Mechanism of acridine orange interaction with phospholipids and proteins in renal microvillus vesicles

The mechanism of interaction of acridine orange (AO), a fluorescent, weak base, with rabbit kidney brush border membrane vesicles (BBMV) has been studied by absorption, and steady-state and time-resolved fluorescence spectroscopy. Equilibrium binding experiments indicate that AO binds to an apparent single class of sites on BBMV with a dissociation constant of 90 microM and site stoichiometry of 810 nmol/mg protein. The absorption spectra AO indicate that BBMV induces aggregation of AO; experiments with lipid vesicles show that the aggregation requires BBMV membrane proteins. Fluorescence stopped-flow experiments in which 0.15 mg/ml BBMV is mixed with increasing concentrations of AO result in a time course of fluorescence enhancement for [AO] less than 1.5 microM, and of fluorescence quenching for [AO] greater than 1.5 microM. Similar stopped-flow experiments with phosphatidylcholine lipid vesicles result only in a fluorescence enhancement time course. These results indicate the presence of two parallel pathways for AO binding to BBMV: one for AO binding to BBMV lipid, the other for AO binding to BBMV protein. Nanosecond lifetime measurements and fluorescence titration experiments confirm the presence of two environments for AO in BBMV. Fluorescence stopped-flow experiments indicate that AO responds to the imposition of an outwardly directed proton gradient by a rapid (less than 0.5 s) decrease in fluorescence, corresponding to re-equilibration of AO into the acidic intravesicular compartment, followed by an increase in fluorescence, corresponding to proton flux across the membrane. These findings have been incorporated into a stepwise mechanism for AO interaction with BBMV which have direct implications for the use of AO as a pH indicator in biological systems.     

Effect of the segregation of neutral red, acridine orange and ammonium chloride by L cells (subline LSM) on lysosomal hydrolase activity

Kinetics of Neutral red (NR) and Acridine orange (AO) uptake by cultured L cells (subline LSM) has been studied. It was found that the uptake of both NR and AO, with their constant concentrations in the medium was characterized as a two-phase process. During 2 hours, these cells concentrated as much as 90% of the total amount of NR and AO taken up during the whole incubation period. The segregation and accumulation of NR, AO as well as NH4Cl took place in lysosomes. NR and AO concentrations within the cells exceed by 600 and 400 times, respectively, those in the medium. NR, AO and NH4+ accumulation in cells resulted in inhibition of the activity of the following lysosomal hydrolases: cathepsins B and D, acid lipase, N-acetyl-beta,D-glucosaminidase, beta-galactosidase, acid phosphatase and galactosyltransferase, the latter being a marker of Golgi apparatus. The effect of lysosomal enzyme activity inhibition on the cell economy, and a possible role of lysosomotropic agents as regulators of the lysosomal apparatus functional activity are discussed.     

Synaptic vesicle acidification and exocytosis studied with acridine orange fluorescence in rat brain synaptosomes

The acidification of synaptic vesicles (SV) in rat brain synaptosomes was studied using acridine orange (AO) as a fluorescent probe. In synaptosomal suspensions the AO fluorescence was partially quenched, indicating the presence of an acidic compartment. In permeabilized synaptosomes, the quenching was augmented by MgATP and was sensitive to concanamycin A, a specific inhibitor of the V-type H⁺-ATPase known to be present in synaptic vesicles. Some ATP-dependent acidification was also observed without permeabilization, suggesting that a fraction of synaptosomes (ca. 15%) was unsealed, irrespective of the method used to prepare the synaptosomes (sucrose or Ficoll density gradient, sedimentation or flotation). Depolarization of synaptosomes with 30 mM KCl resulted in an immediate, albeit small, rise in AO fluorescence that was prevented by the removal of Ca²⁺ or by substituting NaCl for KCl. This response is consistent with depolarization-evoked release of the acidic contents of an exocytosis-competent pool of synaptic vesicles, representing ca. 5% of the total. No further AO release subsequent to the immediate phase was observed in depolarized synaptosomes, which indicates an extremely rapid reacidification. The results demonstrate that AO fluorescence is suitable for monitoring SV acidification within synaptosomes, and may be used to derive an independent estimate of the relative size of the immediately releasable SV pool. In addition, the use of AO might be advantageous for the assessment of synaptosomal integrity by comparing the ATP-dependent acidification in intact and permeabilized synaptosomes.     

