Enhancement Of ANS Fluorescence Research Articles

Tocopheryl Succinate-Induced Structural Changes in DPPC Liposomes: DSC and ANS Fluorescence Studies.

Recent studies show that alpha-tocopheryl succinate (TS) exhibits selective toxicity against cancer cells. In this study, we investigated the effect of TS’s presence on the physico-chemical and structural properties of DPPC liposomes using fluorescence parameters (intensity, lifetime, and position of emission maximum) of 1-anilino-8-naphtalene sulphonate (ANS), differential scanning calorimetry (DSC) and zeta potential methods. Increasing the TS presence in the DPPC gel phase produced ANS fluorescence enhancement with a hypsochromic shift of the maximum. The zeta potential measurements show an increase in the negative surface charge and confirmed that this process is connected with the hydrophobic properties of dye, which becomes located deeper into the interphase region with a progressing membrane disorder. Temperature dependence studies showed that an increase in temperature increases the ANS fluorescence and shifts the ANS maximum emission from 464 to 475 nm indicating a shift from hydrophobic to a more aqueous environment. In the liquid crystalline phase, the quenching of ANS fluorescence occurs due to the increased accessibility of water to the ANS located in the glycerol region. The DSC results revealed that increasing the presence of TS led to the formation of multicomponent DSC traces, indicating the formation of intermediate structures during melting. The present results confirmed that TS embedded into the DPPC membrane led to its disruption due to destabilisation of its structure, which confirmed the measured biophysical parameters of the membrane.

Conformation specificity and arene binding in a peptide composed only of Lys, Ile, Ala and Gly

The first life on Earth is believed to have been based on RNA, but might have taken advantage of amino acids and short peptides which form readily under conditions like those of the primitive Earth. We have shown that simple peptides adopt specifically folded four-helix bundle structures that can recognize and cleave RNA. Here, to explore the limits of conformational specificity, we characterize a simpler peptide composed of just Lys, Ile, Ala, and Gly called KIA7I. Using nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations, we find kinks in the helices of KIA7I and multiple C-terminal conformations. These results suggest that the C-terminal Ile residue does not completely occupy the hydrophobic pocket that is filled by aromatic side-chains in well-folded KIA7 variants. The capacity of arenes to fill this cavity was tested. Using NMR, we show that benzene and phenol can bind KIA7I, but do not bind the well-folded variant KIA7W or hen egg white lysozyme. Benzene also binds Aβ(1-40), a mostly disordered polypeptide implicated in Alzheimer's disease. 8-Anilinonaphthalene-1-sulfonate (ANS) fluorescence is further enhanced in the presence of both KIA7I and arenes relative to KIA7I alone. This ANS fluorescence enhancement is stronger for smaller and less polar arenes and less ordered KIA variants. These results suggest that arenes are not confined to the pocket, but penetrate and loosen the hydrophobic core of KIA7I.

Encapsulation of Anilinonaphthalenesulfonates in Carboxylate-Terminated PAMAM Dendrimer at the Polarized Water|1,2-Dichloroethane Interface

Molecular encapsulation of water-soluble anionic fluorescent dye molecules, 8-anilino-1-naphthalenesulfonic acid (ANS), and its bimolecular derivative (bis-ANS), in the generation 3.5 polyamidoamine (G3.5 PAMAM) dendrimer was investigated in the bulk aqueous phase and at the polarized water|1,2-dichloroethane interface. ANS(-) was electrostatically incorporated in the dendrimer, and the fluorescence enhancement with a blue shift of the emission maximum was observed at pH values <6, where the interior of the dendrimer was positively charged. The fluorescence enhancement of ANS was maximized around pH 3 and then decreased under more acidic conditions. The potential dependences of the molecular encapsulation and the interfacial mechanism were studied in detail by means of potential modulated fluorescence (PMF) spectroscopy. Under acidic conditions, the dendrimer incorporated ANS(-) at the positively polarized interface as well as in the aqueous phase. ANS(-) was released from the dendrimer at the intrinsic transfer potential and independently transferred across the interface. Bis-ANS exhibited relatively strong interaction with the dendrimer over a wide pH range (1 < pH < 8), and a negative shift of the transfer potential was observed under the corresponding pH condition. The PMF analysis clearly demonstrated that the interfacial mechanism of the dendrimer involves transfer and adsorption processes depending on the pH condition and the Galvani potential difference.

