ANS fluorescence. I. Effect of self-association on the spectral properties of the probe]

The dependence of spectral properties of Mg2+ and NH4+ salt of 8-anilino-1-naphthalenesulfonic acid (Mg-(ANS)2 and NH4-ANS, respectively) on the dye concentration and solvent composition was investigated by means of steady-state and time-resolved fluorescence spectroscopy. We have shown that the increase in ANS concentrations leads to changes in the shape of absorption and fluorescence spectra of the dye, accompanied by the decrease in its fluorescence decay time values. Such changes, observed in aqueous and organic solvents for both salts of ANS, reflect the existence of self-association of the dye molecules. The decrease in fluorescence intensity induced by self-association of the probe molecules is too small to explain a weak fluorescence of ANS in water. At the same time, it expounds the difference between the decay times of protein-embedded ANS molecules upon interaction of this probe with native and molten globule proteins.     

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.     

Structural and thermodynamic effects of ANS binding to human interleukin-1 receptor antagonist

Although 8-anilinonaphthalene-1-sulfonic acid (ANS) is frequently used in protein folding studies, the structural and thermodynamic effects of its binding to proteins are not well understood. Using high-resolution two-dimensional NMR and human interleukin-1 receptor antagonist (IL-1ra) as a model protein, we obtained detailed information on ANS-protein interactions in the absence and presence of urea. The effects of ambient to elevated temperatures on the affinity and specificity of ANS binding were assessed from experiments performed at 25 degrees C and 37 degrees C. Overall, the affinity of ANS was lower at 37 degrees C compared to 25 degrees C, but no significant change in the site specificity of binding was observed from the chemical shift perturbation data. The same site-specific binding was evident in the presence of 5.2 M urea, well within the unfolding transition region, and resulted in selective stabilization of the folded state. Based on the two-state denaturation mechanism, ANS-dependent changes in the protein stability were estimated from relative intensities of two amide resonances specific to the folded and unfolded states of IL-1ra. No evidence was found for any ANS-induced partially denatured or aggregated forms of IL-1ra throughout the experimental conditions, consistent with a cooperative and reversible denaturation process. The NMR results support earlier observations on the tendency of ANS to interact with solvent-exposed positively charged sites on proteins. Under denaturing conditions, ANS binding appears to be selective to structured states rather than unfolded conformations. Interestingly, the binding occurs within a previously identified aggregation-critical region in IL-1ra, thus providing an insight into ligand-dependent protein aggregation.     

ANS fluorescence: potential to augment the identification of the external binding sites of proteins

8-anilino-1-naphthalenesulfonic acid (ANS) is believed to strongly bind cationic groups of proteins and polyamino acids through ion pair formation. A paucity of data exists on the fluorescent properties of ANS in these interactions. ANS binding to arginine and lysine derivatives was studied by fluorescence and circular dichroism spectroscopies to augment published information attained by isothermal titration calorimetry (ITC). Fluorescence enhancement with a hypsochromic shift results from the interaction of the charged group of lysine and arginine with the sulfonate group of ANS. Ion pairing between Arg (or Lys) and the sulfonate group of ANS reduce the intermolecular charge transfer (CT) rate constant that leads to enhancement of fluorescence. A positive charge near the -NH group of ANS changes the intramolecular CT process producing a blue shift of fluorescence. The Arg side chain compared to that of Lys more effectively interacts with both the -NH and sulfonate groups of ANS. ANS binding also induces a random coil-alpha helix transition in poly-Arg. Our data, in contrast to ITC results, indicate that electrostatic interactions between ANS derivatives and positively charged side chains do not account for binding affinity in the micromolar range. In addition to ion pairing complementary interactions, such as van der Waals, should be considered for high affinity (K(d)<1 mM) external binding sites of proteins.     