Systematic colocalization errors between acridine orange and EGFP in astrocyte vesicular organelles

Dual-color imaging of acridine orange (AO) and EGFP fused to a vesicular glutamate transporter or the vesicle-associated membrane proteins 2 or 3 has been used to visualize a supposedly well-defined subpopulation of glutamatergic astrocytic secretory vesicles undergoing regulated exocytosis. However, AO metachromasy results in the concomitant emission of green and red fluorescence from AO-stained tissue. Therefore, the question arises whether AO and EGFP fluorescence can be distinguished reliably. We used evanescent-field imaging with spectral fluorescence detection as well as fluorescence lifetime imaging microscopy to demonstrate that green fluorescent AO monomers inevitably coexist with red fluorescing AO dimers, at the level of single astroglial vesicles. The green monomer emission spectrally overlaps with that of EGFP and produces a false apparent colocalization on dual-color images. On fluorophore abundance maps calculated from spectrally resolved and unmixed single-vesicle spectral image stacks, EGFP is obscured by the strong green monomer fluorescence, precluding the detection of EGFP. Hence, extreme caution is required when deriving quantitative colocalization information from images of dim fluorescing EGFP-tagged organelles colabeled with bright and broadly emitting dyes like AO. We finally introduce FM4-64/EGFP dual-color imaging as a remedy for imaging a distinct population of astroglial fusion-competent secretory vesicles.     

Molecular Complexes of acridine orange and nucleosides

Summary The addition of various nucleosides to the aqueous AO solution brings about the red shift of the absorption band of AO monomer and the enhancement of the AO fluorescence emission. These phenomena are attributable to the formation of a kind of molecular complex between AO monomer and nucleoside.The absorption and fluorescence characteristics and their thermal behaviours enable us to determine the association constant and the binding energy. The binding energy of AO with purines is larger than that with pyrimidines, and the association constant between AO and the deoxyribonucleoside is larger than that between AO and the ribonucleoside. For the molecular complexes dealt with, the face-to-face arrangement of AO and nucleoside, linking of AO with sugar by hydrogen bridge, may be more preferential than the side-by-side arrangement with the direct linkage between AO and nucleic acid base by hydrogen bonding. The Van der Waals-London interactions may be one of the essential factors for the binding in these molecular complexes.The association constant and the binding energy for AO-DNA and -RNA systems were also determined. The magnitude of these quantities seems to reflect the difference in the structure of these nucleic acids. The rather open structure of RNA compared with DNA is in favour of affinity with AO and gives the larger association constant than that for AO-DNA. The binding energy is somewhat larger for AO-DNA complex than for AO-RNA complex, probably due to the structural difference of the base arrangement; the stacked base pairs for DNA and the stacked bases for RNA.This may explain the selective degradation [12] of guanine photo-sensitized by dyes either in the free state or when incorporated to DNA or RNA, as discussed elsewhere.     

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.     

Synthesis of an acridine orange sulfonamide derivative with potent carbonic anhydrase IX inhibitory action

Acridine orange (AO) a fluorescent cationic dye used for the management of human musculoskeletal sarcomas, due to its strong tumoricidal action and accumulation in the acidic environment typical of hypoxic tumors, was used for the preparation of a primary sulfonamide derivative. The rationale behind the drug design is the fact that hypoxic, acidic tumors overexpress carbonic anhydrase (CA, EC 4.2.1.1) isoforms, such as CA IX, which is involved in pH regulation, proliferation, cell migration and invasion, and this enzyme is strongly inhibited by primary sulfonamides. The AO-sulfonamide derivative was indeed a potent, low nanomolar CA IX inhibitor whereas its inhibition of the cytosolic isoforms CA I and II was in the micromolar range. A second transmembrane, tumor-associated isoform, CA XII, was also effectively inhibited by the AO-sulfonamide derivative, making this compound an interesting theranostic agent for the management of hypoxic tumors.     

Interaction of poly- ,L-glutamic acid with acridine orange

Interaction of poly-α,L -glutamic acid (PGLA) with acridine orange (AO) was studied with circular dichroism and absorption spectra measurements. The following results were observed: (1) the addition of a comparable amount of AO with the glutamyl residue to the PLGA solution at pH of 4.5 reduced the fraction of helix of the polymer; (2) when AO was added to the PGLA solution, the pH range of the helix-coil transition of the polymer shifted toward higher pH regions; and (3) when the mixture of the same amount of AO and the glutamyl residue was brought to the neutral and alkaline pH region, some induced circular dichroism bands were observed. In this case, it was assumed that PLGA in the system takes a helical form due to the neutralization of the anionized side chains by the cationic species of AO. We concluded that AO molecules bound to the carboxylate groups of the side chains of PLGA arrange to from a righthanded super-helix which surrounds the core of the righthanded α-helix of PLGA.     