Investigations of effects of environmental factors in unfolding/refolding pathway of proteins on 8-anilino-1-naphthalene-sulfonic acid (ANS) fluorescence

The effects of various environmental factors in unfolding/refolding pathway of proteins on ANS fluorescence were investigated. In phosphate buffer of pH 7.4, ANS bound cetyl trimethyl ammonium bromide (CTMAB) and resulted in significant increase of ANS fluorescence and a sudden blue-shift in emission maximum wavelength. Sodium dodecyl sulfate (SDS) caused less increase in ANS fluorescence and less blue-shift in emission maximum wavelength than CTMAB and Tween 20. Tween 20, urea, polyethylene glycol 8000 (PEG 8000) and glycerol which contains hydrophobic groups also enhanced ANS fluorescence and caused their emission maximum wavelength blue-shift. For guanidine hydrochloride (GdmCl), due to its shorter hydrophobic chain, it caused less increase in ANS fluorescence and less decrease in emission maximum wavelength than CTMAB although GdmCl also has a positive charge. The results demonstrated the need for caution in interpreting enhancement of ANS fluorescence as an evidence for the existence of partially folded protein intermediates.

ATP hydrolysis-driven H+ translocation is stimulated by sulfate, a strong inhibitor of mitochondrial ATP synthesis

Sulfate is a partial inhibitor at low and a non-essential activator at high [ATP] of the ATPase activity of F(1). Therefore, a catalytically-competent ternary F(1) x ATP x sulfate complex can be formed. In addition, the ANS fluorescence enhancement driven by ATP hydrolysis in submitochondrial particles is also stimulated by sulfate, clearly showing that the ATP hydrolysis in its presence is coupled to H(+) translocation. However, sulfate is a strong linear inhibitor of the mitochondrial ATP synthesis. The inhibition was competitive (K (i) = 0.46 mM) with respect to Pi and mixed (K (i) = 0.60 and K'(i) = 5.6 mM) towards ADP. Since it is likely that sulfate exerts its effects by binding at the Pi binding subdomain of the catalytic site, we suggest that the catalytic site involved in the H(+) translocation driven by ATP hydrolysis has a more open conformation than the half-closed one (beta(HC)), which is an intermediate in ATP synthesis. Accordingly, ATP hydrolysis is not necessarily the exact reversal of ATP synthesis.

Thermal denaturation of yeast alcohol dehydrogenase and protection of secondary and tertiary structural changes by sugars: CD and fluorescence studies

The present communication reports on changes in the secondary and tertiary structures of native and apo-yeast alcohol dehydrogenase upon heating at 50 °C, as evident from circular dichroism (CD) studies. The presence of sugars provided significant protection with trehalose being the most effective. Exposure of hydrophobic clusters in the protein molecule upon heat denaturation was confirmed by fluorescence studies using 1-anilinonaphthalene-8-sulfonate (ANS) as a hydrophobic reporter probe. All sugars, and especially trehalose, reduced the affinity of both forms of the enzyme for this probe. The effectiveness of sugars in diminishing ANS fluorescence enhancement is in accordance with their ability to protect aggregation of the proteins, reported earlier [Miroliaei M, Nemat-Gorgani M. Sugars protect native and apo yeast alcohol dehydrogenase against irreversible thermoinactivation. Enzyme Microb Technol 2001;29:554–9]. It is concluded that prevention of the mechanisms of irreversible thermoinactivation may occur with retention of the secondary and tertiary structural properties of the proteins.

