Kinetics and Thermodynamics of 1-anilino-8-naphthalene Sulfonate Interactions with Urinary Trypsin Inhibitor

The interactions between Urinary Trypsin Inhibitor (UTI) and 1-anilino-8-naphthalene sulfonate (ANS) were investigated by fluorescence spectra, isothermal titration calorimetry and molecular modeling. The results revealed the presence of four specific binding sites for ANS on UTI, with interactions driven mainly by electrostatic forces. The four specific binding sites indicated the involvement of four hydrophobic patches on UTI. Experimental data also confirmed the presence of a further five nonspecific binding sites that interacted mainly by the formation of salt bridges between the sulfonates of ANS and positive residues on the surface of UTI.     

The interaction of human glycophorin with 8-anilino-1-naphthalene sulfonate.

Both the sialoglycoprotein of human erythrocyte membranes, glycophorin, and the sialic acid free protein, obtained by treatment of glycophorin with neuraminidase (EC 3.2.1.18), increase the fluorescence of 8-anilino-1-naphthalene sulfonate (ANS). Binding of ANS to glycophorin is weak compared with the binding to bovine serum albumin (BSA). equilibrium dialysis gives an apparent binding constant of about 4 X 10(3) M(-1) at neutral pH, but Ka increases 1.75 times when NaCl or CaCl2 are added and 10-fold when the pH is lowered to 3.0. Sialic acid groups do not significantly affect ANS binding, although they have some effect at low ionic strength and neutral pH. Fluorescence studies indicate only one to two binding sites for ANS, with apparent pK = 3.8 +/- 0.2, and located close to aromatic residues in glycophorin. Polarization and quantum efficiency of the fluorescence of ANS associated with glycophorin fail to indicate changes in the vicinity of the binding site when the pH is lowered.     

Membrane effects of aminoglycoside antibiotics measured in liposomes containing the fluorescent probe, 1-anilino-8-naphthalene sulfonate

The mechanism of membrane disturbance by aminoglycoside antibiotics was investigated in liposomes containing the fluorescent probe, 1-anilino-8-naphthalene sulfonate (ANS). Liposomes of PC and different anionic phospholipids (1:1 to 15:1 molar ratios) were challenged with aminoglycosides in the presence of low (1 microM) and high (3 mM) concentrations of calcium. Liposomes containing PIP2 showed the greatest drug-induced changes in ANS fluorescence in the presence of high and low concentrations of calcium and at all PC:PIP2 molar ratios tested. Liposomes containing other anionic phospholipids (PS, PI and PIP) were not reactive toward aminoglycosides in the presence of 3 mM calcium or when the ratio of PC to anionic lipid was increased to 10:1. The aminoglycoside-induced changes of ANS fluorescence were not due to any changes in the emission spectrum of ANS, nor to changes in quantum yield, nor to a change in the binding affinity of ANS. It is concluded that a specific aminoglycoside-PIP2 interaction results in phase separation of PC and PIP2 and thus increases the number of available ANS binding sites in PC:PIP2 liposomes.     

Evidence for protein self-association induced by excluded volume. Myoglobin in the presence of globular proteins

The fluorescence polarization of fluorescent derivatives of hemoglobin and myoglobin was measured as a function of the concentration of added polymers (PEG-6 000, PEG-20 000) and globular proteins (lysozyme, ribonuclease A, beta-lactoglobulin). The results indicated that the effective size and shape of 1-anilino-9-naphthalene sulfonate myoglobin are unaltered in the presence of up to 25 g/dl poly(ethylene glycol), whereas they are significantly altered in the presence of comparable concentrations of other proteins. The results are consistent with the hypothesis that in the presence of high concentrations of added protein, 1-anilino-9-naphthalene sulfonate myoglobin self-associates to form a dimer similar in size and shape to 1-anilino-9-naphthalene sulfonate hemoglobin.     

