Structural basis for the anticoagulant activity of heparin. 2. Relationship of anticoagulant activity to the thermodynamics and fluorescence fading kinetics of acridine orange-heparin complexes.

Complexing heparin or dermatan sulfate with the fluorescent probe acridine orange provides a means of studying electrostatic as well as static and dynamic conformational aspects of these glycosaminoglycans via the thermodynamic and photochemical (fluorescence fading) properties of these complexes. The cooperative binding constants (Kq), fluorescence fading rate parameters (r'), and anticoagulant activities of heparins fractionated according to anionic density all showed qualitatively the same dependence upon anionic density. When Kq and r' were plotted against anticoagulant activity, empirical relationships were observed. Interestingly, the corresponding values for unfractionated dermatan sulfate fell on the lines defined by the heparin fractions. Temperature-dependence, studies demonstrated that differences in fading rate observed for heparins of different anionic densities are entropic in origin and reflect differences in the ability to assume a special configuration. Differences in activation entropy for fluorescence fading can be empirically correlated with anticoagulant activity. The latter correlation suggests a physical similarity in the roles played by anionic density in both fluorescence fading and anticoagulant activity.     

The use of acridine orange for testing blood platelet integrity

Two processes are involved in the accumulation of acridine orange in human blood platelets. One follows a diffusion like kinetics and is independent of the ATP level whereas the second one can be completely abolished by ATP depletion. The acridine orange incorporation rate seems to be a suitable parameter for testing platelet integrity. It reflects very sensitively the influence of the preparation method as well as of anticoagulating substances used on the stability of platelet suspensions. The rates of acridine orange incorporation and of aggregation were measured in platelet-rich plasma and in saline suspended platelets after gel filtration, respectively, over a period of 120 min storage. Both rates are influenced to a different degree by anticoagulating agents such as citrate, heparin and EDTA. When contact with anticoagulating agents during platelet preparation is avoided, platelets show a constant acridine orange incorporation and aggregation during storage and the smallest morphological alteration.     

Fluorescence of acridine orange in the composition of blood and tissues

The interaction of acridine orange with blood albumins and tissue cells from different organs of white mouse has been studied by the spectral luminescence method. It was shown that acridine orange, by penetrating cells or organelles, is able to intercalate between base pairs in the DNA molecule. It was found that the application of acridine orange as a fluorescent probe can influence the metabolic activity of organs.     

10N-nonyl acridine orange interacts with cardiolipin and allows the quantification of this phospholipid in isolated mitochondria.

The acridine orange derivative, 10N-nonyl acridine orange, is an appropriate marker of the inner mitochondrial membrane in whole cells. We use membrane model systems to demonstrate that 10N-nonyl acridine orange binds to negatively charged phospholipids (cardiolipin, phosphatidylinositol and phosphatidylserine). The stoichiometry has been found to be 2 mol 10N-nonyl acridine orange/mol cardiolipin and 1 mol dye/mol phosphatidylserine or phosphatidylinositol, while, with zwitterionic phospholipids, significant binding could not be detected. The affinity constants were 2 x 10(6) M-1 for cardiolipin-10N-nonyl-acridine-orange association and only 7 x 10(4) M-1 for that of phosphatidylserine and phosphatidylinositol association. The high affinity of the dye for cardiolipin may be explained by two essential interactions; firstly an electrostatic interaction between the quaternary ammonium of nonyl acridine orange and the ionized phosphate residues of cardiolipin and secondly, hydrophobic interactions between adjacent chromophores. A linear relationship was demonstrated between the cardiolipin content of model membranes and the incorporated dye. Consequently, a convenient and rapid method for cardiolipin quantification in membranes was established and applied to the cardiolipin-containing organelle, the mitochondrion.     

lectrochemical and conformational study of the interactions of fibrinogen with acidic polysaccharides

The interactions of fibrinogen wth acidic polysaccharides have been studied in connection with anticoagulant properties of heparin. Despite the high charge density of heparin, a polyelectrolyte complex of fibrinogen and heparin could not be detected at any mixing ratio by the measurement of turbidity, metachromasis with acridine orange, circular dichroism, and viscosity of their mixture solutions. Sodium cellulose sulphate and dextran sulphate, however, which have similar charge densities, formed precipitates of polyelectrolyte complexes with fibrinogen. This difference was presumed to be due to the secondary structure characteristics of heparin in solution as well as the relatively low molecular weight of heparin.     

