Spectroscopic studies on the interaction mechanisms of safranin T with herring sperm DNA using acridine orange as a fluorescence probe

Under the condition of physiological pH environment (pH = 7.40), the interactions of safranin T (ST) with herring sperm DNA were studied by means of spectral methods using acridine orange (AO) as a fluorescence probe. The spectroscopic characteristics of DNA–AO in the case of ST (along with the increase of concentration) were observed in an aqueous medium. The binding constants for ST stranded DNA and competitive bindings of ST interacting with DNA–AO systems were examined by fluorescence spectra, and the binding mechanism of ST with DNA was researched via viscosity measurements. All the testimony manifested that bonding modes between ST and DNA were evidenced to be intercalative binding and electrostatic binding, and the combining constant of ST with DNA was obtained. The binding of ST to DNA was driven by entropy and enthalpy through the calculated thermodynamic parameters (Δ_rH_m^?, Δ_rS_m and Δ_rG_m^?). Copyright © 2014 John Wiley & Sons, Ltd.     

Characterization of the binding of neomycin/paromomycin sulfate with DNA using acridine orange as fluorescence probe and molecular docking technique

The binding of neomycin sulfate (NS)/paromomycin sulfate (PS) with DNA was investigated by fluorescence quenching using acridine orange (AO) as a fluorescence probe. Fluorescence lifetime, FT-IR, circular dichroism (CD), relative viscosity, ionic strength, DNA melting temperature, and molecular docking were performed to explore the binding mechanism. The binding constant of NS/PS and DNA was 6.70 × 10³/1.44 × 10³ L mol^?1 at 291 K. The values of ΔH^θ, ΔS^θ, and ΔG^θ suggested that van der Waals force or hydrogen bond might be the main binding force between NS/PS and DNA. The results of Stern–Volmer plots and fluorescence lifetime measurements all revealed that NS/PS quenching the fluorescence of DNA–AO was static in nature. FT-IR indicated that the interaction between DNA and NS/PS did occur. The relative viscosity and melting temperature of DNA were almost unchanged when NS/PS was introduced to the solution. The fluorescence intensity of NS/PS–DNA–AO was decreased with the increase in the ionic strength. For CD spectra of DNA, the intensity of positive band at nearly 275 nm was decreased and that of negative band at nearly 245 nm was increased with the increase in the concentration of NS/PS. The binding constant of NS/PS with double-stranded DNA (dsDNA) was larger than that of NS/PS with single-stranded DNA (ssDNA). From these studies, the binding mode of NS/PS with DNA was evaluated to be groove binding. The results of molecular docking further indicated that NS/PS could enter into the minor groove in the A–T rich region of DNA.     

A simple and sensitive fluorescence method for the determination of trace ozone in air using acridine red as a probe

The ozone in an air sample was trapped by H₃BO₃‐LK solution to produce iodine (I₂) that interacted with excess I^– to form I₃^–. In pH 4.0 acetate buffer solutions, the I₃^– reacted with acridine red to form acridine red–I₃ ion association particles that resulted in the fluorescence peak decreased at 553 nm. The decreased value ΔF_{553 nm} is linear to the O₃ concentration in the range 0.08–53.3 × 10^–6 mol/L, with a detection limit of 4 × 10^–8 mol/L. This fluorescence method was used to determine ozone in air samples, and the results were in agreement with that of indigo carmine spectrophotometry. Copyright © 2014 John Wiley & Sons, Ltd.     

Mutagenesis of acridine orange in mitotic cleavage nuclei of the silkworm, Bombyx mori

The present study was done to determine whether acridine orange (AO) is mutagenic for the mitotic cleavage nuclei in the silkworm. The mutation frequency was estimated by the specific locus method using egg-color genes. AO was injected into the body cavity of marked female pupae (homozygous for pe and re genes) in active vitellogenesis (prophase I oocytes). The moths emerging from the treated pupae were mated to wild type male moths. AO increased the frequency of mosaic type mutations, indicating that AO has a positive mutagenic action on the paternal chromosomes in the mitotic cleavage nuclei in the silkworm.     