Thermal transitions in erythrocyte membranes revealed by their permeability to ANS

A number of breaks were recorded on the curve of Arrhenius relationship of the rate constant of the dye 1-anilino-8-naphthalenesulphonate sodium salt (ANS) input into human erythrocytes of 20, 28, 36, 42 and 46 degrees C. Variations in the values of activation energies within the temperature range of 28-36 degrees and 42-46 degrees C obtained in various blood samples allow to consider these temperatures as those at which structural changes of the membranes take place. The values of activation energy of the process for temperature "conformers" of the erythrocyte membrane are 12(10-20 degrees C), 26.5 (20-28 degrees C), 34.2(36-42 degrees C) and 47 kcal/mol (t is greater than 46 degrees C). Within the temperature range of 28-36 degrees and 42-46 degrees C an irreversible decrease of permeability to ANS of the erythrocyte ghost after their incubation for 10 min at increased temperatures were observed. Thus the temperature regions of the change in erythrocyte permeability correspond to those at which the resealing of ghost takes place. The break in Arrhenius graph at 20 degrees C seems to characterize a highly cooperative "point" transition. The lipid nature of the initiator of structural transition within 28-36 degrees C is proved by a sharp increase of the permeability of liposomes prepared from erythrocyte membrane lipids to ANS at 28 degrees C. The nature of the initiators of two other thermal transitions is discussed.     

ANS fluorescence detects widespread perturbations of protein tertiary structure in ice

Freeze-induced perturbations of the protein native fold are poorly understood owing to the difficulty of monitoring their structure in ice. Here, we report that binding of the fluorescence probe 1-anilino-8-naphthalene sulfonate (ANS) to proteins in ice can provide a general monitor of ice-induced alterations of their tertiary structure. Experiments conducted with copper-free azurin from Pseudomonas aeruginosa and mutants I7S, F110S, and C3A/C26A correlate the magnitude of the ice-induced perturbation, as inferred from the extent of ANS binding, to the plasticity of the globular fold, increasing with less stable globular folds as well as when the flexibility of the macromolecule is enhanced. The distortion of the native structure inferred from ANS binding was found to draw a parallel with the extent of irreversible denaturation by freeze-thawing, suggesting that these altered conformations play a direct role on freeze damage. ANS binding experiments, extended to a set of proteins including serum albumin, alpha-amylase, beta-galactosidase, alcohol dehydrogenase from horse liver, alcohol dehydrogenase from yeast, lactic dehydrogenase, and aldolase, confirmed that a stressed condition of the native fold in the frozen state appears to be general to most proteins and pointed out that oligomers tend to be more labile than monomers presumably because the globular fold can be further destabilized by subunit dissociation. The results of this study suggest that the ANS binding method may find practical utility in testing the effectiveness of various additives employed in protein formulations as well as to devise safer freeze-drying protocols of pharmaceutical proteins.     

高通量脂肪酶稳定性测定法-ANS法的建立

根据蛋白质变性后结构变化的特点和荧光探针8-苯胺基-1-萘磺酸 (8-Anilino-1-naphthalenesulfonic acid,ANS) 的特性,建立了一种快捷而准确的脂肪酶热稳定性 (Tm) 测定的新方法——ANS脂肪酶稳定性测定法,即将脂肪酶在25 ℃~65 ℃的不同温度下保温30 min后,加入到ANS反应体系 (0.2 mg/mL酶蛋白,0.05 mmol/L ANS,20 mmol/L Tris-HCl (pH 7.2),100~500 mmol/L NaCl) 中。在激发波长378     

Photophysics of ANS. III: Circular dichroism of ANS and anilinonaphthalene in I-FABP

We investigate the circular dichroism of the I-FABP system with the ligands ANS (1,8-anilinonaphthalene sulfonate) and AnN (anilinonaphthalene) as previously reported in our earlier publications in the series (referred to as I and II here) on ANS photophysics. We employ our semi-empirical calculated spectral functions (from II) to compute the actual CD spectra, without any additional assumptions or data except what we have previously presented with respect to binding geometry (in I). The common mechanisms fail to produce the observed spectra. However, we identify a novel mechanism of induced CD activity, which does succeed. This new mechanism also suggests how it is that near UV CD can often show extreme sensitivity to local 'order' effects.     

ANS fluorescence. II. Use of the method of fluorescent probe decay for studying the structural transformations in globular proteins]

Changes in ANS fluorescence decay parameters induced by the interaction of the probe with proteins have been investigated. The existence of at least two different modes of interactions between the ANS and protein was established. The interactions of the first type are connected with binding of an ANS molecule with the surface of a protein molecule. In this case ANS molecules are well acceptable for a solvent. The interactions of the second type are characteristic of the protein-embedded ANS molecules. The decay time values of the second type complexes change considerably (> 1.5-fold) during the protein molecule transformation into the molten globule-like conformation. The molecular model explaining such a behaviour is suggested.     

