Alignment of anilinonaphthalene-sulfonate and related fluorescent probe molecules in squid axon membrane and in synthetic polymers

As a fluorescent probe for the squid axon membrane, the behavior of 1-anilinonaphthalene-8-sulfonate (1,8-ANS) was found to be very different from that of its positional isomer, 2,6-ANS, or of the methylated derivative, 2,6-TNS. The degree of polarization of the fluorescent light contributing to a transient intensity reduction during nerve excitation was larger than about 0.7 for both 2,6-ANS and 2,6-TNS, while the corresponding value for 1,8-ANS in a squid axon was about 0.35.The physicochemical basis of this difference was investigated by measuring the fluorescence polarization of these probe molecules incorporated into poly(vinyl alcohol) sheets. In a stretched sheet of this synthetic polymer, 1,8-ANS showed poor alignment, while the 2,6-derivatives were highly oriented with their transition moments aligned approximately in the direction of stretching. Based on these findings, the experimental results obtained from squid axons were interpreted as an indication of the existence, at or near the membrane, of a longitudinally oriented macromolecular structure, bringing about a high degree of alignment of 2,6-ANS or 2,6-TNS molecules.It is clear that, as a probe for fluorescence polarization studies of macromolecular structures, 2,6-TNS is far superior to 1,8-ANS.     

The interaction of calmodulin with the C-terminal M5 peptide of myosin light chain kinase

The interaction with calmodulin of the 17-residue C-terminal fragment M5 of myosin light chain kinase has been studied by several physical techniques. Circular dichroism measurements suggest that M5 exists within the complex primarily as an alpha-helix. Fluorescence intensity measurements of the single tryptophan of M5 (Trp-4) indicate that it is in a relatively nonpolar environment and is shielded from solvent. Dynamic measurements of fluorescence anisotropy decay indicate that Trp-4 changes from a freely rotating fluorophore to one which is largely immobilized upon complex formation. Static fluorescence measurements show that 2,6-TNS is displaced from its binding site on calmodulin by M5. The binding of M5 also partially inhibits the proteolytic scission by trypsin of the bond between residues 77 and 78.     

A comparative analysis of extrinsic fluorescence in nerve membranes and lipid bilayers

Changes in extrinsic fluorescence intensity, associated with step changes in membrane potential, have been studied in intracellularly or extracellularly stained squid axons, and in lipid bilayers, using six different aminonaphthalene dyes: 1,8-TNS; 2,6-TNS; 1,8-MANS; 2,6-MANS; 2,6-ANS and NPN. In all preparations the optical signals were found to be roughly proportional to the voltage applied. All signals had a very fast initial component, which was followed in some case by a slower change in the same direction. The slow component was observed only in intracellularly stained axons, and not for all chromophores studied. 1,8-TNS, 1,8-MANS and 2,6-MANS yielded the largest fluorescence signals in all preparations. The sign of these signals was independent of the type of membrane studied. However, the fluorescence changes of 2,6-MANS were opposite to those of 1,8-TNS and 1,8 MANS. Staining of both sides of the axolemma with 1,8-MANS or 2,6-MANS showed that these dyes yield larger signals when applied to the extracellular face. The changes in fluorescence light intensity of 2,6-TNS, 2,6-ANS and NPN were smaller and their sign depended on the membrane preparation studied. The comparison of the extrinsic fluorescence signals from the nerve membrane and the phosphatidylcholine bilayer suggests strong similarities between the basic structures of the two systems. The variety of observed signals cannot be easily interpreted in terms of changes in membrane structure. A possible alternative interpretation in terms of electrically induced displacements, rotations and changes in partition coefficient of bound chromophores, is discussed.Abbreviations 1,8-TNS 1-toluidinonaphthalene-8-sulfonate, and similarly, 2,6-TNS - 1,8-MANS 1-N-methylanilinonaphthalene-8-sulfonate, and similarly, 2,6-MANS - 1,8-ANS 1-anilinonaphthalene-8-sulfonate, and similarly, 2,6-ANS - NPN N-phenyl-1-naphthylamine     