Fluorescent probe studies of mixed micelles of phospholipids and bile salts. Effect of cholesterol incorporation

The binding of the fluorescent alkylamines, $\text{N-(2-}\text{aminoethyl}\text{)-5-}\text{dimethylamino-1-naphthalene}$ sulfonamide, $\text{N-(5-}\text{aminopentyl}\text{)-5-}\text{dimethylamino-1-naphthalene}$ sulfonamide (dansyl cadaverine) and $\text{N-(10-}\text{aminodecyl}\text{)-5-}\text{dimethylamino-1-napthalene}$ sulfonamide with phospholipid and phospholipid-deoxycholate micelles, has been shown to increase with the length of the alkyl spacer chain. The probes bind more effectively to micelles containing unsaturated phospholipids and do not interact strongly with bile salt solutions at low concentrations. Cholesterol incorporation into mixed micelles results in a quenching of probe fluorescence due to displacement of probe molecules. The enhanced rigidity of the mixed micelles on solubilizing cholesterol is established by a decrease in pyrene excimer fluorescence and by the less effective perturbation of the micellar structure by 1-anilino-8-naphthalene sulfonate. The anionic probe 1-anilino-8-naphthalene sulfonate is also displaced from the mixed micelles when cholesterol is incorporated, suggesting a dominant role for packing and hydrophobic effects in binding both positively and negatively charged probes.     

Hepatic microsomal glucose-6-phosphatase of normal and alloxan-diabetic rats. Thermotropic effects on kinetics and interaction with deoxycholate and 1-anilino-8-naphthalene sulfonate

Thermotropic effects on the kinetics of glucose-6-phosphatase (D-glucose-6-phosphate phosphohydrolase, EC 3.1.3.9) activity of hepatic microsomes from normal and alloxan-diabetic rat liver were investigated by determining V, Km and Ki (substrate inhibition) values. Influence of deoxycholate (0.1%) and 1-anilino-8-naphthalene sulfonate (2.5 mM) on the kinetics was also evaluated. 1. Substrate inhibition occurred at 0.06 M for the enzyme from normal rats and at 0.0-0.025 M for the enzyme from diabetic rats. 2. The enzyme from diabetic rats showed a transition that extended between 22.7 and 27 degrees C in the Arrhenius plot (log V vs. T-1) instead of at 19.5 degrees C. 3. Deoxycholate increased the V value of both enzymes without affecting substrate inhibition at all the temperatures but did not completely abolish the transition in the Arrhenius plot of the enzyme from diabetic rats. 4. 1-Anilino-8-naphthalene sulfonate eliminated substrate inhibition and activated the enzyme of normal rats above 27.5 degrees C by increasing both V and Km values. Below this temperature, the enzyme showed biphasic or allosteric kinetics. At low substrate concentrations it was activated as both V and Km values were increased. The enzyme from diabetic rats, on the other hand, was activated at all the temperatures and exhibited linear kinetics. 5. Binding of 1-anilino-8-naphthalene sulfonate to the microsomal fraction increased with decreasing temperature as revealed by the increase of relative fluorescence. The microsomal fraction of diabetic rats showed a more anomalous fluorescence response between 13-18 degrees C. 6. Enthalpy changes for glucose 6-phosphate binding to the inhibition site were slightly larger than binding to the active site. Calculated entropies of activation for transition state complex of glucose-6-phosphatase reaction were fairly large and negative. The free energy of activation (28-30 kcal/mol) was independent of temperature and experimental conditions. 7. In the microsomal fraction (total as well as rough), phospholipid content and fatty acid unsaturation index of phospholipids were decreased after diabetes. The level of free cholesterol remained unchanged but the molar ratio of cholesterol to phospholipid increased. The different thermal response and 1-anilino-8-naphthalene sulfonate interaction to the enzyme from diabetic rat and liver could be ascribed to the altered lipid environment of the enzyme on the endoplasmic reticulum membrane.     