Use of acridine orange for histologic analysis of the central nervous system

The application of the fluorescent dye acridine orange (AO) to the staining of histologic sections of the brain, and its use for automatic cyto- and histophotometric evaluation are described and compared with the results obtained using cresyl violet. The most suitable procedure for aldehyde-fixed brain tissue, embedded in paraffin and sectioned at 5 micron, proved to be treatment of the sections with an aqueous solution of AO (1:50,000) at pH 1.2 for 30 min, followed by rinsing in distilled water for 10 min. This procedure revealed the morphology in a highly acceptable manner, clearly differentiating various cell components; its characteristics included exact reproducibility and high contrast. The degree of fading was calculable, with a very gradual decrease in fluorescent intensity. The AO procedure appears to be compatible with most other staining procedures that do not rely on the same binding mechanisms. Thus, AO staining has two advantages over the classical cytoarchitectural staining methods: first, it is more suitable for automated morphometric analysis, and second, it can be used in conjunction with immunologic and other techniques.     

Vacuolar accumulation of acridine orange and neutral red in zygotic and somatic embryos of carrot (Daucus carota L.)

Summary The accumulation of neutral red and acridine orange, to indicate differences in vacuolar pH, was studied during embryogenesis of carrot. Neutral red accumulated barely in proembryogenic masses, but was present conspicuously in globular-shaped somatic embryos. From the late globular to the torpedo-shaped stage, it was mainly found in the root side of the somatic embryo. Here, neutral red was predominantly present in large dark-red to purple stained vesicles. In the cotyledons neutral red was found in small orange vesicles. In zygotic embryos of carrot, the dye was uniformly distributed with no specific localization in organelles. During germination, however, neutral red accumulated mainly in regions in the root side and the hypocotyl of the germling. Acridine orange was dispersed erratically in proembryogenic masses with a great variety in intensity. It was quite obviously present in early stages of somatic embryogenesis and restricted to the root side in late globular to torpedo-shaped embryos. Confocal images revealed the vacuolar presence of the fluorescence and the predominant presence in the protoderm. During germination of zygotic embryos the signal changed from uniform to localized, with sharp borders between fluorescent and non-fluorescent regions. Two to three days after the beginning of germination, acridine orange accumulated preferentially in the root tip of the germling. Differences between somatic and zygotic embryos and similarities between somatic embryogenesis and zygotic embryo germination are discussed.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - pH_c cytosolic pH - pH_e extracellular pH - pH_v vacuolar pH     

Spectrometric and voltammetric investigation of interaction of neutral red with calf thymus DNA: pH effect

The interaction of neutral red (NR) with calf thymus DNA (CT DNA) was investigated by spectrometric (UV-vis, circular dichroism and fluorescence) and voltammetric techniques. It was shown that the interaction of NR with DNA depended on the values of R (R is defined as the ratio of the concentration of NR to that of CT DNA) and pH of the solution. NR intercalated into CT DNA base pairs at lower R value (R < 2.4) and following by NR aggregating along the helical surface of DNA at higher R value (R > 2.4) in pH 6.0 solution. Interestingly, we found that at lower R value, NR intercalated into CT DNA with its long axis perpendicular or parallel to the dyad axis of DNA in the solution of pH 6.0. While in pH 7.0 solution, NR bound with CT DNA through intercalation and electrostatic interactions. The electrochemical inactive complexes, NR-2CT DNA, 3NR-CT DNA, and NR-CT DNA were formed when NR interacted with nucleic acids in pH 6.0 and 7.2 solutions, respectively. The corresponding intrinsic binding constants for these complexes were obtained by UV-vis and fluorescence spectrometric methods, respectively. The CD spectra showed that the conformation of CT DNA was converted from right-handed B-DNA to left-handed Z-DNA due to the aggregating of NR along the surface of DNA in pH 6.0 solution, whereas a conversion from B-DNA to C-DNA was induced due to the interaction of DNA with NR in pH 7.2 solution. Finally, two binding modes of NR with CT DNA in aqueous with different values of pH were shown in the scheme.     