Inhibition of the mitochondrial ATP synthesis by polygodial, a naturally occurring dialdehyde unsaturated sesquiterpene

Polygodial is a naturally occurring sesquiterpene dialdehyde that exhibits several pharmacologically interesting activities. Among them, its antifungal properties have been more thoroughly studied. The mitochondrial ATPase has been suggested as one of the possible targets for polygodial action. However, its mechanism of action is not well defined yet. The effect of polygodial on the mitochondrial energy metabolism is described in this paper. Polygodial inhibited ATP synthesis coupled to succinate oxidation in beef-heart submitochondrial particles at concentrations (IC 50 = 2.4 ± 0.1 μM) which marginally affected electron transport and ATPase activity (IC 50 = 97 ± 4 μM). A transitory stimulation of the electron transport in intact rat liver mitochondria in state 4 was also obtained at low polygodial concentrations (EC 50 = 20 ± 4 μM). These results suggest that polygodial uncouples ATP synthesis from electron transport at low concentrations. Similar concentrations of polygodial partially abolished the ANS fluorescence enhancement (IC 50 = 2.2 ± 0.4 μM) induced by succinate oxidation in submitochondrial particles but did not collapse the ΔpH. We postulate that polygodial uncouples mitochondrial ATP synthesis by affecting the electrical properties of the membrane surface and consequently collapsing the membrane potential (Δ ψ) and/or the localized transmembrane pH difference (ΔpH S) without affecting the ΔpH between the two bulk aqueous phases (ΔpH B). The relevance of these findings for the understanding of the biochemical basis of the antifungal activity of polygodial and the evaluation of its potentiality as a therapeutic agent are discussed.

Hymenolepis diminuta:Catalysis of Transmembrane Proton Translocation by Mitochondrial NADPH → NAD Transhydrogenase

Mercer, N. A., McKelvey, J. R., and Fioravanti, C. F. 1999.Hymenolepis diminuta:Catalysis of transmembrane proton translocation by mitochondrial NADPH → NAD transhydrogenase.Experimental Parasitology91,52–58. The mitochondrial, inner-membrane-associated, reversible NADPH → NAD transhydrogenase of adultHymenolepis diminutaphysiologically couples matrix-localized, NADP-specific “malic” enzyme with NADH-dependent anaerobic electron transport. Employing submitochondrial particles (SMP) as the source of enzyme activity and both spectrophotometric and fluorometric assessments, the present study made evident that in its catalysis of transhydrogenation between NADPH and NAD, the cestode enzyme engages in the concomitant transmembrane translocation of protons. As assessed spectrophotometrically, the catalysis of NADPH-dependent NAD reduction byH. diminutaSMP was stimulated significantly by carbonyl cyanide 3-chlorophenylhydrazone (CCCP), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), as well as by the protonophoric anthel mintic, niclosamide. In addition,N, N′-dicyclohexylcarbodiimide (DCCD) markedly diminished SMP-catalyzed hydride ion transfer between NADPH and NAD. The catalysis by SMP of concomitant, transhydrogenase-mediated proton translocation was evaluated more directly via fluorometric assays using 8-anilino-1-napthalenesulfonic acid (ANS) as the probe. These latter evaluations revealed a transhydrogenase-dependent enhancement of ANS fluorescence in accord with an intravesicular accumulation of protons. ANS fluorescence was quenched rapidly when the assay system was supplemented with CCCP, FCCP, or niclosamide. Consistent with the helminth transhydrogenase acting as a proton pump, transhydrogenase-mediated enhanced fluorescence also was inhibited by DCCD. Considered collectively, these data indicated, apparently for the first time for any invertebrate system, that the transhydrogenase, in catalyzing the NADPH → NAD reaction, acts in the translocation of protons from the matrix to the intermembrane space mitochondrial compartment.

Probing of DNA-binding sites of Escherichia coli RecA protein utilizing 1-anilinonaphthalene-8-sulfonic acid.