8-anilino-1-naphthalene sulfonic acid (ANS) induces folding of acid unfolded cytochrome c to molten globule state as a result of electrostatic interactions.

Hydrophobic interaction of 8-anilino-1-naphthalene sulfonic acid (ANS) with proteins is one of the widely used methods for characterizing/detecting partially folded states of proteins. We have carried out a systematic investigation on the effect of ANS, a charged hydrophobic fluorescent dye, on structural properties of acid-unfolded horse heart cytochrome c at pH 2.0 by a combination of optical methods and electrospray ionization mass spectroscopy (ESI MS). ANS was found to induce, a secondary structure similar to native protein and quenching of fluorescence of tryptophan residue, in the acid-unfolded protein. However, the tertiary structure was found to be disrupted thus indicating that ANS stabilizes a molten globule state in acid-unfolded protein. To understand the mechanism of ANS-induced folding of acid-unfolded cytochrome c, comparative ESI MS, soret absorption, and tryptophan fluorescence studies using nile red, a neutral hydrophobic dye, and ANS were carried out. These studies suggested that, at low pH, electrostatic interactions between negatively charged ANS molecules and positively charged amino acid residues present in acid-unfolded cytochrome c are probably responsible for ANS-induced folding of acid-unfolded protein to partially folded compact state or molten globule state. This is the first experimental demonstration of ANS induced folding of unfolded protein and puts to question the usefulness of ANS for characterization/determination of partially folded intermediates of proteins observed under low pH conditions.     

Fluorescence lifetime distribution of 1,8-anilinonaphthalenesulfonate (ANS) in reversed micelles detected by frequency domain fluorometry.

The fluorescence emission decay of ANS (1,8-anilinonaphthalenesulfonate) in reversed AOT (sodium bis-(2-ethyl-1-hexy)sulfosuccinate) micelles at different water contents was investigated by frequency domain fluorometry. The whole ANS emission decay in reversed AOT micelles could not be fitted in terms of discrete lifetime values, i.e., mono-exponential and bi-exponential models. Better fits were obtained when using continuous unimodal Lorentzian lifetime distributions. This was interpreted as arising from the reorientation processes of water molecules around the excited state of ANS or probe exchange among different probe locations, occurring on a time scale longer than fluorophore lifetime. The dependence of ANS fluorescence anisotropy on the emission wavelength was consistent with the existence of a great emission heterogeneity especially for inverted micelles having reduced H2O/AOT molar ratio. Finally, the observation that the distribution width decreases with increasing temperature and/or micelle size suggested that fast processes of water dipolar reorganization around the fluorophore are facilitated under these conditions.     

Photophysics of ANS. IV. Electron transfer quenching of ANS in alcoholic solvents and mixtures

ANS is observed to decay from the fluorescent state with distributed kinetics in nearly pure ethanol solvent, notwithstanding that in mixed ethanol/water solvents the decay is discrete and biexponential. The origin of this behavior is investigated. In particular, a theory of electron transfer theory in the adiabatic regime is adduced, with specific involvement of solvent cage structure in the form of the solvent-electron polaron wave function. Properties of various polarons for various solvent systems are predicted and, for the case of ethanol and cyclohexanol, employed to generate the observed Arrhenius-type decay parameters in a quantitative fashion.     

Interaction of 8-anilinonaphthalene 1-sulphonate (ANS) and human matrix metalloproteinase 7 (MMP-7) as examined by MMP-7 activity and ANS fluorescence

Human matrix metalloproteinase 7 (MMP-7) is the smallest matrix metalloproteinase. It plays important roles in tumour invasion and metastasis. 8-Anilinonaphthalene 1-sulphonate (ANS) is a fluorescent probe widely used for the analysis of proteins. It emits large fluorescence energy when its anilinonaphthalene group binds with hydrophobic regions of protein. In this study, we analysed the interaction of ANS and MMP-7. At pH 4.5-9.5, ANS inhibited MMP-7 activity in the hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH(2). The inhibition was a non-competitive manner and depended on the time for pre-incubation of ANS and MMP-7. At pH 4.5-9.5, the fluorescence of ANS was not changed by the addition of MMP-7. At pH 3.5, MMP-7 lacked activity, and the fluorescence of ANS was increased by the addition of MMP-7. These results suggest that at pH 4.5-9.5, the sulphonic group of ANS binds with MMP-7 through electrostatic interaction, whereas at pH 3.5, the anilinonaphthalene group of ANS binds with MMP-7 through hydrophobic interaction.     