The comparative analysis of interaction between acridine orange and DNA-containing systems]

The interaction between acridine orange (AO) and diluted and concentrated solutions of DNA, DNP systems and chromatin suspension at the physiologic ionic strength was investigated. The effect of AO on DNP systems was also investigated. It was shown that highest possible number of AO molecules bound to DNA made up 70% of the total number of nucleotides. The model of AO binding to DNA is proposed and used for calculation of constants of stronger and weaker AO-binding capacities equal to 6-10(6) M-1 and 1,7-10(5) M-1, respectively. The AO-DNA binding constants in DNP-complex are five as low. The primary number of binding sites in chromatin suspension made up 10% of the corresponding sites in DNA and increased as AO was adsorbed. AO induced the supercontraction of oriented DNP systems at the physiologic ionic strength and the appearance of the low-temperature melting hump.     

De-intercalation of ethidium bromide and acridine orange by xanthine derivatives and their modulatory effect on anticancer agents: a study of DNA-directed toxicity enlightened by time correlated single photon counting

Time Correlated Single Photon Counting (TCSPC) was used for the first time to analyze the effect/changes in the mode of intercalation of ethidium bromide (EtBr) and acridine orange (AO) to calf thymus DNA brought about due to interaction of naturally occurring methylxanthines such as theophylline (X1), theobromine (X2) and caffeine (X3). UV absorption and fluorescence studies were also carried to observe the behaviour of these xanthines on the modulation of the binding mode of anticancer agents (cisplatin, novantrone, and actinomycin D) and certain intercalating dyes (EtBr and AO) to DNA. In TCSPC analysis we found that when the concentration of the drugs (X1, X2 and X3) increased from 0.025 mM to 2 mM i.e. P/D 2.4 to P/D 0.03 reduction in intercalation of EtBr and AO was observed, suggesting that xanthine derivatives could play very important role in reducing the DNA-directed toxicity in a dose dependent manner. In TCSPC, the amplitude of smaller lifetime component A(1) and higher lifetime component A(2) are attributed to free and intercalated dye concentration and their variation could indicate the process of intercalation or reduced intercalation of EtBr and AO by xanthine derivatives. We found that at the maximum drug concentration the smaller lifetime component A(1) was increased by 7-8% and 17-37% in EtBr and AO intercalated complex respectively. Also the changes in lifetime and fluorescence decay profile were observed for the DNA-intercalated dyes before and after treatment with xanthines. Especially, at maximum P/D 0.03 the lifetime of DNA-intercalated EtBr and AO reduced by 1-2 ns. The present analysis reveals that xanthines are able to interact with free dyes and also with intercalated dyes, suggesting that when they interact with free dyes they might inhibit the further intercalation of dye molecules to DNA and the interaction with intercalated dyes might lead to displacement of the dyes resulting in de-intercalation. The results obtained from UV and fluorescence spectroscopy also support the present investigation of probable interaction of xanthines with the DNA damaging agents in modulating/reducing the DNA-directed toxicity.     

The effect of acridine orange on the structure of ribosomes

Exposure of Escherichia coli MRE-600 ribosomes to acridine orange (AO) at low ionic strength (1mM Tris-acetate pH 7.4) results in quantitative binding of the dye. Under our experimental conditions about a few hundred dye molecules can be bound to any one of the 30, 50, or 70-S particles. AO causes the 30 and the 50-S subunits to form ribosomal aggregates of approximate sedimentation constants of 70 and 100-S.     

Highly sensitive and surface-renewable electrochemical quartz crystal microbalance assays of heparin and chondroitin sulfate based on their effects on the electrodeposition of neutral red

The electrochemical quartz crystal microbalance (EQCM) technique was used to investigate the electrochemistry of neutral red (NR) in phosphate buffer solution (PBS) and the effects of coexisting heparin (Hep) or chondroitin sulfate (CS) for the first time. The pH dependence of the electrochemistry of NR was examined, and a V-shaped frequency response (versus time) was observed during the cyclic voltammetric experiment of NR in a nearly neutral medium (pH ca. 6.10-7.00), being due to the electrodeposition and stripping of the poorly soluble reduced product of NR (NR(Red)) at these pH values. The effects of potential scan rate, the concentration of NR, and several supporting electrolytes were examined at pH 6.80. The V-shaped response to the redox switching of NR was weakened by the introduction of Hep or CS, being due to the increased inhibition of the NR(Red) electrodeposition probably via the electrostatic interaction of the NR and especially the NR(Red) with Hep or CS. The height of the V-shaped response decreases with the increase of Hep or CS concentration, with limits of detection down to 3 nmol L(-1) for Hep and 2 nmol L(-1) for CS, respectively. The novel and surface-regenerable EQCM assay protocol based on the electrochemically switchable deposition of a dye is highly recommended for wide biosensing applications.     