Acridine orange distribution and fluorescence spectra in myoblasts and single muscle fibers

Acridine orange (AO) fluorescence spectra in nuclei and cytoplasm of living myoblasts L6J1 and frog single muscle fibers have been studied using spectral scanning system of Leica TCS SL confocal microscope. AO fluorescence spectra in salt solutions dependent on free AO concentrations or in complex with DNA have also been obtained. Myoblast nuclei fluoresced in the green spectral region with maximum at about 530 nm; nucleoli had the brightest fluorescence. The fluorescence of nuclear chromatin was not uniform. Similar fluorescence of nuclei and nucleoli was observed in frog single muscle fibers. Uniform, weak, green fluorescence was observed in the myoblast cytoplasm. In the sarcoplasm of muscle fibers, AO green fluorescence was seen in A discs. In the cytoplasm of myoblasts and muscle fibers stained with AO, different red, yellow, and green fluorescent granules, which were acidic organelles, were visualized. The comparison of AO fluorescence spectra in living cells with AO fluorescence spectra in buffer solutions with different AO concentrations and AO in complex with DNA enables the estimation of the AO concentration in acidic granules. It is important for the evaluation of these cellular organelles functions in intracellular transport, adaptation, and apoptosis, as well as in a number of pathological processes.     

Study on the interaction between human serum albumin and a novel bioactive acridine derivative using optical spectroscopy

The interaction of a novel bioactive agent N‐{[N‐(2‐dimethylamino) ethyl] acridine‐4‐carboxamide}‐α‐alanine [N‐(ACR‐4‐CA)‐α‐ALA] with human serum albumin (HSA) was investigated by fluorescence spectroscopy, UV–vis absorption and circular dichroism spectrophotometric techniques under simulative physiological conditions. The fluorescence quenching of HSA by addition of N‐(ACR‐4‐CA)‐α‐ALA is due to static quenching and hydrogen bonding. Moreover, hydrophobic interactions play a role in the binding of N‐(ACR‐4‐CA)‐α‐ALA to HSA as well. The number of binding sites, n, and the binding constant values, K_A, were noted to be 0.88 and 3.4 × 10⁴ L mol^?1 for N‐(ACR‐4‐CA)‐α‐ALA at 293 K. The binding distances and the energy transfer efficiency between N‐(ACR‐4‐CA)‐α‐ALA and protein were determined. The negative value of enthalpy change and positive value of entropy change in the present study indicated that both hydrogen bonding and hydrophobic forces played a major role in the binding of N‐(ACR‐4‐CA)‐α‐ALA to HSA. Copyright © 2010 John Wiley & Sons, Ltd.     

Intracellular distribution of the fluorescent dye nonyl acridine orange responds to the mitochondrial membrane potential: implications for assays of cardiolipin and mitochondrial mass

Cardiolipin, a polyunsaturated acidic phospholipid, is found exclusively in bacterial and mitochondrial membranes where it is intimately associated with the enzyme complexes of the respiratory chain. Cardiolipin structure and concentration are central to the function of these enzyme complexes and damage to the phospholipid may have consequences for mitochondrial function. The fluorescent dye, 10 nonyl acridine orange (NAO), has been shown to bind cardiolipin in vitro and is frequently used as a stain in living cells to assay cardiolipin content. Additionally, NAO staining has been used to measure the mitochondrial content of cells as dye binding to mitochondria is reportedly independent of the membrane potential. We used confocal microscopy to examine the properties of NAO in cortical astrocytes, neonatal cardiomyocytes and in isolated brain mitochondria. We show that NAO, a lipophilic cation, stained mitochondria selectively. However, the accumulation of the dye was clearly dependent upon the mitochondrial membrane potential and depolarisation of mitochondria induced a redistribution of dye. Moreover, depolarisation of mitochondria prior to NAO staining also resulted in a reduced NAO signal. These observations demonstrate that loading and retention of NAO is dependant upon membrane potential, and that the dye cannot be used as an assay of either cardiolipin or mitochondrial mass in living cells.     

Kinetic studies of interaction between acridine orange and DNA

The interaction between acridine orange (AO) and deoxyribonucleic acid (DNA) was studied by the stopped-flow method. The spectral change of AO due to interaction with DNA was followed over the wavelength range 350–600 nm at various concentration ratios of DNA phosphate to dye. The spectral change observed by the stopped-flow method was found distinctly different from that, during the dead-time, leading to a conclusion that the binding of AO to the outside of DNA occurs much faster than the intercalation into base pairs of DNA. The dependence of the rate of reaction on the reactant concentration and on the salt, concentration of the solution was also studied. The results are consistent with the mechanism that the intercalation proceeds via the outside bound state.     