The effect of medium on functional and structural properties of serum albumins. II. The effect of temperature on the N-form of human serum albumin

In order to investigate effects of temperature in the physiological range (from 10 to 50 degrees C) on structural, physical and functional properties of the N-form of human serum albumin (HSA), the temperature dependences of fluorescence parameters of Trp-214 residue of HSA and of the specifically bound dye ANS, as well as of association constants of ANS binding in the primary and secondary binding sites on HSA molecule were measured. The temperature-induced changes of these properties of HSA are essentially dependent on pH (7.0 or 5,6) and ionic strength (0.001-0.008 or 0.2 M NaCl). At pH 7.0 and 0.2 M NaCl the environment of Trp-214 remained invariant at temperature changes between 10 and 50 degrees C. On the other hand, the affinity to ANS of a primary binding site doubled and that of secondary ones halved. These affinity changes seem to be due, are least partly, to the heating-induced dissociation of Cl-ions, which are inhibitors of the primary dye binding. By lowering pH (to 5.6) and ionic strength the temperature-induced changes in the Trp-214 environment were observed. The changes are interpreted as indole group transition into the buried region, inaccesible to water (the "closing" of a structural slit). The affinity of secondary binding sites of ANS was halved.     

Dynamics of ANS binding to tuna apomyoglobin measured with fluorescence correlation spectroscopy.

The dynamics of the binding reaction of ANS to native and partly folded (molten globule) tuna and horse apomyoglobins has been investigated by fluorescence correlation spectroscopy and frequency domain fluorometry. The reaction rate has been measured as a function of apomyoglobin and ANS concentrations, pH, and temperature. Examination of the autocorrelation functions shows that the reaction rate is fast enough to be observed in tuna apomyoglobin, whereas the reaction rate in horse apomyoglobin is on the same time scale as diffusion through the volume or longer. Specifically, for tuna apomyoglobin at pH 7 and room temperature the on rate is 2200 microM(-1) s(-1) and the off rate is 5900 s(-1), in comparison with k(on) = 640 microM(-1) s(-1) and k(off) = 560 s(-1) for horse myoglobin as measured previously. The independence of the reaction rate from the ANS concentration indicates that the reaction rate is dominated by the off rate. The temperature dependence of the on-rate shows that this rate is diffusion limited. The temperature dependence of the off rates analyzed by Arrhenius and Ferry models indicates that the off rate depends on the dynamics of the protein. The differences between horse and tuna apomyoglobins in the ANS binding rate can be explained in terms of the three-dimensional apoprotein structures obtained by energy minimization after heme removal starting from crystallographic coordinates. The comparison of the calculated apomyoglobin surfaces shows a 15% smaller cavity for tuna apomyoglobin. Furthermore, a negative charge (D44) is present in the heme cavity of tuna apomyoglobin that could decrease the strength of ANS binding. At pH 5 the fluorescence lifetime distribution of ANS-apomyoglobin is bimodal, suggesting the presence of an additional binding site in the protein. The binding rates determined by FCS under these conditions show that the protein is either in the open configuration or is more flexible, making it much easier to bind. At pH 3, the protein is in a partially denatured state with multiple potential binding sites for ANS molecule, and the interpretation of the autocorrelation function is not possible by simple models. This conclusion is consistent with the broad distribution of ANS fluorescence lifetimes observed in frequency domain measurements.     