Comparison of auramine-rhodamine B and acridine orange for staining of acid-fast bacteria.

In cooperation of 6 laboratories in Czechoslovakia and in the GDR, the efficiency of auramine-rhodamine B (AR) and acridine orange (AO) (short-time method) for staining of acid-fast bacilli was compared. Whereas a former comparison of AR and AO (original method) pointed out the superiority of AR, the investigation of both methods used as short-time procedures showed significantly more acid-fast rods after using AO. The number of "false positive" results was somewhat higher on AR staining. However the results depend not only on the method used but also on the procedure of staining and the optical equipment, and they are essentially influenced by the experience and proficiency of the microscopist. Taking into account the results of both studies both auramine-rhodamine B and acridine orange can be proposed for the staining of slides for microscopical detection of acid-fast rods. In case of AO, the short-time method is superior to the original long-time procedure.     

2-colored fluorescence of differentially condensed chromosomes stained with acridine orange]

Metaphase chromosomes of the Chinese hamster differentially-condensed under the influence of a) 5-bromdeoxyuridine, b) colcemide, and c) cold, were stained with acridine-orange (AO) in concentrations of 1.5 X 10(-7) to 3 X 10(-5) g/ml at pH 4.1 to 8.5. It was found that stretched chromosomal segments fluoresced in the orange/red part of the spectrum, whereas normally condensed ones--were green. The colour distribution along the chromosome depended mainly on the AO concentration and the exposure in the UV-light, and was independent of pH and molarity of the buffer. Apparently this phenomenon cannot be attributed to the uneven denaturation of the chromosomal DNA, but rather depends on structural and/or chemical differences between euchromatin and heterochromatin.     

Induced optical activity of chondroitin sulfate c–acridine orange complexes

Chondroitin sulfate C (CSC) and acrdiine orange (AO) formed two types of complexes at neutral pH, depending upon the order of mixing. The induced optical activity of AO was much more pronounced when the polysaccharide was added to dye than the dye to polymer (final concentration of dye was 5 × 10⁵M). The difference in aggregation of the dye molecules is believed to be responsible for the observed peculiarities. The Cotton effects of the CSC-to-dye solution displayed a sharp inversion near 59°C. and the profile at 76°C. was almost a mirror image of that at room temperature. At pH 1.3, however, the order of mixing became unimportant, suggesting that the carboxylate on the polysaccharide way involved more intimately than were sulfates in the peculiarities of the Cotton effects.     

Some photometric and fluorimetric characteristics of acridine orange and of its complexes with glycosaminoglycans: proposed method for their spectrophotofluorimetric evaluation. I. Spectrophotometric study

In spite of the wide use of AO as basic fluorochrome in cytology, histology and histochemistry, however there still persist some queries on the meaning of fluorescence due to the encounter of a such molecule with anionic polymers. From the spectrophotometric data exposed in the present paper, AO is stressed as a metachromatic colouring and therefore, as such, may be used in histochemical investigation. Besides, because of the reliable identificaton of the single types of GAG, screening may be performed at different pH, since metachromasia appears at different pH for the three different types of GAG. A useful counterproof may be performed by means of selective elutions of the AO/GAG complexes, being solutions with increasing ionic power.     

Selective staining of pericentromeric heterochromatin regions in chromosomes of spontaneously dividing cells with the use of the acridine orange fluorochrome

A novel phenomenon of unusual selective acridine orange (AO) staining ofpericentromeric heterochromatin regions (HRs) in chromosomal preparations from tissue with known spontaneous mitotic activity (chorionic villi, placenta, embryonic tissues, bone marrow, and testes), as well as embryonic stem cells, is described. Staining with 0.01% AO in a citric-phosphate (pH 5.5) or sodium phosphate (pH 7.0) buffer solution allows the HRs of human chromosomes (1q12, 9q12, 13p11.2, 14p11.2, 15p11.2, 16q11.2, 21p11.2, 22p11.2, and Yq12) and pericentromeric HRs of mouse chromosomes to be reliably detected by the red fluorescence of AO. This method of AO staining does not require any pretreatment. Explanations for metachromatic AO staining of polymorphic pericentromeric HRs in chromosomes of spontaneously dividing cells are suggested. A high reproducibility of the specific AO staining makes it possible to suggest its use as a reliable quick method for detection of polymorphic HRs of human chromosomes in cytogenetic prenatal diagnosis and oncohematology.