Fluorescence studies of the binding of amphiphilic amines with phospholipids

The binding characteristics of several amine drugs with dispersed phospholipids (phosphatidylcholine, phosphatidylserine, and phosphatidylglycerol) have been studied using the fluorometric method and 1-anilino-8-naphthalene sulfonate and 1,6 diphenyl-1,3,5-hexatriene as fluorescence probes. The results show that amphiphilic amines, such as chlorphentermine, interact with phospholipids via both ionic and hydrophobic forces. The ionic interaction, which occurs between the protonated amine group of the drug and the phosphate oxygen of the lipid, changes the amphiphilic characteristics of the lipid by reducing the number of negative charges on the lipid vesicles, and inhibits the Ca2+-dependent lipid hydrolysis by blocking the Ca2+ binding sites on the lipid vesicles. The hydrophobic interaction, which involves the nonpolar moieties of the drug and the lipid, is of primary importance to the overall drug-lipid binding stability. Drugs without a strong hydrophobic moiety, such as dopamine, do not interact with phospholipids.     

EFFECT OF OVIDUCAL PROTEINASE UPON BUFO ARENARUM VITELLINE ENVELOPE. A FLUORESCENCE APPROACH

The intrinsic fluorescence of vitelline envelope from amphibian eggs was enhanced after having been treated with pars recta secretion fluid. At the same time the total number of binding sites for 8-anilino-1-naphthalene sulfonate (ANS) was increased, with the affinity constant remaining unchanged. These observations are interpreted in terms of a structural change of the vitelline envelope submitted to pars recta fluid effect.     

Fluorescence studies on binding of amphiphilic drugs to isolated lamellar bodies: relevance to phospholipidosis

Lysosomal phospholipid storage disorder in lung tissue was observed during chronic treatment with amphiphilic amine drugs. The prevailing and widely accepted mechanism of phospholipidosis is that amphiphilic drugs bind to phospholipids and make the phospholipids unsuitable substrates for the action of phospholipases. We investigated hydrophobic and hydrophilic binding of fifteen drugs to the phospholipid storage organelle, lung lamellar bodies, isolated from male Sprague-Dawley rats. Hydrophobic interactions were studied using 1,6-diphenyl-1,3,5-hexatriene as a fluorescent probe and hydrophilic binding was studied using 1-anilino-8-naphthalene sulfonate as a fluorescent probe. The binding parameters were calculated using Scatchard equations. Of the fifteen drugs used, nine drugs bound to the hydrophobic moiety of lamellar bodies. The order of binding capacities was promethazine greater than chloramphenicol greater than amiodarone = desethylamiodarone greater than promazine greater than chlorpromazine greater than trimipramine greater than propranolol greater than imipramine much greater than chlorphentermine, phentermine, chloroquine, chlorimipramine, cyclizine and chlorcyclizine. Two binding affinities were calculated for all the bound drugs. Binding affinities to hydrophilic sites of lamellar bodies were calculated in terms of emission coefficients for 1-anilino-8-naphthalene sulfonate in the presence of drugs. Hydrophilic binding was in the order chlorpromazine greater than chlorimipramine greater than promazine greater than trimipramine greater than imipramine greater than chlorcyclizine greater than propranolol greater than promethazine greater than chlorphentermine greater than cyclizine greater than phentermine greater than chloroquine much greater than chloramphenicol, amiodarone and desethylamiodarone. The binding affinities of chlorinated analogs were stronger to hydrophilic sites when compared to the parent compound. Amiodarone, which is known to induce pulmonary phospholipidosis and its major non-polar metabolite, desethylamiodarone, bound strongly to lamellar bodies. These two drugs also inhibit phospholipases in vitro. The drugs with weak phospholipidosis-inducing capacity and extensive in vivo metabolism, namely, imipramine, chlorpromazine and promazine, also bound strongly to lamellar bodies with hydrophilic as well as hydrophobic interactions. On the other hand, chloroquine, which is known to induce phospholipidosis and to inhibit phospholipases, did not bind to lamellar bodies. Two major conclusions could be drawn from this study: one is that the drug interactions with isolated lamellar bodies could be studied using membrane fluorescence probes, 1,6-diphenyl-1,3,5-hexatriene and 1-anilino-8-naphthalene sulfonate; second is that the amphiphilic drugs bind to lamellar bodies, as reported for phospholipid vesicles, and the binding of drugs to lamellar bodies could be correlated with their phospholipidosis-inducing capacity only if     