Acridine orange, an inhibitor of protein kinase C, abolishes insulin and growth hormone stimulation of lipogenesis in rat adipocytes

To determine whether protein kinase C plays a role in the actions of insulin and growth hormone in rat adipocytes, we tested the effect of acridine orange, a potent inhibitor of kinase C, on the lipogenic activity of both hormones. This compound completely inhibited the effects of insulin, growth hormone and phorbol ester 12-myristate 13-acetate, whereas 9-acridine carboxylic acid, an analog of acridine orange which does not inhibit kinase C, had no effect. Acridine orange did not act through inhibition of hormone binding. These data are consistent with the involvement of kinase C in the action of insulin and growth hormone on lipogenesis in rat fat cells.     

Comparison of the interaction of antineoplastic aminoanthraquinone analogs with DNA using competitive fluorescence polarization

1,4-dihydroxy-5, 8-bis{{2-{(2-hydroxyethyl)amino}ethyl}amino} -9,10-anthracenedione (NSC 287836) and 1,4-bis{{2-{(2-hydroxyethyl) amino}ethyl}amino}-9, 10-anthracenedione diacetate (NSC 287513) have shown activity against solid tumors and are now in Phase I clinical trials. Fluorescence polarization was used to determine the extent of inhibition of the binding of acridine orange to DNA (Richardson, Roboz, Holland, Res. Comm. Chem. Pathol. Pharmac. $\text{27}$, 497, 1980). Displacement of 50% of acridine orange from calf thymus DNA was obtained with 0.18 uM of NSC 287836 while 0.52 uM of NSC 287513 was needed to displace an equivalent amount of acridine orange. NSC 287513 showed preference for polynucleotides of high adenine+thymine content while NSC 287836 did not. Analogs lacking both hydroxyethylaminoethyl-amino side chains did not displace acridine orange.     

Aminoacridines, potent inhibitors of protein kinase C

Acridine orange, acridine yellow G, and related compounds potently inhibited protein kinase C (Ca2+/phospholipid-dependent enzyme) activity and phorbol dibutyrate binding. Inhibition was investigated in vitro using Triton X-100 mixed micellar assays (Hannun, Y. A., Loomis, C. R., and Bell, R. M. (1985) J. Biol. Chem. 260, 10039-10043 and Hannun, Y. A., and Bell, R. M. (1986) J. Biol. Chem. 261, 9341-9347). Inhibition by the acridine derivatives was subject to surface dilution; therefore, the relevant concentration unit is mol % rather than the bulk molar concentration. Fifty percent inhibition of protein kinase C activity occurred at concentrations of these compounds comparable to concentrations of sn-1,2-diacylglycerol (DAG) and phosphatidylserine (PS) required for enzyme activation (i.e. 1-6 mol %). The mechanism of inhibition appeared to be complex: both the catalytic and regulatory sites of protein kinase C were affected. Acridine orange was a competitive inhibitor with respect to MgATP when the catalytic fragment of protein kinase C was employed. Inhibition at the active site was overcome by the addition of Triton X-100 micelles or phospholipid vesicles. When the activity of intact protein kinase C was measured, inhibition was noncompetitive with respect to MgATP. Further kinetic analysis suggested a competitive type of inhibition with respect to PS and DAG implying an interaction of acridine compounds with the regulatory lipid cofactors or with the regulatory domain of protein kinase C. This was further supported by demonstrating inhibition of phorbol dibutyrate binding to both protein kinase C and the lipid-binding domain generated by trypsin hydrolysis. Acridine orange and acridine yellow G also inhibited thrombin-induced 40-kDa phosphorylation in human platelets and phorbol dibutyrate binding to platelets. These effects were also subject to surface dilution. These results suggest that acridine derivatives have multiple interactions with protein kinase C with the predominant effect being inhibition of activation within the regulatory domain of the enzyme. Some of the biologic effects of acridine derivatives including anti-tumor action may occur as a consequence of protein kinase C inhibition.     

Classification of larval circulating hemocytes of the silkworm,<Emphasis Type="Italic"> Bombyx mori</Emphasis>, by acridine orange and propidium iodide staining

Circulating hemocytes of the silkworm can be classified by fluorescence microscopy following staining with acridine orange and propidium iodide. Based on their fluorescence characteristics, three groups of circulating hemocytes can be distinguished. The first group, granulocytes and spherulocytes, is positive for acridine orange and contain bright green fluorescent granules when observed by fluorescence microscopy. In granulocytes, these green granules are heterogeneous and relatively small. In contrast, in spherulocytes, the green granules appear more homogenous and larger. The second group of hemocytes consists of prohemocytes and plasmatocytes. These cells appear faint green following staining with acridine orange and do not contain any green fluorescent granules in the cytoplasm. Prohemocytes are round, and their nuclei are dark and clear within a background of faint green fluorescence. Inside the nucleus there are one or two small bright green fluorescent bodies. Plasmatocytes are irregularly shaped and their nuclei are invisible. Oenocytoids belong to the third group, and their nuclei are positive for propidium iodide. Therefore, all five types of circulating hemocytes of the silkworm, including many peculiar ones that are difficult to identify by light microscopy, can now be easily classified by fluorescence microscopy following staining with acridine orange and propidium iodide. In addition, we show that hemocytes positive for acridine orange and propidium iodide are in fact living cells based on assays for hemocyte composition, phagocytosis, and mitochondrial enzyme activity.     