Study on the interaction between ginsenoside Rh2 and calf thymus DNA by spectroscopic techniques

The interaction between ginsenoside Rh2 (G‐Rh2) and calf thymus DNA (ctDNA) was investigated by spectroscopic methods including UV–vis absorption, fluorescence and circular dichroism (CD) spectroscopy, coupled with DNA melting techniques and viscosity measurements. Stern–Volmer plots at different temperatures proved that the quenching mechanism was a static quenching procedure. The thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS) were calculated to be –22.83 KJ · mol^–1and 15.11 J · mol^–1 · K^–1by van ’t Hoff equation, suggesting that hydrophobic force might play a major role in the binding of G‐Rh2 to ctDNA. Moreover, the fluorescence quenching study with potassium iodide as quencher indicated that the K_SV (Stern–Volmer quenching constant) value for the bound G‐Rh2 with ctDNA was lower than the free G‐Rh2. The relative viscosity of ctDNA increased with the addition of G‐Rh2 and also the ctDNA melting temperature increased in the presence of G‐Rh2. Denatured DNA studies showed that quenching by single‐stranded DNA was less than that by double‐stranded DNA. The observed changes in CD spectra also demonstrated that the intensities of the positive and negative bands decreased with the addition of G‐Rh2. The experimental results suggest that G‐Rh2 molecules bind to ctDNA via an intercalative binding mode. Copyright © 2015 John Wiley & Sons, Ltd.     

Lymphocyte stimulation by concanavalin a studied by the fluorescent probe acridine orange

Summary Viable mouse thymocytes or spleen leucocytes stained with acridine orange (AO) were divided into one part used for stimulation, and the other part for control. Analysis of cellular green-fluorescence emission enabled physicochemical changes in lymphocytes to be detected after 30 min stimulation with the mitogens concanavalin A (Con A) and pokeweed mitogen (PWM). No change in fluorescence was observed with the nonmitogenic reagent wheat germ lectin (WGL) or with allogeneic cell stimulation (MLR). When green fluorescence intensity of individual cells was monitored by microfluorimetry, 30 min stimulation with Con A induced an increase, whereas PWM induced a decrease. When analysed by fluorescence spectrophotometry, Con A induced a 2 nm blue shift in emission maximum and a decrease in polarization values.Supported by grants from the Anti-Cancer Council of Victoria. We are grateful to Dr. H.A. Ward for helpful discussion     

Mutagenic effect of acridine orange on the expression of penicillin G acylase and beta-lactamase in Escherichia coli

AIMS: The present work aimed to improve the production of penicillin G acylase (PGA) and reduce the beta-lactamase activity through acridine orange (AO) induced mutation in Escherichia coli. METHODS AND RESULTS: Three wild E. coli strains BDCS-N-FMu10, BDCS-N-S21 and BDCS-N-W50, producing both the enzymes PGA and beta-lactamase were treated by AO. Minimum inhibitory concentration of AO was 10 microg ml(-1) and it was noted that bacterial growth was gradually suppressed by increasing the concentration of AO from 10 to 100 microg ml(-1). The highest concentration that gave permissible growth rate was 50 microg ml(-1). The isolated survivals were screened on the bases of PGA and beta-lactamase activities. Among the retained mutants, the occurrence of beta-lactamase deficient ones (91%) was significantly higher than penicillin acylase deficient ones (27%). CONCLUSIONS: In seven of the mutants, PGA activity was enhanced with considerable decrease in beta-lactamase activity. One of the mutant strains (BDCS-N-M36) exhibited very negligible expression of beta-lactamase activity and twofold increase in PGA activity [12.7 mg 6-amino-penicillanic acid (6-APA) h(-1) mg(-1) wet cells] compared with that in the wild-type strain (6.3 mg 6-APA h(-1) mg(-1) wet cells). SIGNIFICANCE AND IMPACT OF THE STUDY: The treatment of E. coli cells with AO resulted in mutants with enhanced production of PGA and inactivation of beta-lactamase. These mutants could be used for industrial production of PGA.     

Asymmetric inclusion by de novo designed proteins: fluorescence probe studies on amphiphilic alpha-helix bundles

The inclusion feature and supersecondary structure of the de novo designed proteins which are constructed with several amphiphilic alpha-helices and flexible linkage parts were investigated with fluorescence probes. Five types of small proteins (or peptides) have been designed, which are composed of 2, 3, 4, 4, and 6 helices, respectively, and are linked with only linear junctions except for one of 4-helix proteins. All of these proteins have inclusion ability for hydrophobic fluorophores. Further, by the analysis of fluorescence polarization anisotropy, it was suggested that these proteins include guest molecules in compact helix bundles constructed with about 4 helices. Asymmetric inclusion of both monomer and stacked dimer of acridine orange derivatives was found by means of induced circular dichroism except for the 4-helix protein with cross-junction. The chirality of the included dimer proved to be in accordance with the chiral sense of alpha-helical coiled-coil. The 6-helix protein has especially high efficiency in inclusion for any fluorophores examined in this study and brings about a significant blue-shift of maximal emission for 8-anilino-1-naphthalenesulfonate.     

