Effect of air ions on L 1210 cells: changes in fluorescence of membrane-bound 1,8-aniline-naphthalene-sulfonate (ANS) after in vitro exposure of cells to air ions

The ability of air ions to produce changes in the electrical properties of L 1210 mouse leukemia cells was tested. The fluorescence of ANS incorporated into a membrane lipid bilayer (measured microphotometrically) was used as a probe. It was shown that the action of air ions of both signs could change (negative ions by increasing and the positive ones by decreasing) the fluorescence intensity of ANS in the cell surface structures or to an imbalance of ions inside and outside the cell. Both possibilities are discussed in the light of the results of experiments using ouabain or biguanide as factors diminishing the intensity of ANS fluorescence.     

The influence of diffusion potentials across liposomal membranes on the fluorescence intensity of 1-anilinonaphthalene-8-sulphonate

The influence of diffusion potentials across different phospholipid membranes on the fluorescence intensity of 1-anilinonaphthalene-8-sulphonate (ANS) was studied. With liposomes or chloroform spheres covered with a monolayer of egg lecithin, no specific effects were found. With liposomes of soy-bean phospholipids, generation of a diffusion potential leads to an enhancement or decrease, depending on the direction of the potential, of the intensity of ANS fluorescence. This effect is mainly due to a change in quantum yield of the bound ANS. These data support a mechanism according to which ANS molecules are pushed into or pulled out of the membrane by a potential, but not an electrophoretic one in which the potential causes movement of ANS across the membrane.     

Interaction of the fluorescent probe, 1-anilino-8-napthalene sulfonate, with the sulfate transport system of Ehrlich ascites tumor cells.

The addition of the fluorescent dye, ANS, to intact ascites tumor cells results in an enhancement of fluorescence intensity. The increase in fluorescence intensity as a function of time is biphasic which suggests that at least two processes occur. The first associated with the rapid initial rise in fluorescence represents binding to the cell surface while the second or slower phase reflects entrance of ANS into the intracellular phase. The relationship between bound and free ANS in 0.50 mM sulfate medium was used to calculate the apparent dissociation constant of ANS-membrane complex (Kd = 6.53 times 10(-5) M) and the total number of ANS binding sites (4.49 nmoles/mg dry weight). Kinetic analysis of steady state sulfate transport in the presence and absence of ANS suggests that (1) sulfate exchange can be described by Michaelis Menten type kinetics (Km = 2.05 times 10(-3) M), (2) a small fraction of surface associated ANS competitively inhibits sulfate exchange (Ki = 4.28 times 10(-6) M) and (3) the transport system has a higher affinity for ANS than for sulfate. These data are consistent with the hypothesis that inhibition of sulfate exchange is related to the direct, reversible interaction of the negatively charged sulfonate group of ANS with superficial positively charged membrane sites.     

The interaction of 1-anilino-8-naphthalenesulfonate with thyroid lipids and membranes: A nuclear magnetic resonance study

The proton magnetic resonance (PMR) spectra of thyroid cell membranes and their total lipid extracts, in the presence of 1-anilino-8-naphthalenesulfonate (ANS), have been studied. The addition of ANS causes a shifting of the head group PMR signal, a splitting of the signal into two components and an increase in total spectral intensity. The data suggest that ANS interacts with phospholipid in the membrane as it does in total lipid vesicles. Evidence is also presented for the removal of lipids from the membrane, by ANS, and the subsequent formation of micelles. The membrane results are compatred with our earlier work on the interaction of ANS with egg phosphatidycholine (P.C.) vesicles and the results are used in explaining the inhibition of iodide transport in isolated thyroid slices.     

Acetylcholine promotes the proliferation and collagen gene expression of myofibroblastic hepatic stellate cells

The mechanisms that initiate and perpetuate the fibrogenic response, during liver injury, are unclear. Animal studies, however, strongly support a role for the autonomic nervous system (ANS) in wound healing. Therefore, the ANS may also mediate the development of cirrhosis. Hepatic stellate cells (HSC), the liver's major matrix-producing cells, are activated by injury to become proliferative, fibrogenic myofibroblasts. HSC respond to sympathetic neurotransmitters by changing phenotype, suggesting that HSC may be the cellular effectors of ANS signals that modulate hepatic fibrogenesis during recovery from liver damage. We show here that the parasympathetic neurotransmitter acetylcholine markedly stimulates the proliferation of myofibroblastic HSC and induces HSC collagen gene expression in these cells. By extending evidence that HSC are direct targets of the ANS, these results support the proposed neuroglial role of HSC in the liver and suggest that interrupting ANS signalling may be useful in constraining the fibrogenic response to liver injury.     