Induction of cytoplasmically inherited respiration-deficient ('petite') mutants by photodynamic action of acridine compounds

All acridines used (acriflavine, proflavine, acridine orange and 3-azido-10-methylacridinium chloride) produced killing in yeast cells when activated with visible light. Acriflavine, proflavine and 3-azido-10-methylacridinium chloride, but not acridine orange, produced petite and sectored colonies. Both cell killing and petite induction by light activation of acriflavine resulted apparently from photodynamic action mediated by singlet oxygen (1O2) since the effect were prevented by either sodium azide or anaerobiosis. The biological effects of 3-azido-10-methylacridinium chloride, which was developed as a potential photoaffinity probe for studying the binding and biological effects of acridines, appeared to be due to a photodynamic action analogous to that of acriflavine. Sodium azide or anaerobiosis prevented the light-activated effects of 3-azido-10-methylacridinium chloride despite the fact that the initial chemical breakdown of the azido derivative induced by light was not affected. Cells suspended in D2O demonstrated an enhanced response to 3-azido-10-methylacridinium chloride with irradiation. These results indicate that singlet oxygen mediates the light-activated biological effects of both acriflavine and 3-azido-10-methylacridinium chloride.     

Na+/H+ exchange is present in basolateral membranes from rabbit small intestine

Fluorescence quenching of the pH gradient sensitive dye acridine orange and that of the membrane potential sensitive dye Di-S-C3(5) have been studied in purified basolateral membrane vesicles obtained from rabbit small intestine. Basolateral membranes contain an electroneutral, carrier mediated, Na+/H+ exchange activity. They also appear to contain an electrogenic pathway for H+ movement. Based on the comparison of acridine orange fluorescence quenching in the presence of an outwardly directed Na+ gradient and in the presence of known K+ diffusion gradients it can be estimated that at least 50% of the observed proton fluxes are due to the activity of the exchanger. Acridine orange fluorescence recovery measurements have been used to assess the kinetic properties of the exchanger.     

Acridine orange binding to chromatin of individual cells and nuclei under different staining conditions. Thermadenaturation of Chromatin.

The thermodenaturation of chromatin in situ was studied by staining heat-treated nuclei with acridine orange. It was found that formaldehyde, which under the present conditions had to be used to prevent extensive renaturation of DNA, seriously affects the results of standard acridine orange staining in an unspecific manner. In particular the acetylation step involved in this staining method is strongly inhibited. Thus the standard method of staining can only give qualitative information about the effects of thermodenaturation. On the other hand, acridine orange staining at defined equilibrium, without prior acetylation is insensitive to formaldehyde and multiphasic thermodenaturation profiles are obtained with this method. At low temperatures these profiles mainly reflect changes in the protein-DNA interaction whereas at higher temperatures DNA denaturation also contributes to the curves. Although these two processes cannot be separately quantitated by simple measurements of dye binding, the thermodenaturation profiles still contain biologically significant information about the properties of chromatin in situ.     

Thermodynamics of mucopolysaccharide–dye binding. II. Binding constant and cooperativity parameters of acridine orange–dermatan sulfate system

In the acridine orange–dermatan sulfate system, free and bound dye can be distinguished from each other spectroscopically. This permits the use of fluorometric methods to study the binding of acridine orange to the acid mucopolysaccharide dermatan sulfate. Experiments were conducted at 24°C in 10^?3 M citrate/phosphate buffer at pH = 7.0. The binding of the dye is highly cooperative, as evidenced by considerable interaction between adjacent bound dye molecules. Analysis of the data indicates that dermatan sulfate binds 2.3 ± 0.3 mol of acridine orange per dermatan sulfate uronic acid residue with a cooperative binding constant, K_q ranging from 4.9 to 6.0 × 10⁵ M^?1 which corresponds to a free energy of 7.74 ? ΔG° ? 7.86. The cooperativity parameter q apparently increases with increasing polymer-to-dye ratio.     

Millisecond fading and recovery phenomena in fluorescent biological objects.