Fluorescent derivative of cysteine-10 reveals thyroxine-dependent conformational modifications in human serum prealbumin

Fluorescence studies on the N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine-labeled cysteine-10 residue of human prealbumin were carried out to detect conformational changes induced by the binding of the ligand thyroxine to the two structurally identical binding sites. A red shift of the spectrum was observed and the total change was confined to the first ligand. This was interpreted as resulting from a conformational change which increases the exposure of the fluorescent probe moiety. Thyroxine also alters the effect of the collisional quencher, acrylamide, confirming the greater exposure of the probe. This modification in structure is associated with changes in relaxation time which indicate that when thyroxine is bound there is an increase in the rotational freedom of the segment or domain of prealbumin which contains the fluorescent probe.     

Spectral analyses of extrinsic fluorescence of the nerve membrane labeled with aminonaphthalene derivatives

A fluorescence spectrophotometer was constructed to determine the emission spectrum of a nerve labeled with various fluorochromes. Using this spectrophotometer, the spectra of 2-p-toluidinylnaphthalene 6-sulfonate (2,6-TNS) and other aminonaphthalene derivatives in squid giant axons were determined at the peak of nerve excitation, as well as in the resting state of the axons. During nerve excitation the fluorescent light deriving from the 2,6-TNS-stained nerve undergoes a transient change in intensity. The spectrum of the light contributing to this change in intensity was found to be much narrower and sharper than the fluorescent spectrum of the light arising from labeled axons at rest. This narrow and sharp spectrum is interpreted as being derived from a transient variation in the polarity of the 2,6-TNS binding sites in the axon. In the Appendix, the results of a physicochemical investigation into the factors affecting the fluorescence of 2,6-TNS in vitro are described.     

Binding of hydrophobic ligands by Pseudomonas aeruginosa PA-I lectin

The ability of Pseudomonas aeruginosa PA-I lectin to bind the fluorescent hydrophobic probe, 2-(p-toluidinyl) naphthalene sulfonic acid (TNS), and adenine was examined by spectrofluorametry and equilibrium dialysis. Interaction of TNS with PA-I caused significant enhancement of TNS fluorescence. The Hill coefficient (3.8+/-0.3) and the dissociation constant (8.7+/-0.16 microM) showed that TNS probably bound to four high affinity hydrophobic sites per PA-I tetramer. Interactions between PA-I and adenine were examined by equilibrium dialysis using [3H] adenine. The results indicated the presence of at least two classes of binding sites--one high and four lower affinity sites per tetramer with dissociation constants of 3.7+/-1.5 and 42.6+/-1.2 microM, respectively. These were distinct from the TNS sites as titration of TNS-equilibrated PA-I with adenine caused TNS fluorescence enhancement. The titration curve confirmed the existence of two classes of adenine-binding sites. Conversely, when PA-I was first equilibrated with adenine and then titrated with TNS, no TNS-binding was registered. This may indicate that conformational rearrangements of the lectin molecule caused by adenine prevent allosterically TNS binding.     

Effect of pulse electromagnetic radiation on erythrocyte ghosts

The pulse microwave radiation has been shown to increase the fluorescence intensity of 2-toluidinonaphthanene-6-sulfonate (2,6-TNS) and 1-anilinonaphthalene-8-sulfonate (1,8-ANS) built-in membranes of erythrocyte ghosts. In experiments with 2,6-TNS a frequency dependence of the effect of microwave radiation with maximum within the frequency range of 55-65 Hz has been found. It is suggested that the changes registered with fluorescent probes are induced by mechanical oscillations generated by the pulse microwave radiation.     

Malate dehydrogenase, anticooperative NADH, and L-malate binding in ternary complexes with Supernatant pig heart enzyme.

Supernatant malate dehydrogenase from pig heart, a dimeric protein containing two very similar or identical subunits, shows negatively cooperative (anticooperative) interactions between NADH binding sites in the presence, but not in the absence, of 0.1 M L-malate. This behavior is observed consitently whether the technique used employs protein fluorescence quenching, NADH fluorescence enhancement, or ultrafiltration dialysis. Fluorescence titration shows that L-malate is also anticooperatively bound in the presence of saturating concentrations of NADH. The data are consistent with an "induced asymmetry" model in which conformational change accompanies the formation of the ternary complex. Two of the three chromatographically resolvable forms of the enzyme have been tested and found to have anticooperative behavior.     