RecA protein of Escherichia coli plays an essential role in homologous recombination of DNA strands. To analyze the interaction of RecA with single-stranded DNA (ssDNA), we performed a fluorescence competition assay employing 1-anilinonaphthalene-8-sulfonic acid (ANS) as an extrinsic fluorescent probe. ANS bound to RecA at three sites, leading to enhancement of ANS fluorescence. Addition of synthetic polynucleotides to the RecA-ANS complex in the absence of a nucleotide quenched the ANS fluorescence, indicating displacement of ANS molecules by ssDNA. Less effective quenching by poly(dA) suggests that the nucleoprotein filament on poly(dA) may differ from those on poly(dT) and poly(dC). A titration experiment with poly(dT) and poly(dA) showed clear stoichiometric binding of 3.5 nucleotides per protein. The site size for poly(dC) was 7.0, which could be explained by the formation of a double helix of poly(dC). ATP and other nucleotides also displaced the ANS. To identify ANS-binding sites, ANS was incorporated into RecA by UV irradiation, and fluorescent peptides were isolated from the proteolytic digest. Sequence analysis suggested that ANS binds to or near the ATP-binding region. These results suggest that the fluorescence quenching and photoincorporation assay using ANS may be useful for the analysis of the interaction of a protein and its ligand.

The fluorescence enhancement of 1-anilinonaphthalene-8-sulfonate (ANS) by modified β-cyclodextrins

The fluorescence enhancement of the probe molecule 1-anilino-8-naphthalenesulfonate (ANS) by a number of modified β-cyclodextrins has been studied. Alkyl- and hydroxyalkyl-substituted β-cyclodextrins show significantly greater enhancement of ANS fluorescence than does the parent unmodified β-eyclodextrin (β-CD). In the cases of methyl-β-cyclodextrin (Me-β) and hydroxypropyl-β-cyclodextrin (HP-β), enhancements by a factor of 120 and 180, respectively, were observed for ANS fluorescence, compared to a factor of only 8.4 in the case of β-CD. The binding constant for formation of the 1:1 ANS:CD complex was determined to be 370 ± 80 M tor Me-β and 430 ± 70 M for HP-β. The large increase in enhancement ability was shown to be a result of the relatively less polar environment experienced by the ANS probe incorporated in the modified as compared to the unmodified cyclodextrin cavities. The dielectric constants of the HP-β and Me-β cavities experienced by the included ANS were found to be 22 and 25, respectively. These polarities are similar to that of ethanol. whereas that of β-CD was found to be 54. a polarity similar to that of a 3:1 methanol:water mixture.

Beta-Site covalent reactions trigger transitions between open and closed conformations of the tryptophan synthase bienzyme complex.

The tryptophan synthase bienzyme complex (alpha2beta2) from Salmonella tryphimurium catalyzes the final steps in the biosynthesis of L-Trp. To investigate the roles played by conformational change in tryptopthan synthase catalysis, the fluorophore 8-anilino-1-naphthalensulfonate (ANS) is used to identify conformational states. The binding of ANS to the alpha2beta2 bienzyme complex is accompanied by a dramatic enhancement of ANS fluorescence and a shift of the emission maximum from 520 to 482 nm. The ANS binding isotherm is biphasic and consists of a class of moderately high-affinity, noninteracting sites with a stoichiometry of 1 site/alpha beta dimeric unit (Kd' = 62 + or - 15 micrometer) and a much weaker set of non-specific interactions with K'd>1mM. Our findings show that the affinity of the enzyme for ANS is strongly decreased (> 10-fold) by interactions at two loci 30 angstroms apart: (i) the binding of the alpha-site ligands, 3-indole-D-glycerol 3'-phosphate or alpha-glycerol phosphate (GP) or (ii) reaction at the beta-subunit to form either the alpha-aminoacrylate Schiff base, E(A-A), or quinonoid species, E(Q). In contrast, formation of the L-Ser and L-Trp external aldimines E(Aex1) and E(Aex2) at the beta-site causes a 2-3 fold decrease in the affinity of the enzyme for ANS. The combination of E(A-A)or E(Q) with GP brings about almost complete displacement of ANS, indicating that these interactions drive a conformation change in alphabeta subunit pairs which prevents the binding of ANS. These results are consistent with a model which postulates that alphabeta subunit pairs undergo ligand-mediated transitions between open and closed conformations during the catalytic cycle. Consistent with the kinetic data showing that binding of alpha-site ligands increases the affinity of the beta site for L-Ser and that formation of E(A-A) activates the alpha reaction [Brzović, P. S., Ngo, K., & Dunn, M. F. (1992) Biochemistry 31, 3831-3839], while mutations in alpha subunit loops 2 and 6 prevent the ligand- mediated transition to a closed structure [Brzović, P.S., Hyde, C.C., Miles, E.W., & Dunn, M.F. (1993) Biochemistry 32, 10404-10413], we conclude that reciprocal ligand-mediated allosteric interactions between the heterologous subunits promote conformational transitions between open and closed structures in alphabeta subunit pairs which function to coordinate catalytic activities and facilitate the channeling of indole between the two catalytic sites.