Cytofluorometric signals derived from some frequently used fluorophores were studied during illumination times in the millisecond range. These rapid signals were recorded on a storage oscilloscope. The objects studied included (1) Berberine sulphate stained mast cell heparin, (2) Acriflavine-Feulgen stained DNA, (3) Acridine orange stained mast cell heparin, (4) Acridine orange stained DNA and (5) Fluorescein isothiocyanate-conjugated IgG in an antinuclear factor test. A new rapid fading phenomenon, appearing as an initial peak upon the familiar slowly declining fluorescence signal, is reported. This fading, which had a duration of about 10 ms, also showed a very rapid recovery. The influence of this phenomenon on fluorometric measurement techniques is discussed. The millisecond fading phenomenon occurred in all the fluorophores studied except Fluorescein isothiocyanate-conjugated IgG. In the case of acridine orange the phenomenon was present when the dye was bound to nuclear DNA but absent when the dye was bound to mast cell heparin. This suggests that the millisecond fading and recovery phenomenon may be used in fluorescent microprobe studies.     

Competitive interaction of serotonin and the dye acridine orange with DNA

The interaction of serotonin and acridine orange dye with DNA isolated from bacterium Escherichia coli and the yeast Candida utilis has been analysed by spectrofluorimetric method. Using data on competitive binding to DNA of serotonin and acridine orange, known as DNA intercalator, a conclusion concerning the formation of intercalated complex between serotonin and DNA has been made. It is shown that for yeast DNA the constant of intercalated binding of serotonin is 3,5-fold smaller than for the bacterial one.     

Thermodynamics of mucopolysaccharide–dye binding. I. Identification of free and bound dye via membrane filtration: Acridine orange–dermatan sulfate system

Stoichiometric mixtures of acridine orange with dermatan sulfate at total dye concentrations ? 1 × 10^?5 M show fluorescence maxima at 540 nm and 660 nm on excitation at 436 nm. By means of membrane filtration, it is directly demonstrated that the species emitting at 540 nm is due only to unbound dye whereas the 660-nm emitting species is due to bound dye. It is, therefore, possible to differentiate unbound acridine orange from its dermatan sulfate complex solely by spectroscopic methods. Thermodynamic binding parameters can be calculated from rapid spectroscopic measurements without disturbing the system.     

Induced optical activity of acridine orange bound to poly-S-carboxymethyl-L-cysteine

The absorption and rotatory properties of acridine orange-poly-S-carboxymethyl-L -cysteine system in water and in 0.2 M NaCl have been measured at different pH and polymer-to-dye mixing ratios. The absorption spectra indicate that the dyes are bound to the polymer in dimeric or highly aggregated forms. At neutral pH where the polymer is randomly coiled, no optical activity is induced on the absorption bands of bound acridine orange. At acid pH where the polymer has the β-conformation, a pair of positive and negative circular dichroic bands occur at each of the absorption bands, centered around 458 and 261 mμ. The signs of those bands are opposite to those found for α-helical poly-L -glutamic acid. A model for the binding of dye to the β-form polymer is presented, in which dimeric dyes are attached to ionized carboxyl groups and slack one another to form linear arrays on both sides of an extended polypeptide chain. The observed circular dichroism spectra can be explained by the Tinoco's exciton mechanism, based on this model. Low molecular weight poly-S-carboxymethyl-L -cysteine induces quite a different circular dichroism on bound acridine orange.     

Disruption of Ion Homeostasis by Verrucosin and a Related Compound

We have found that (?)-virgatusin and related compounds have antimicrobial and antifungal activity. To identify further biological activities of these compounds, we tested the activity of acridine orange efflux, which shows ionophore-like disruption of cellular ion homeostasis activity. After testing 31 compounds, we found that verrucosin and a related compound had disruption activity.     

Disruption of ion homeostasis by verrucosin and a related compound

We have found that (-)-virgatusin and related compounds have antimicrobial and antifungal activity. To identify further biological activities of these compounds, we tested the activity of acridine orange efflux, which shows ionophore-like disruption of cellular ion homeostasis activity. After testing 31 compounds, we found that verrucosin and a related compound had disruption activity.     

Specific fluorescent bands on chromosomes produced by acridine orange after prestaining with base specific non-fluorescent DNA ligands

Metaphase chromosomes stained with acridine orange exhibit uniform yellow-green fluorescence. Chromosome preparations treated with the non-fluorescent A-T specific antibiotic distamycin A prior to acridine orange staining exhibit longitudinal fluorescent banding patterns similar to those produced by a number of fluorescent R-band techniques. Similarly, chromosome preparations treated with the non-fluorescent G-C specific antibiotic actinomycin D followed by acridine orange staining exhibit Hoechst-type banding patterns. Interactions of various ligand-DNA combinations in solution indicate that the base pair specific antibiotics induce banding patterns by selectively altering acridine orange binding sites in chromosomal regions rich in the particular base pair for which the antibiotic exhibits specificity.