Monitoring of chemical and enzymatic hydrolysis of water-soluble proteins using flow-injection analysis with fluorescence detection and an aqueous eluant containing 2-p-toluidinylnaphthalene-6-sulfonate as the fluorescent probe

The exposed hydrophobicity of proteins, which is due to the hydrophobic regions located on their surfaces, enhances the fluorescence intensity of the probe 2-p-toluidinylnaphthalene-6-sulfonate (2,6-TNS) by the formation of a complex. During the hydrolysis of a protein, the average exposed hydrophobicity of the substrate continuously changes with incubation time, and these changes are immediately reflected by a corresponding change in the fluorescence intensity of the 2,6-TNS/substrate complex. Therefore, 2,6-TNS seems to be a good probe to monitor the course of the depolymerization processes of proteins. In this work, bovine serum albumin and alpha-casein have been hydrolyzed both chemically and enzymatically, and the course of the reactions is monitored by using flow-injection analysis (FIA) with fluorescence detection and a buffered aqueous eluant containing 2,6-TNS as the fluorescent probe. Results indicate that the time evolution of the fluorescence intensity of the 2,6-TNS/substrate complex can be correlated with the initial concentration of the parent protein, in mass per unit volume, the hydrolytic activity added, and the time evolution of the mean chain length of the substrate. In addition, because the time elapsed between injection of the sample into the FIA system and measurement of the corresponding fluorescence intensity is only a few seconds, this methodology could be a useful tool for on-line monitoring of processes for the production of protein hydrolysates.     

Does fluorescence of ANS reflect its binding to PAMAM dendrimer?

The analysis of binding between cationic PAMAM G5 dendrimer and anionic fluorescent probe using fluorescence and equilibrium dialysis has been made. It was found that at low concentrations of ANS the double fluorimetric titration technique can be successfully used for quantitative analysis of binding of ANS to dendrimer. Based on fluorescence and dialysis data the constants of binding and the number of binding centers were calculated for binding of ANS to PAMAM G5 dendrimer: K(b) is approx. (0.5-1)x10(5)M(-1) and n is (0.5-0.7).     

Fluorescence depolarization of actin filaments in reconstructed myofibers: the effect of S1 or pPDM-S1 on movements of distinct areas of actin

Fluorescence polarization measurements were used to study changes in the orientation and order of different sites on actin monomers within muscle thin filaments during weak or strong binding states with myosin subfragment-1. Ghost muscle fibers were supplemented with actin monomers specifically labeled with different fluorescent probes at Cys-10, Gln-41, Lys-61, Lys-373, Cys-374, and the nucleotide binding site. We also used fluorescent phalloidin as a probe near the filament axis. Changes in the orientation of the fluorophores depend not only on the state of acto-myosin binding but also on the location of the fluorescent probes. We observed changes in polarization (i.e., orientation) for those fluorophores attached at the sites directly involved in myosin binding (and located at high radii from the filament axis) that were contrary to the fluorophores located at the sites close to the axis of thin filament. These altered probe orientations suggest that myosin binding alters the conformation of F-actin. Strong binding by myosin heads produces changes in probe orientation that are opposite to those observed during weak binding.     

Hydrophobic interaction of fluorescent probes with fetuin, ovine submaxillary mucin, and canine tracheal mucins.

The presence of hydrophobic sites in fetuin, ovine submaxillary mucin and two homogeneous canine tracheal mucins was established by fluorescence probe techniques. The interaction between the above-mentioned glycoproteins and two hydrophobic fluorescent compounds, sodium mansate and mansylphenylalanine, was accompanied by an enhancement in fluorescence and a shift of the fluorescence maxima to shorter wavelengths. The introduction of a phenylalanine residue to the mansyl group enhanced the binding affinity of the probe for the hydrophobic sites of these glycoproteins as evidenced by lower values for the dissociation constants. The high molecular weight (581 600) tracheal mucin, which had the highest carbohydrate content (80%) of all the glycoproteins investigated, exhibited the highest fluorescence enhancement and the largest number of binding sites for these fluorescent probes.     