Localization of a 1-anilinonaphthalen-8-sulfonate fluorescent probe on the surface monolayer of low density lipoproteins

A decay of fluorescence probe 1-anilino-8-naphtalene sulfonate (ANS) sorbed on low density lipoproteins (LDL) surface obtained from human plasma was described. It was demonstrated that on the LDL surface the ANS probe is allocated among two pools of molecules with the time of fluorescence decay 4-7 ns and 12-16 ns. One may conclude that 75-90% of the probe is connected with lipid LDL matrix.     

Spectroscopic studies on Erythrina lysistemon seed lectin: effect of denaturing agents on lectin quaternary structure

The equilibrium unfolding of ElysL, a homodimeric legume lectin, was studied using different denaturing agents such as guanidinium chloride (GdnHCl), temperature and pH. Simultaneously, changes in the secondary as well as tertiary structure of lectin were followed by CD spectroscopy examination in both far and near-UV region, respectively. The hydrophobic cluster binding dye, 1-anilino-8-naphthalene sulfonate (ANS), was used to further explore intermediates and to follow the unfolding pathway of lectin. The adenine binding ability of lectin was examined and monitored via absorption spectra and the intrinsic tryptophan fluorescence. Our findings indicate that the ElysL unfolding process occurs via a three state pathway with an intermediate state. We also showed that ElysL binds adenine in a manner that involves a hydrophobic binding pocket that is independent of the carbohydrate binding sites.     

Competitive partial inhibitors of serum albumin-catalyzed sulfur cyanolysis

Efforts to locate the active site for sulfur cyanolysis catalyzed by bovine serum albumin have led to systematic tests of several compounds that inhibit the catalyzed reaction. Hexanoate and 5-dimethylaminonaphthalene-1-sulfonate bind at the same site and are partial inhibitors competitive with cyanide, uncompetitive with respect to sulfur. Various dansyl amino acids and 1-anilino-8-naphthalene sulfonate display the same inhibitory behavior but bis (1-anilino-8-naphthalene sulfonate) is a total inhibitor competitive with cyanide. These findings are interpreted to indicate that the cyanolysis active site is near, but not at, one of the short-chain fatty acid binding sites on albumin subdomain 2-AB or 3-AB. Both ionic repulsion and steric considerations are implicated in the mechanisms of inhibition.     

Effect of metal ions and EGTA on the optical properties of concanavalin A at alkaline pH

In our earlier communications, we reported the effect of salts and alcohols on alpha-chymotrypsinogen [1] and the existence of stable intermediates at low pH in bromelain [2] and glucose oxidase [3]. In the present study, the role of metal ions and EGTA on the conformation of concanavalin A at alkaline pH was studied by near- and far-UV circular dichroism, fluorescence emission spectroscopy and binding of a hydrophobic dye, 1-anilino-8-naphthalene sulfonate (ANS). Far-UV CD spectra showed the transition from an ordered secondary structure at pH 7 with a trough at 223 nm to a relatively unordered state at pH 12. Near-UV CD spectra showed the loss of signal at 290 nm, thereby indicating the disruption of native three dimensional structure. Maximum ANS binding occurred at pH 12 suggesting the presence of an intermediate or molten globule-like state at alkaline pH.     