Membrane permeability transition promoted by phosphate enhances 1-anilino-8-naphthalene sulfonate fluorescence in calcium-loaded liver mitochondria.

Phosphate and a number of other compounds induce membrane permeability transition (MBT) in Ca(2+)-loaded mitochondria. 1-Anilino-8-naphthalene sulfonate (ANS) was used as a fluorescent probe to investigate perturbations on the inner membrane during MBT. Induction of MBT caused ANS fluorescence enhancement with a biphasic rate that reached a plateau. The enhancement is analogous to that reported for de-energization of mitochondria. The fluorescence level was independent of whether ANS was added before or at different times after phosphate. In the absence of ANS, fluorescence was low and remained unchanged. The initial time course of MBT, as followed by large-amplitude swelling, was similar to that of fluorescence enhancement. Ruthenium red, EGTA, ADP, and cyclosporin A inhibited the enhancement. Only EGTA + ADP (or ATP) reversed the enhancement when added after phosphate. Efflux of matrix Ca2+ by sodium acetate or A23187 did not alter ANS fluorescence. The binding parameters (Kd and number of binding sites) were not significantly different, but the fluorescence maximum was more than doubled after MBT. Although the fluorescence of bound ANS showed a nonlinear relationship, it was always higher (73.0 +/- 19.0%) after reaching the plateau. Since ANS binding to membranes is nonspecific, the exact mechanism of the enhanced fluorescence is not apparent. The dependence of the initial rate of fluorescence enhancement on Ca2+ concentration was nonlinear, with 45 microM at half-maximal rate. The dependence on phosphate was hyperbolic with 0.7 mM at half-maximal rate, which is close to the Km value of phosphate carrier. The kinetics is compatible with Ca2+ binding to some membrane component(s) during MBT and cause ANS fluorescence enhancement.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydrophobicity gradient on silica surfaces: A study using total internal reflection fluorescence spectroscopy

A silica surface with hydrophobicity gradient was characterized using total internal reflection fluorescence spectroscopy and ANS, 1-anilinonaphthalene-8-sulfonate, as a surface probe. The probe responded to the hydrophobicity gradient of the surface. Strong enhancement of ANS fluorescence was found at the hydrophobic side of the gradient and no fluorescence enhancement at the hydrophilic side. Similar results were obtained with ANS binding to bovine serum albumin adsorbed on the gradient surface.

Fluorescence emission from adsorbed bovine serum albumin and albumin-bound 1-anilinonaphthalene-8-sulfonate studied by TIRF

Total internal reflection fluorescence (TIRF) spectroscopy was used to analyse the conformational changes of bovine serum albumin (BSA) upon adsorption to silica surfaces. The intrinsic fluorescence emitted by BSA tryptophans and the fluorescence of BSA-bound ligand 1-anilinonaphthalene-8-sulfonate (ANS) were analysed prior to and after the removal of nonadsorbed protein. It was found that the irreversibly adsorbed BSA emits a blue-shifted intrinsic fluorescence (11 nm) and that the surface-adsorbed BSA-bound ANS fluorescence emission is red-shifted (12 nm). It was concluded that the conformational change in adsorbed BSA involves the whole BSA molecule: tryptophans become exposed to a less polar environment while the binding site polarity is increased. The conformation of irreversibly adsorbed BSA is not similar to the unfolded conformation of heat-denatured BSA. The red-edge excitation of adsorbed BSA-bound ANS indicates a slower spectral relaxation in the adsorbed protein as compared with the protein in solution. The fluorescence intensity contributions from irreversibly adsorbed species (BSA with and without bound ANS) suggest some changes in either ANS-BSA binding affinity and/or ANS fluorescence enhancement.