Perturbation of protein tertiary structure in frozen solutions revealed by 1-anilino-8-naphthalene sulfonate fluorescence

Although freeze-induced perturbations of the protein native fold are common, the underlying mechanism is poorly understood owing to the difficulty of monitoring their structure in ice. In this report we propose that binding of the fluorescence probe 1-anilino-8-naphthalene sulfonate (ANS) to proteins in ice can provide a useful monitor of ice-induced strains on the native fold. Experiments conducted with copper-free azurin from Pseudomonas aeruginosa, as a model protein system, demonstrate that in frozen solutions the fluorescence of ANS is enhanced several fold and becomes blue shifted relative free ANS. From the enhancement factor it is estimated that, at -13 degrees C, on average at least 1.6 ANS molecules become immobilized within hydrophobic sites of apo-azurin, sites that are destroyed when the structure is largely unfolded by guanidinium hydrochloride. The extent of ANS binding is influenced by temperature of ice as well as by conditions that affect the stability of the globular structure. Lowering the temperature from -4 degrees C to -18 degrees C leads to an apparent increase in the number of binding sites, an indication that low temperature and /or a reduced amount of liquid water augment the strain on the protein tertiary structure. It is significant that ANS binding is practically abolished when the native fold is stabilized upon formation of the Cd(2+) complex or on addition of glycerol to the solution but is further enhanced in the presence of NaSCN, a known destabilizing agent. The results of the present study suggest that the ANS binding method may find practical utility in testing the effectiveness of various additives employed in protein formulations as well as to devise safer freeze-drying protocols of pharmaceutical proteins.     

Effect of chlorpromazine on the relation between temperature and the binding of ANS by human erythrocytes

It has been shown that under the effect of chlorpromazin (concentration to 2.10(-5) M) breaks observed on Arrhenius graphs of the rate constant of ANS binding with erythrocytes (k) at 28 and 36 degrees C are shifted to the region of low temperatures approximately to a similar interval (14-15 degrees C). The value k measured at 22 degrees C is not changed within the pH range of 5-7. It is concluded that breaks characterised the initial and final stages of temperature transition initiated in the zone of acid phospholipid of membranes.     

Misconceptions over Förster resonance energy transfer between proteins and ANS/bis-ANS: Direct excitation dominates dye fluorescence

Our aim was to disprove the widespread misconception that Förster resonance energy transfer (FRET) is the only explanation for observing fluorescence from ANS (8-anilino-1-naphthalenesulfonic acid) and bis-ANS (4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid, dipotassium salt) following excitation at 280 nm in the presence of protein. From ultraviolet (UV) absorption spectra and fluorescence emission spectra of bis-ANS and ANS in buffer and ethanol, direct excitation at 280 nm was found to be the dominant mechanism for the resulting dye fluorescence. Furthermore, Tyr/Trp quenching studies were performed for solutions of N-acetyl-l-tryptophanamide, heat-stressed immunoglobulin G (IgG), and bovine serum albumin (BSA) by monitoring changes in steady state fluorescence spectra and time-resolved fluorescence decays as a function of dye concentration. Stronger quenching of the intrinsic BSA and IgG fluorescence in steady state than in time-resolved fluorescence by bis-ANS and ANS pointed toward static quenching being the dominant mechanism in addition to dynamic quenching and/or FRET. In conclusion, one should consider the role of direct excitation of ANS and bis-ANS at 280 nm to ensure a proper interpretation of fluorescence signals resulting from dye-protein interactions. When ANS or bis-ANS is to be used for protein characterization, we recommend selectively exciting the dyes at the higher absorption wavelength maximum (370 or 385 nm, respectively).     

Characterization of the binding of 8-anilinonaphthalene sulfonate to rat class Mu GST M1-1

Molecular docking and ANS-displacement experiments indicated that 8-anilinonaphthalene sulfonate (ANS) binds the hydrophobic site (H-site) in the active site of dimeric class Mu rGST M1-1. The naphthalene moiety provides most of the van der Waals contacts at the ANS-binding interface while the anilino group is able to sample different rotamers. The energetics of ANS binding were studied by isothermal titration calorimetry (ITC) over the temperature range of 5-30 degrees C. Binding is both enthalpically and entropically driven and displays a stoichiometry of one ANS molecule per subunit (or H-site). ANS binding is linked to the uptake of 0.5 protons at pH 6.5. Enthalpy of binding depends linearly upon temperature yielding a DeltaC(p) of -80+/-4 cal K(-1) mol(-1) indicating the burial of solvent-exposed nonpolar surface area upon ANS-protein complex formation. While ion-pair interactions between the sulfonate moiety of ANS and protein cationic groups may be significant for other ANS-binding proteins, the binding of ANS to rGST M1-1 is primarily hydrophobic in origin. The binding properties are compared with those of other GSTs and ANS-binding proteins.