ANS fluorescence: Potential to discriminate hydrophobic sites of proteins in solid states

In the current study, ANS fluorescence was established as a powerful tool to study proteins in solid-state. Silk fibroin from Bombyx mori cocoons was used as a paradigm protein. ANS incorporated into the films of silk fibroin exhibits fluorescence with two-lifetime components that can be assigned to the patches and/or cavities with distinct hydrophobicities. Decay associated spectra (DAS) of ANS fluorescence from both sites could be fit to the single log-normal component indicating their homogeneity. ANS binding sites in the protein film are specific and could be saturated by ANS titration. ANS located in the binding site that exhibits the long-lifetime fluorescence is not accessible to the water molecules and its DAS stays homogeneously broadened upon hydration of the protein film. In contrast, ANS from the sites demonstrating the short-lifetime fluorescence is accessible to water molecules. In the hydrated films, solvent-induced fluctuations produce an ensemble of binding sites with similar characters. Therefore, upon hydration, the short-lifetime DAS becomes significantly red-shifted and inhomogeneously broadened. The similar spectral features have previously been observed for ANS complexed with globular proteins in solution. The data reveal the origin of the short-lifetime fluorescence component of ANS bound to the globular proteins in aqueous solution. Findings from this study indicate that ANS is applicable to characterize dehydrated as well as hydrated protein aggregates, amyloids relevant to amyloid diseases, such as Alzheimer's, Parkinson, and prion diseases.     

Interaction of N-phenyl-1-amino-8-sulfonaphthalene (ANS) with albumin in blood in hyperbilirubinemia

The fluorescent intensity of the N-phenyl-1-amino-8-sulfonaphthalene (ANS) probe significantly decreases in hyperbilirubinemic serum. A decrease of the albumin concentration and absorption of ANS fluorescence by bilirubin cannot explain such a considerable reduction of the probe fluorescence intensity. Measurements of the fluorescence decay kinetics has shown two types of sites occupied by ANS in albumin. ANS quantum yields in hyperbilirubinemic and normal serum are practically identical. The coupling parameters for ANS decrease, but the coupling constant increases under hyperbilirubinemia. As a result the coupling of organic anions with serum albumin significantly decreases if there is high anion concentration, and it does not decrease at low anion concentration. Bilirubin is not a main cause of a decrease of the albumin binding capacity.     

The interaction of 1-anilino-8-naphthalenesulfonate with the membranes of the sarcoplasmic reticulum and various lipid compounds

Summary (1) The enzymatic removal of lipids from the vesicular membranes of the sarcoplasmic reticulum does not interfere with the fluorescence of the 1-anilino-8-naphthalenesulfonate (ANS) vesicular complex. (2) The fluorescence intensity of the ANS vesicular complex is considerably (50%) reduced by oleic acid (0.5mm) because it displaces ANS from its binding sites. (3) Stearic acid, which also combines with the membranes, interferes neither with ANS binding nor with ANS fluorescence. (4) Of all lipid compounds tested, oleylamine produces the most pronounced fluorescence enhancement of ANS. (5) The complexes formed between oleic acid and cetyltrimethyl ammonium salts or between oleic acid and polylysine produce a much higher fluorescence enhancement than the isolated components. (6) Low concentrations of ether added to ANS-containing vesicular suspensions reduce their fluorescence intensity. It returns to the initial intensity when the ether is removed. (7) A small cyclic change of the fluorescence of the vesicular ANS complex takes place during active calcium uptake.     

Interaction of a hydrophobic fluorescent probe with mouse embryo fibroblasts

Fluorescence photomicrographs show that the hydrophobic fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS) binds to hydrophobic components of intact 3T3 cells. Cells exposed to ANS exhibit fluorescence in the cytoplasm, intense nuclear membrane fluorescence, and well-defined fluorescent nucleoli. Fluorescence titrations of 3T3 cells with ANS show a decrease in fluorescence intensity, a blue shift of native cell emission with increasing ANS concentration and the appearance of a new peak due to ANS fluorescence. These fluorescence effects are ascribed to energy transfer processes involving bound ANS and the tryptophan and tyrosine residues of cellular proteins. ANS bound to 3T3 cells appears to quench the long wavelength component of the cellular tryptophan fluorescence, resulting in an unmasking of tryptophan and tyrosine emission at shorter wavelengths.     

