/mol; 依据Forster非辐射能量转移机制,得到授体-受体间的结合距离(r1=1.67 nm, r2=1.46 nm)和能量转移效率(E1=0.41, E2=0.66). 金霉素与牛血清白蛋白分子间有较强的结合作用, 且结合力以疏水作用为主.     

Fluorescence studies on the interaction of ethidium bromide with duplex, triplex and quadruplex DNA structures

Under different conditions, oligonucleotides can form several alternative DNA structures such as duplex, triplex and quadruplex. All these structures can interact with ethidium bromide (EB) and make its fluorescence intensity change. The fluorescence spectra and other related parameters provided by static fluorescence techniques showed that the interaction mechanisms between EB and these structures were not always the same. Among them, B type duplex and triplex DNA adopt an intercalative mode when binding to the EB, which has a relatively high efficiency of energy transfer and the fluorescence of EB cannot be quenched easily. While for the parallel duplex DNA, the interaction mode is an outside binding in which energy transfer can hardly happen and its fluorescence intensity as well as Stern-Volmer constant is almost the same to the free EB. For the quadruplex, the binding mechanism to EB is more complex. Results from the energy transfer and quenching studies indicate that the two interaction modes note     

MECHANISM OF CONCENTRATION QUENCHING OF A XANTHENE DYE ENCAPSULATED IN PHOSPHOLIPID VESICLES

Encapsulating a xanthene dye in phospholipid vesicles produces vesicle solutions that contain dye at very high microscopic concentrations, but have a low overall optical density, thereby eliminating reabsorption. Using this system, we have studied the effects of concentration on the fluorescence lifetime of one such dye, sulforhodamine 101. We have observed that the lifetime decreases as a function of encapsulated dye concentration, which is indicative of collisional quenching. The lifetime decreases from 4.5 nsec for sulforhodamine in dilute aqueous solution to 0.69 ns at an encapsulated concentration of 33 m M . The bimolecular rate constant for this event is 2.6 10¹⁰ M ^-1 s^-1, consistent with a diffusion controlled event. However, the quenching constant calculated from changes in intensity is 2.2 10¹¹ M ^-1 s^-1. Thus, collisional quenching is not the predominant mechanism of quenching. The absorption spectra of dye in vesicles indicate an important contribution from static complex formation. Förster distance calculations indicate that energy transfer can also occur to a significant extent, with a predicted efficiency of transfer of 34% at a dye concentration of only 1 m M     

Environments of ethidium binding to allosteric Dna: II. Accessibility, mobility and mode of binding

DNA binding compounds were previously shown to bind to the right-handed DNA forms and hybrid B-Z forms in a highly cooperative manner and indicate that structural specificity plays a key role in a ligand binding to DNA. In this study, the modes of binding and structural specificity of agents to unusual DNA are examined by a variety of fluorescence techniques (intensity, polarization and quenching, etc.) to explore a reliable method to detect the association environment of ligands to deoxyoligonucleotides initially containing a B-Z junction between the left-handed Z-DNA and right-handed B-DNA. The results of fluorescence energy transfer measurement demonstrated that the ligand molecules bind to the allosterically converted DNA structures by intercalation. In the absence of high-resolution structural data, this fluorescence energy transfer measurement allowed reliable measures and infer the binding environment of ligands to the allosteric DNA structures.     

ENERGY TRANSFER IN THE ACCESSORY PIGMENTS R-PHYCOERYTHRIN AND C-PHYCOCYANIN

Abstract— The photosynthetic accessory pigments R-phycoerythrin and C-phycocyanin were extracted from a red sea-weed and a filamentous blue-green alga. Investigation of quenching of phycoerythrin fluorescence by phycocyanin at room and liquid nitrogen temperatures yielded rates for the SternVolmer quenching constants ( K_q From these values, rate constants k_(D–A) were calculated for the transfer of energy from phycoerythrin to phycocyanin.
The transfer rate constants obtained were such that a collisional transfer mechanism was ruled out, and were of similar order of magnitude as the rate constants for transfer of electronic energy in mixed organic crystals.     

Multi-spectroscopic Investigations of Aspirin and Colchicine Interactions with Human Hemoglobin: Binary and Ternary Systems

The interactions of colchicine (COL) and aspirin (ASA) with human hemoglobin (HB) was studied by fluorescence, UV/vis absorption, resonance light scattering, synchronous fluorescence and circular dichroism (CD) spectroscopic techniques under physiological conditions. The inherent binding information, including the quenching mechanism, binding constants, number of binding sites, effective quenching constant, fraction of the initial fluorescence and thermodynamic parameters were determined by the fluorescence quenching technique at different temperatures. The results proved that the mechanism of fluorescence quenching of HB by COL and ASA is due to formation of HB–drug complexes in the binary and ternary systems. The distance between the acceptor drugs and HB was estimated by Förster’s equation on the basis of fluorescence energy transfer. In addition, according to the synchronous fluorescence spectra of HB, the results showed that the fluorescence quenching of HB originated solely from the tryptophan residues and indicated a conformational change for HB caused by addition of the drugs. Far-UV CD spectra of HB were recorded before and after the addition of ASA and COL both as binary and ternary systems. An increase in intensity of the positive CD peak of HB was observed in the presence of these drugs. The results were interpreted as excited state interactions between the aromatic residues of the HB binding sites and the drugs bound to them.     