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 μg of histone per band.

Nucleophilic modification of human complement protein C3: correlation of conformational changes with acquisition of C3b-like functional properties.

Inactivation of C3 by enzymatic cleavage, nucleophilic addition, or slow freezing and thawing resulted in the acquisition of similar end-state conformations as judged by near-UV circular dichroism. Although inactivation by the two nonenzymatic processes involves no peptide bond scission, the inactivated C3 resembled C3b in that it possessed a free sulfhydryl group not present in the native protein and an increased surface hydrophobicity as evidenced by enhanced binding of the fluorophore 8-anilino-1-naphthalensulfonate (ANS). The C3b-like functional properties of modified C3 [Pangburn, M. K., & Müller-Eberhard, H. J. (1980) J. Exp. Med. 152, 1102-1114] may thus be understood in terms of the similarity of its conformation to that of C3b. The rate of the conformational change following proteolytic cleavage was fast and appeared to be limited by the rate of the enzymatic reaction. In contrast, the rate of conformational change following addition of methylamine was slow and rate limited by the conformational rearrangement itself, not by the chemical modification. A kinetic analysis of the changes in circular dichroism and ANS fluorescence enhancement suggested that the nucleophilic addition was spectroscopically undetectable and was followed by a minimally biphasic, spectroscopically demonstrable conformational rearrangement. The appearance of C3b-like functional activity in nucleophile-modified C3 largely parallels the time course of the spectroscopically detectable conformational change but is distinctly slower than the rate at which hemolytic activity is lost. While fully transconformed methylamine-inactivated C3 can bind factor B and is susceptible to cleavage by C3b inactivator and its cofactor beta 1H, this cleavage occurs at a substantially slower rate than the equivalent process in C3b. The implications of these findings in terms of the mechanism through which the alterative pathway of complement is initiated are discussed.

THE EFFECT OF TREATMENT OF CHLOROPLAST MEMBRANES WITH GUANIDINE HC1 AND AQUEOUS ACETONE ON THE FLUORESCENCE OF BOUND ANS AND CHLOROPHYLL‐a†

Abstract The fluorescence intensity of the extrinsic chromophore 1‐anilino‐naphthalene‐8‐sulfonate (ANS) bound to pea chloroplast fragments shows a sigmoidal rise as the pH of the suspending medium is decreased by the addition of HC1. The abrupt increase occurs at pH – 4.5. A 70% decrease in the maximal fluorescence intensity (pH range 3.5‐4.5) of bound ANS was observed when soluble chloroplast proteins were removed by washing with water. Extraction of chloroplast membranes with 6 M guanidine‐HC1 abolishes the acid–induced enhancement of ANS fluorescence. However, the subsequent removal of lipids (by 80% acetone extraction) from the guanidine‐HC1‐extracted naked membranes restores the acid‐induced fluorescence increase. These results suggest that ANS binds mainly to the surface of the chloroplast membrane and the fluorescence changes of ANS by acidification mainly reflect the changes in the associated proteins. The lack of enhancement of the fluorescence of ANS by acidification of the guanidine‐HCl treated membranes and the recovery of the acid‐induced fluorescence rise after extraction of the lipids from the guanidine‐HCl treated membranes suggest that the boundary lipids somehow prevent the entry of the ANS molecules into the hydrophobic interior of the naked membrane. The lipid‐depleted, guanidine‐HCl extracted naked membrane fragments do not show any shift in the position of the peak of emission of ANS (λ= 470 nm) upon acidification as the lipid‐depleted preparations without guanidine‐HCl treatment do (shift from 460 to 470 nm). Divalent cations (Mn2+, Ca2+, Mg2+) also increased ANS fluorescence intensities when added to both types of lipid‐depleted chloroplast preparations. A comparative analysis of ANS fluorescence bound to the lipid‐depleted and guanidine‐HCl treated chloroplast fragments with that of just lipid‐depleted fragments shows that the acidification of the latter brings about a greater change in the value of the relative binding sites (n) and the dissociation constant kd of ANS than the protonation of the former. The role of chloroplast protein and lipid components in the structural changes of the thylakoid membrane imposed by external perturbations is discussed.