A thermodynamic characterization of the interaction of 8‐anilino‐1‐naphthalenesulfonic acid with native globular proteins: the effect of the ligand dimerization in the analysis of the binding isotherms

8‐Anilino‐1‐naphthalenesulfonic acid (ANS) is a popular fluorescence probe, broadly used for the analysis of proteins, but the nature of its interaction with proteins and the high increase in the fluorescence intensity that takes place upon such process are still unclear. In the last few years, isothermal titration calorimetry has been used to characterize the nature of the interaction of this dye with proteins. The analysis of the binding isotherms of these studies has not considered the dimerization equilibrium of ANS, which is pH dependent, and it can result in serious errors in the data analysis. In the present work we have developed a suitable data analysis by which this process is taken into account. To study the binding of the dye to proteins at different pH values, we have used the Abl‐SH3 domain. Our results suggest that at pH 3 and 5, where the dimerization of the ANS is important, electrostatic interactions are significant for the binding of ANS to the Abl‐SH3 domain. However, at pH 7, ANS behaves mostly as monomer and the interaction with the protein is mainly hydrophobic. The pH dependent behavior of the ANS binding to proteins can be explained in terms of ionization states of both, the protein and the ANS. Copyright © 2010 John Wiley & Sons, Ltd.     

Flow cytometric detection of lymphocyte alterations in Huntington's disease

Several recent reports indicate that patients with Huntington's Disease (HD) may manifest membrane abnormalities in a wide variety of cells including peripheral blood lymphocytes. In this study, flow cytometry is used in conjunction with the fluorescent membrane probe, 8-anilino-1-naphthalene sulfonate (ANS), to examine peripheral blood lymphocytes from 16 HD patients and 14 age- and diet-matched control subjects. Increased ANS fluorescence intensity of lymphocytes (p less than 0.02) was found in HD patients as compared to control subjects. These differences are masked when the mean fluorescence of the total leukocyte population is measured, possibly explaining conflicting data of other investigators. These observed differences in ANS fluorescence intensity between HD patients and control subjects support the concept of a gene defect which may be expressed as membrane alterations in non-neural as well as neural cells. The selective alterations of lymphocytes may also reflect altered immunological activity reported in HD.     

1-anilino-8-naphthalene sulfonate as a protein conformational tightening agent

1-Anilino-8-naphthalene sulfonate (ANS) anion is conventionally considered to bind to preexisting hydrophobic (nonpolar) surfaces of proteins, primarily through its nonpolar anilino-naphthalene group. Such binding is followed by an increase in ANS fluorescence intensity, similar to that occurring when ANS is dissolved in organic solvents. It is generally assumed that neither the negative sulfonate charge on the ANS, nor charges on the protein, participate significantly in ANS-protein interaction. However, titration calorimetry has demonstrated that most ANS binding to a number of proteins occurs through electrostatic forces, in which ion pairs are formed between ANS sulfonate groups and cationic groups on the proteins (D. Matulis and R. E. Lovrien, Biophys. J., 1998, Vol. 74, pp. 1-8). Here we show by viscometry and diffusion coefficient measurements that bovine serum albumin and gamma-globulin, starting from their acid-expanded, most hydrated conformations, undergo extensive molecular compaction upon ANS binding. As the cationic protein binds negatively charged ANS anion it also takes up positively charged protons from water to compensate the effect of the negative charge, and leaves the free hydroxide anions in solution thus shifting pH upward (the Scatchard-Black effect). These results indicate that ANS is not always a definitive reporter of protein molecular conformation that existed before ANS binding. Instead, ANS reports on a conformationally tightened state produced by the interplay of ionic and hydrophobic characters of both protein and ligand.     