Luminescent study on the binding interaction of bioactive imidazole with bovine serum albumin-A static quenching mechanism

Novel bioactive imidazole derivatives were synthesized and characterized by NMR spectra, mass and CHN analysis. The interaction between the imidazole derivative and bovine serum albumin (BSA) was investigated by fluorescence and UV-vis absorption spectroscopy. The fluorescence quenching of BSA by the imidazole derivatives may be due to the formation of imidazole-BSA complex. The fluorescence quenching mechanism of BSA by imidazole was analyzed and the binding constant has been calculated. The binding distance between imidazole and BSA was obtained based on Forester's non-radiation energy transfer (FRET). The effect of some common ions on the binding constant between imidazole and BSA was also examined.     

HOW HYDROGEN BONDING OF CARBAZOLE TO ETHANOL AFFECTS ITS FLUORESCENCE QUENCHING RATE BY ELECTRON ACCEPTOR QUENCHER MOLECULES

Abstract— Absorption spectra and fluorescence quenching of carbazole (C) and N-ethyl carbazole (EC) by some aliphatic halocarbons have been studied in 3-methylpentane (3MP) and ethanol (EtOH). Both steady-state and transient kinetic measurements were used to determine the quenching rate constants ( k _q). These rate constants were found to be sensitive to the polarity and also to the hydrogen bonding ability of the solvents used. Endothermic electron transfer reactions were shown to take place in these systems. When the electron donor C is involved in hydrogen bonding with EtOH, the net effect is a decrease in k _q. This is explained by taking into account the red-shifted (0,0) transition energy (E_s) of the ¹ L _b absorption band when the reaction is nearly diffusion controlled. For reaction far from diffusion controlled, the E _s value correction to k_q is not enough to explain the low k _qvalue observed.     

MIGRATION OF EXCITATION ENERGY FROM THIONINE TO METHYLENE BLUE IN MICELLES

Abstract— The main absorption bands of thionine (Th⁺) and methylene blue (MB⁺) in aqueous solution lie at 598 nm and 664 nm, respectively. This position permits excitation energy transfer from Th⁺ to MB⁺, but not vice versa. We describe here studies of such transfer between these molecules adsorbed on micelles of sodium lauryl sulfate (SLS), imitating, at least to some extent, the state of pigments in chloroplasts.
The SLS concentration was varied from 3.0 to 11 × 10^-3 M. In the presence of dye, aggregation to micelles, each containing 70–100 detergent molecules, begins at about 3.0 × 10^-3 M SLS. Practically all dye ions are adsorbed on these micelles as soon as their formation begins.
Energy transfer from adsorbed Th⁺ ions to adsorbed MB⁺ ions can be demonstrated by observing the quenching of the fluorescence of thionine and the sensitization of that of methylene blue.
At [Th⁺] = [MB⁺] = 1 × 10^-5 M , the most efficient energy transfer (82 per cent efficiency, as derived from measurements of the quenching of Th⁺ fluorescence, or 90 per cent, as derived from sensitization of MB⁺ fluorescence) is observed at the lowest SLS-concentration (3.0 × 10^-3 M ), when the only micelles present are those formed by aggregation of dye-carrying low molecular complexes of SLS with dye cations. Each micelle carries, under these conditions, 10–14 molecules of the two dyes, and the distance between two closest dye ions is about 16 A. Transfer becomes less efficient as the SLS-concentration increases, causing pigment molecules to distribute themselves among a greater number of micelles.     

Study on the Synergism Effect of Lomefloxacin and Ofloxacin for Bovine Serum Albumin in Solution by Spectroscopic Techniques

Both lomefloxacin (LOM) and ofloxacin (OFL) have a powerful ability to quench the fluorescence of bovine serum albumin (BSA). The fluorescence quenching action is much stronger when the two drugs coexist. The synergism between LOM and OFL was studied using fluorescence and ultraviolet spectroscopy under imitated physiological conditions. The results show that static quenching and non-radiation energy transfer are the main reasons for the fluorescence quenching. The synergism results in both the reduction of the binding stability between drugs and BSA and an increase of the free drug concentration, which will increase the efficacy of drugs. The thermodynamic parameters at different temperatures were calculated and the binding distances r between the drugs and BSA were obtained based on Försters theory of non-radiation energy transfer. The synchronous fluorescence spectra indicated that the effect of synergism affected the conformation of BSA.     

Characterization of the interaction between farrerol and bovine serum albumin by fluorescence and circular dichroism

The binding of farrerol to bovine serum albumin (BSA) in aqueous solution was investigated by fluorescence quenching spectra, synchronous fluorescence spectra, circular dichroism (CD) and the three-dimensional (3D) fluorescence spectra at pH 7.40. The results of fluorescence titration indicated that farrerol could quench the intrinsic fluorescence of BSA in a static quenching way. The cause of showing upward curvy patterns in Stern-Volmer plots was analyzed. The binding sites number n and binding constant K using fluorescence quenching equation at 310 K were calculated. The binding distance and the energy transfer efficiency between farrerol and BSA were also obtained according to the theory of F?rster's non-radiation energy transfer. The effect of some metal ions on the binding constant of farrerol with BSA was also studied. The effect of farrerol on the conformation of BSA was analyzed using CD, synchronous fluorescence spectra and three-dimensional (3D) fluorescence spectra under experimental conditions. Furthermore, the fluorescence displacement experiments indicated that farrerol could bind to the site I of BSA.