Exposed hydrophobic regions in histone oligomers studied by fluorescence

The interaction of the fluorescent hydrophobic probe 1-anilinonaphthalene-8-sulfonate (ANS) with histone oligomers and with histone H1 has been studied. The enhancement of ANS fluorescence produced by histones Hν (a roughly equimolar mixture of histones H2A, H2B, H3 and H4) in 2.0 M NaCl, pH 7.5, is higher than that produced by histone H1 under identical conditions. In addition, the fact that the wavelength of maximum emission of the H1-ANS complex is larger than that of the Hν-ANS complex indicates that histone H1 has a weaker hydrophobic character than histones Hν. The increase in ANS concentration produces a red shift of the emission maximum of the Hν-ANS complex, indicating some heterogeneity in the ANS binding sites. Both the H2A-H2B and the H3-H4 complexes cause a similar enhancement of the ANS fluorescence. Trypsin digestion of N-terminal sequences of histones Hν produces only small changes in the intensity of ANS fluorescence. This result produces only small changes in the intensity of ANS fluorescence. This result indicates that the hydrophobic regions of histones Hν to which ANS binds are not located in the N-terminal portions of histone sequences. It is suggested that these exposed hydrophobic regions may be important in the maintenance of chromatin structure.

Formation of micelles and membrane action of the local anesthetic tetracaine hydrochloride

The formation of micelles of the local anesthetic tetracaine hydrochloride in aqueous phosphate buffer solution of pH 6.5 and ionic strength ( I) 0.10 was examined at 22°C by surface tension and using the fluorescent indicators perylene (peri-dinaphthalene) and 8-anilino-1-naphthalene sulfonic acid, sodium salt (ANS). The critical micelle concentration was located at 0.069, 0.071 and 0.063 M by measurements of surface tension, perylene solubilization and enhancement of ANS fluorescence, respectively. In contrast to other cationic surfactants, the anesthetic monomer did not show evidence of forming a fluorescent molecular complex with ANS under the experimental conditions of this study. The formation of micelles by tetracaine-HCl showed a pronounced effect on lipid membranes by inducing an abrupt decrease in the scattered light of egg lecithin liposomes at an anesthetic concentration roughly similar to its critical micelle concentration. This optical behaviour is characteristic of liposome damage and can be interpreted to mean that the lipids become solubilized into tetracaine-HCl micelles. The ability of this local anesthetic to form micelles can be taken as a manifestation of the same hydrophobic forces that lead to partitioning of the drug into membranes.

Interaction of .alpha.-chymotrypsin with the fluorescent probe 1-anilinonaphthalene-8-sulfonate in solution

The binding of the fluorescence probe 1-anilinonaphthalene-8-sulfonate (Ans) to alpha-chymotrypsin (alpha-CHT) at pH 3.6 is accompanied by a dramatic enhancement of Ans fluorescence and a shift of the emission maximum to shorter wavelengths. Our study reveals that one Ans molecule binds to alpha-CHT at a site different from either the active site of alpha-CHT or the 2-p-toluidinylnapthalene-6-sulfonate binding site. the binding constant of Ans is about the same (10(4) M-1) at pH 3.6 and 6.4. Nanosecond fluorescence depolarization data indicate that Ans is rigidly bound to alpha-CHT. The fluorescence enhancement due to binding of Ans to alpha-CHT at low pH could be due to binding either to a hydrophobic site or to a site where local dipoles do not relax during the excited-state lifetime of Ans. As the pH is increased, fluorescence intensity of the Ans-alpha-CHT complex decreases appreciably; and the emission maximum shifts to longer wavelengths. The fluorescence decay curves exhibit a corresponding sensitivity to pH. The pH effect on the fluorescence of Ans-alpha-CHT can be interpreted in terms of a pH-dependent equilibrium between alpha-CHT conformers differing in the degree of mobility of polar residues and water molecules at the Ans binding site or structural changes in the Ans binding site.