Crystallographically mapped ligand binding differs in high and low IgE binding isoforms of birch pollen allergen bet v 1

The ability of pathogenesis-related proteins of family 10 to bind a broad spectrum of ligands is considered to play a key role for their physiological and pathological functions. In particular, Bet v 1, an archetypical allergen from birch pollen, is described as a highly promiscuous ligand acceptor. However, the detailed recognition mechanisms, including specificity factors discriminating binding properties of naturally occurring Bet v 1 variants, are poorly understood.Here, we report crystal structures of Bet v 1 variants in complex with an array of ligands at a resolution of up to 1.2 Å. Residue 30 within the hydrophobic pocket not only discriminates in high and low IgE binding Bet v 1 isoforms but also induces a drastic change in the binding mode of the model ligand deoxycholate. Ternary crystal structure complexes of Bet v 1 with several ligands together with the fluorogenic reporter 1-anilino-8-naphthalene sulfonate explain anomalous fluorescence binding curves obtained from 1-anilino-8-naphthalene sulfonate displacement assays. The structures reveal key interaction residues such as Tyr83 and rationalize both the binding specificity and promiscuity of the so-called hydrophobic pocket in Bet v 1.The intermolecular interactions of Bet v 1 reveal an unexpected complexity that will be indispensable to fully understand its roles within the physiological and allergenic context.     

3,5-Di-t-butyl-4-hydroxytoluene (BHT) and probucol stimulate selectively the reaction of mammalian 15-lipoxygenase with biomembranes

The lipophilic antioxidant 3,5-di-t-butyl-4-hydroxytoluene (BHT) and the structurally-related antiatherogenic drug probucol stimulate the oxygenation of mitochondrial membranes and erythrocyte ghosts by the rabbit 15-lipoxygenase as indicated by an increase in oxygen consumption as well as by an enhanced loss of polyenoic fatty acids and by the formation of specific lipoxygenase products in the membrane phospholipids. The oxygenation of linoleic acid, phospholipids and human low-density lipoproteins was not stimulated. With mitochondrial membranes, BHT causes a quenching of the 1-anilino-8-naphthalene sulfonate fluorescence. Thus, it is suggested that the stimulation of membrane oxygenation may be due to structural changes in the membranes leading to a better susceptibility of the polyenoic fatty acid residues towards lipoxygenase attack. Owing to this unexpected effect of the antioxidants, which is not related to their radical-scavenger capacity, care should be taken in interpreting experimental data on effects of BHT and probucol.     

Detection of conformational changes in immunoglobulin G using isothermal titration calorimetry with low-molecular-weight probes

Proteins for therapeutic use may contain small amounts of partially misfolded monomeric precursors to postproduction aggregation. To detect these misfolded proteins in the presence of an excess of properly folded protein, fluorescent probes such as 8-anilino-1-naphthalene sulfonate (ANS) are commonly used. We investigated the possibility of using isothermal titration calorimetry (ITC) to improve the detection of this type of conformational change using hydrophobic probes. As a case study, conformational changes in human polyclonal immunoglobulin G (IgG) were monitored by measuring the enthalpies of binding of ANS using ITC. Results were compared with those using fluorescence spectroscopy. IgG heated at 63 °C was used as a model system for “damaged” IgG. Heat-treated IgG can be detected already at levels below 5% with both ITC and fluorescence. However, ITC allows a much wider molar probe-to-protein ratio to be sampled. In particular, using reverse titration experiments (allowing high probe-to-protein ratios not available to fluorescence spectroscopy), an increase in the number of binding sites with a K_d > 10 mM was observed for heat-treated IgG, reflecting subtle changes in structure. Both ITC and fluorescence spectroscopy showed low background signals for native IgG. The nature of the background signals was not clear from the fluorescence measurements. However, further analysis of the ITC background signals shows that a fraction (8%) binds ANS with a dissociation constant of approximately 0.2 mM. Measurements were also carried out at pH 4.5. Precipitation of IgG was induced by ANS at concentrations above 0.5 mM, interfering with the ITC measurements. Instead, with the nonfluorescent probes 4-amino-1-naphthalene sulfonate and 1-naphthalene sulfonate, no precipitation is observed. These probes yield differences in the enthalpies of binding to heated and nonheated IgG similar to ANS. The data illustrate that ITC with low-molecular-weight probes is a versatile tool to monitor conformational changes in proteins with a wider application potential than fluorescence measurements.     