能量化时线粒体内膜表面电荷的变化

本文报告用荧光探剂1,8—ANS和电泳激光光散射技术,研究鼠肝线粒体内膜在加入ATP的能量化过程中其膜表面电荷的变化。实验结果表明在加入ATP后线粒体内膜的能量化使其膜表面的负电荷减少。作者论讨了用上述二种方法研究线粒体内膜在能量化时表面电荷变化的有关问题。     

Rapid fluorescent staining of histones in sodium dodecyl sulfate-polyacrylamide gels

The increase in the fluorescence intensity of 1-anilinonaphthalene-8-sulfonate (ANS) produced by core histones is higher than that produced by very lysine-rich histones (H1 and H5). In the presence of the anionic detergent sodium dodecyl sulfate (SDS) the enhancement of ANS fluorescence caused by these two groups of histones is roughly the same, but much lower than that observed for core histones in the absence of this detergent. However, the increase of ANS fluorescence produced by histone-SDS complexes is high enough to use it for the staining of these proteins separated in SDS-polyacrylamide gels. Histone bands are stained with ANS after electrophoresis and visualized by transillumination of the gel with a uv light source. The method described in this work allows the rapid detection of less than 0.5 microgram of histone per band.     

Temperature effects on 1,8-anilinonaphthalene sulfonic acid fluorescence with sarcolemma vesicles.

Increased temperature produces a red shift and decreased fluorescence intensity of the emission peak of 1,8-anilinonaphthalene sulfonic acid (ANS) in suspensions of biomembrane vesicles. These changes have been attributed to a conjectured increase in polarity of the microenvironment of ANS. If the conjecture is correct, fluorescence lifetimes must be decreased with warming. We showed than ANS binds to both protein and lipid protein of sarcolemma, that there are two kinds of sarcolemma-lipid ANS-binding sites, and that there are three fluorescence lifetimes of excited sarcolemma-bound ANS. The three fluroescence lifetimes were unchanged on warming, or decreased too little to account for the observations. Fluorescence lifetime data were consistent with the notion that the effect of increasing temperature is to decrease the amount of ANS bound to sarcolemma. From studies of liposomes prepared from lipid extracts of sarcolemma, and of proteins from sarcolemma it was deduced that warming reducted the amount of ANS bound to both of these sarcolemma components, probably mainly by reducing binding capacity. There might also be a shift of affinities such that the ratio, KA sarcolemma lipid/KA sarcolemma protein, is larger at higher temperature. Except at very small concentration ratios of ANS/sarcolemma, more than twice as much ANS was bound to sarcolemma lipids as to proteins.     

Detection of the metastable rippled gel phase in hydrated phosphatidylcholine by fluorescence spectroscopy

Steady-state and time-resolved emission spectroscopy of 1-anilinonaphthalene-8-sulfonic acid (ANS) have been used for characterization of the metastable rippled gel phase, Pbeta'(mst), formed in fully-hydrated dipalmitoylphosphatidylcholine (DPPC) upon cooling from the liquid crystalline phase Lalpha [Tenchov et al., Biophys. J. 56 (1989) 757]. The Pbeta'(mst) phase of DPPC clearly differs from the stable Pbeta' phase by increased (approximately 27%) ANS emission intensity, by enhanced (approximately 23%) average radiative rate constant, and by reduced (approximately 18%) non-radiative quenching rate constant. The fluorescence intensity peak at the Pbeta'-->Lalpha transition temperature is replaced by a large, reversible stepwise intensity drop at the Pbeta'(mst)-->Lalpha transition. No such effects have been found for dimiristoylphosphatidylcholine (DMPC) dispersions confirming previous results that DMPC does not form a Pbeta'(mst) phase. Since ANS is known to predominantly reside in the interfacial region, the observed effects indicate differences between the stable and metastable rippled phases in the organization and dynamics of their lipid/water interfaces. The data demonstrate that the metastable rippled phase manifests its appearance also through interactions with small molecules (ANS size approximately 8 A).     

Effect of free fatty acid anion on the in vitro binding of 1-anilino-8-naphthalene sulfonate to cytoplasmic proteins from rat liver.

The influence of oleate ion, a free fatty acid anion, on the binding characteristics of 1-anilino-8-naphthalene sulfonate (ANS) with the cytoplasmic proteins (Y and Z) from rat liver has been examined using fluorescence spectroscopy. ANS binds strongly with both ligandin (Y) and Z protein at a single binding site with dissociation constants of 0.6 and 1.4 micron respectively. Increasing concentrations of oleate ion decreased the ANS binding with either protein by competing with the ANS binding site. Relative binding constant of oleate ion for the hepatic ligandin or Z protein was about 2 micron as determined from the competitive inhibition of ANS binding. These results suggest that variations in the hepatic cytoplasmic free fatty acid concentration may be important in regulating the capacity of Y and Z proteins to transport other organic anions.     