Caffeine and other xanthines as cytochemical blockers and removers of heterocyclic DNA intercalators from chromatin

Caffeine (CAF) and other xanthines non-covalently bind with the cationic fluorescent dye acridine orange (AO) and with other heterocyclic mutagens and carcinogens that are known to intercalate into double-stranded DNA (dsDNA). Fluorescence microscopy and spectrofluorometry studies were employed to test the ability of caffeine and certain other methyl substituted xanthines, with different binding affinities for AO, to inhibit and to reverse the intercalation of AO and other heterocyclic agents from intercalation with the DNA of nuclear chromatin of air-dried cells. Results indicated that xanthines with binding affinity for AO greater than 150 m(-1) block the AO molecule in a concentration dependent manner and comply with mass action kinetics. Thus CAF and other xanthines can be used to either inhibit intercalation of AO into nuclear DNA or to remove AO once intercalated into nuclear DNA. The interactions between other planar heterocyclics, xanthines, and nuclear chromatin dsDNA were also found to be non-covalent. Studies are needed to determine the ability of CAF and other xanthines to block and/or remove polyaromatic hydrocarbon (PAH) intercalators from the DNA of living cells.     

Rapid assessment of islet viability with acridine orange and propidium iodide

Summary A simple, rapid method for estimating the viability of isolated islets of Langerhans with fluorescent dyes is described. Low concentrations of acridine orange and propidium iodide (AO/PI) were used to visualize living and dead islet cells simultaneously. AO/PI-stained islets can be divided into three distinct groups. Group A islets fluoresce green, contain insulin, and have normal ultrastructure; group C islets fluoresce primarily red, contain little or no insulin, and have cells with disrupted cellular membranes. Group B islets fluoresce red, green, and yellow. The yellow color is due to the addition of two primary colors from the superimposed red and green fluorescing cells. In this assay, the interpretation that red islet cells are dead and green islet cells are alive was confirmed by sequentially staining single islet cells with AO/PI and trypan blue. The observation that red islets are dead was confirmed by heat-killing, enzymatically damaging, treating with ethanol, or depriving islets of nutrients and observing the red fluorescence. This assay should be useful in studies where the assessment of islet viability is essential. Preliminary reports of this work were presented at two meetings and were published in abstract form (24,25). This research was supported in part by the National Institutes of Health, Bethesda, MD, grant DK 18115.     

Studies on the formation and stability of triplex DNA using fluorescence correlation spectroscopy

Triplex DNA has become one of the most useful recognition motifs in the design of new molecular biology tools, therapeutic agents and sophisticated DNA‐based nanomaterials because of its direct recognition of natural double‐stranded DNA. In this paper, we developed a sensitive and microscale method to study the formation and stability characterization of triplex DNA using fluorescence correlation spectroscopy (FCS). The principle of this method is mainly based on the excellent capacity of FCS for sensitively distinguishing between free single‐strand DNA (ssDNA) fluorescent probes and fluorescent probe–double‐strand DNA (dsDNA) hybridized complexes. First, we systematically investigated the experimental conditions of triplex DNA formation. Then, we evaluated the equilibrium association constants (K_a) under different ssDNA probe lengths, composition and pH. Finally, we used FCS to measure the hybridization fraction of a 20‐mer perfectly matched ssDNA probe and three single‐base mismatched ssDNA probes with 146‐mer dsDNA. Our data illustrated that FCS is a useful tool for the direct determination of the thermodynamic parameters of triplex DNA formation and discrimination of a single‐base mismatch of triplex DNA without denaturation. Compared with current methods, our method is characterized by high sensitivity, good universality and small sample and reagent requirements. More importantly, our method has the potential to become a platform for triplex DNA research in vitro. Copyright © 2015 John Wiley & Sons, Ltd.     

Interaction between tryptophan‐Sm(III) complex and DNA with the use of a acridine orange dye fluorophor probe

The interaction of the Trp–Sm(III) complex with herring sperm DNA (hs‐DNA) was investigated with the use of acridine orange (AO) dye as a spectral probe for UV‐vis spectrophotometry and fluorescence spectroscopy. The results showed that the both the Trp–Sm(III) complex and the AO molecule could intercalate into the double helix of the DNA. The Sm(III)–(Trp)₃ complex was stabilized by intercalation into the DNA with binding constants: K^?_25°C = 7.14 × 10⁵ L·mol^?1 and K^?_37°C = 5.28 × 10⁴ L·mol^?1, and it could displace the AO dye from the AO–DNA complex in a competitive reaction. Computation of the thermodynamic functions demonstrates that Δ_rH_m^? is the primary driving power of the interaction between the Sm(III)(Trp)₃ complex and the DNA. The results from Scatchard and viscometry methods suggested that the interaction mode between the Sm(III)(Trp)₃ complex and the hs‐DNA is groove binding and weak intercalation binding. Copyright © 2015 John Wiley & Sons, Ltd.