Characterization of the binding of Cu(II) and Zn(II) to transthyretin: effects on amyloid formation

Although metal ions can promote amyloid formation from many proteins, their effects on the formation of amyloid from transthyretin have not been previously studied. We therefore screened the effects of Cu(II), Zn(II), Al(III), and Fe(III) on amyloid formation from wild-type (WT) transthyretin as well as its V30M, L55P, and T119M mutants. Cu(II) and Zn(II) promoted amyloid formation from the L55P mutant of transthyretin at pH 6.5 but had little effect on amyloid formation from the other forms of the protein. Zn(II) promoted L55P amyloid formation at pH 7.4 but Cu(II) inhibited it. Cu(II) gave dose-dependent quenching of the tryptophan fluorescence of transthyretin and the fluorescence of 1-anilino-8-naphthalene sulfonate bound to it. Zn(II) gave dose-dependent quenching of the tryptophan but not the 1-anilino-8-naphthalene sulfonate fluorescence. Apparent dissociation constants for Cu(II) and Zn(II) binding at pH 7.4 of approximately 10 nM and approximately 1 microM (approximately 0.4 microM and approximately 5 microM at pH 6.5), respectively, were obtained from the quenching data. Zn(II) enhanced urea-mediated the dissociation of the L55P but not the WT transthyretin tetramer. Cu(II), depending on its concentration, either had no effect or stabilized the WT tetramer but could enhance urea-mediated dissociation of L55P.     

The interaction of 1-anilino-8-naphthalene sulfonate with tubulin: A site independent of the colchicine-binding site

Rat brain tubulin binds 1 mole of 1-anilino-8-naphthalene sulfonate (ANS) per dimer (110,000 daltons) with an association constant of 3.2 × 10⁵m^?1. The quantum yield of ANS fluorescence is increased 120-fold over that in water to φ = 0.48 and there is a hypsochromic shift of 56 nm to an emission maximum of 460 nm. There is energy transfer from tryptophan to bound ANS. Vinblastine and Ca²⁺ enhance ANS fluorescence in tubulin by 35%–40%; this can be ascribed to an increased quantum yield, rather than changes in the affinity constant or number of binding sites. The ANS binding site shows minimal decay at 37 °C when colchicine binding has decreased to 50%. It is concluded that the colchicine- and ANS-binding sites occupy different regions of the tubulin molecule.     

Trypanosoma brucei: a fluorescence and spin label study of the membranes of the bloodstream form

Fluidity of the plasma membrane of Trypanosoma brucei brucei has been examined with fluorescence and electron spin resonance spectroscopy. Fluorescent probes 1,6-diphenyl-1,3,5-hexatriene and 8-anilino-1-naphthalene sulfonate and the spin label probe 5-doxyl stearate have been employed to examine fluidity under a variety of conditions. The temperature dependence of 8-anilino-1-naphthalene sulfonate polarization and of the order parameter S for 5-doxyl stearate reveals phase alterations near 30 C. 1,6-Diphenyl-1,3,5-hexatriene polarization shows that proteolysis of the surface glycoprotein with trypsin increases fluidity but treatment with human serum which is trypanocidal produces no detectable change in membrane fluidity.     

Characterization of the effects of propranolol on the physical state of platelet membrane

The direct membrane effect of propranolol was studied in vitro on washed platelet preparations. Propranolol has been reported to inhibit platelet aggregation by mechanisms unrelated to its beta-blocking activity. In the present study, the drug was found to enhance 1-anilino-8-naphthalene sulfonate binding to platelet membrane by increasing the number of binding sites. Steady-state anisotropy was studied by labeling the platelets with the hydrophobic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene. Propranolol was observed to decrease the equivalent microviscosity of the membrane. When the infinitely slow decaying component of fluorescence anisotropy (r infinity), which is proportional to the square of lipid order parameter, was calculated from the anisotropy data, a decrease in these parameters was also indicated. A higher fusion activation energy for viscosity in the propranolol-treated platelets reflected a lesser degree of order of the hydrocarbon chains in the lipid bilayer.