Age characteristics of the interaction between fluorescent probes and thyroxine with the membranes of rat liver microsomes

By means of fluorescent probes, 1-anilino naphthalene-8-sulfonate (ANS), methoxybenzanthrone (MBA) and pyrene, an increase of membrane affinity to ANS, shift of MBA fluorescence maximum to the short-wave spectrum region and a change of the membrane microviscosity were observed in the liver microsome membranes during aging of rats. No significant changes of fluorescent parameters of the probes with rats aging were found in mitochondria. ANS was concurrently displaced by thyroxine, affinity to which significantly increased with aging, and in young animals during hyperthyroidism. The increase of microsome membranes affinity to thyroxine with age is considered as an intracellular mechanism which is involved in the metabolism changes of hypothyroid pattern in rat liver during aging.     

Effect of protons and cations on chloroplast membranes as visualized by the bound ANS fluorescence

Structural changes in the chloroplast membranes caused by acidification and heat-treatment are studied by observing the changes in the fluorescence of ANS bound to thylakoid membranes. On addition of acids to buffered suspension of isolated pea chloroplasts, the fluorescence intensity of bound ANS shows a sigmoidal rise on reaching a pH value of about 4.5. A part of the fluorescence enhancement of bound ANS brought about by protons is not reversible on back titration with alkali. The reversible part of acid induced rise in ANS fluorescence possibly reflects structural changes expected to be associated with photophosphorylation. Divalent cations enhance the fluorescence of ANS bound to chloroplasts between a pH range 4.5–7.0 but diminish it if the pH is below 4.5.Addition of acid to heat-treated chloroplasts shows similar sigmoidal rise in ANS fluorescence intensity on lowering the pH to about 4.5. On addition of acid upto a pH of 3.1, the ANS fluorescence is greater than that of untreated chloroplasts, however, at pH below 3.1, the fluorescence of bound ANS is lower than the control chloroplasts. This observation indicates that heat-treatment caused some alteration of the microstructure of thylakoid membranes of chloroplasts besides the usual loss in the O₂ evolving capacity.This is further confirmed from the studies of Hill-activity and ANS binding to chloroplasts incubated at various temperatures in the absence and presence of aliphatic alcohol. Hill-activity (DCPIP reduction) of chloroplasts incubated at temperatures between 25 C and 55 C first increases reaching a maximum at 45 C and then declines rather sharply, when the chloroplasts are heated beyond 45 C (Tmax). The presence of 200 mM n-butyl alcohol or 40 mM n-amyl alcohol during the warming treatment lowers the temperature by 8 C at which the decline in the Hill-activity is observed. An enhancement in the fluorescence intensity and a blue shift of the emission spectrum of bound ANS are noted if the chloroplasts are heated beyond the Tmax either in absence or presence of alcohol. The changes in the fluorescence of ANS bound to heat-treated chloroplasts plausibly reflect the nature of the structural changes in chloroplasts during the heating upto 55 C.Abbreviations ANS 1-anilino-8-naphthalene sulphonate - DCPIP 2,6-dichlorophenol indophenol     

Anion-binding centers of human serum albumin during an N-F conformation transition and in isolated albumin and blood serum

Fluorescent probe N-phenyl-1-amino-8-sulfonaphthalene (ANS) was used for studying pH-dependent structural N-F-transition in human serum albumin of two kinds: in commercial albumin and in natural blood serum. The kinetics of ANS fluorescence decay in albumin solutions was measured. There were found two types of the sites occupied by ANS in albumin under physiological conditions (pH 7.4). In the first binding site ANS fluorescence decay time was 16.6 +/- 0.3 nsec and it was not significantly changed at N-F transition (pH 4.0). In the second binding site the decay time was dependent on pH in commercial albumin and was not significantly changed in serum. In the second binding site there were individual differences of ANS decay time (4.3 +/- 0.6 nsec). The observed ANS fluorescence intensity enhancing (about 40-50%) in N-F transition may be explained by an increase of albumin binding sites capacity for ANS.