Circular dichroism of N-phenylnaphthylamine derivatives complexed with the β-form of poly(L-lysine)

Complex formation of 1-anilinonaphthalene-8-sulfonate (ANS) and 2-p-toluidinonaphthalene-6-sulfonate (TNS) with the β-form of poly(L -lysine) [(β-Lys)_n] has been studied by circular dichroism (CD) and absorption spectra measurements. Not only hydrophobic interactions but also hydrogen-bonding and electrostatic interactions contribute to complex formation. The relative importance of these stabilizing factors depends on the relative position of the arylamino group and the sulfonate. For example, ionic interactions play a significant role in the binding of 1,8-ANS and 1,8-TNS, but not in the case of 2,6-TNS. The induced CD of the complexes of (β-Lys)_n with 1,8-ANS and 1,8-TNS is consistent with theoretical calculations for nonplanar conformations of these dyes, twisted in a left-handed sense. As expected for steric reasons, the dominant isomer is one in which the arylamino group is oriented away from the 8-sulfonate (α₁). The induced CD of complexes with 2,6-TNS can be accounted for by an equimolar mixture of left-handed isomers in which the arylamino group is oriented toward the 1-position (β₂) and toward the 3-position (β₁). Our results demonstrate that (β-Lys)_n is capable of chiral discrimination and suggest its general utility for CD studies of racemic anionic dyes.     

Detection of energy transfer between tryptophan residues in the tubulin molecule and bound bis(8-anilinonaphthalene-1-sulfonate), an inhibitor of microtubule assembly, that binds to a flexible region on tubulin

The fluorescent apolar probe bis(8-anilinonaphthalene-1-sulfonate) (Bis-ANS) is a potent inhibitor of microtubule assembly that binds to tubulin at a hitherto uncharacterized site distinct from those of the antimitotic drugs. We have found that energy transfer between tryptophan residues and bound Bis-ANS leads to quenching of the intrinsic tubulin fluorescence. The quenching is biphasic, implying two types of Bis-ANS binding sites. The estimated Kd values are 2.7 and 22.2 microM, consistent with reported values for the primary and secondary Bis-ANS binding sites. Preincubation of tubulin at 37 degrees C results in increased quenching of tryptophan fluorescence without any effect on the Kd values, suggesting localized structural change in the protein around the Bis-ANS binding sites. Concentration-dependent depolarization of Bis-ANS fluorescence was observed, suggesting energy transfer among bound Bis-ANS molecules. Such a concentration-dependent decrease in fluorescence polarization was not observed with 8-anilinonaphthalene-1-sulfonate (1,8-ANS), the monomeric form of Bis-ANS. Perrin-Weber plots were obtained for bound Bis-ANS and 1,8-ANS by varying the viscosity with sucrose. The rotational relaxation times calculated for Bis-ANS and 1,8-ANS are 18 and 96 ns, respectively. Comparison with the theoretical value (125 ns) suggests that Bis-ANS binds to a flexible region of tubulin. This, coupled with the fact that Bis-ANS, but not 1,8-ANS, inhibits microtubule assembly, suggests that the region in the tubulin molecule responsible for microtubule assembly is relatively flexible.     

Nuclear magnetic resonance of protamines. A 1H-NMR study of the interaction of clupeine fractions with mononucleotides

The interaction of the three clupeine fractions, YI, YII, Z, and salmine fraction AI with mononucleotides has been examined by means of 1H nuclear magnetic resonance. The results obtained are interpreted in terms of electrostatic interactions between positive arginine guanidinyl groups and negative nucleotide phosphates. In addition, clupeine fraction YI and salmine fraction AI exhibit with guanine and adenine nucleotides a more specific interaction that leads to the formation of large aggregates in solution. The experimental data presented in this work demonstrate that the strength of interaction between clupeine YI and salmine AI with mononucleotides follows the order: 5'-dTMP approximately equal to 5'-dCMP much less than 5'-dAMP less than 5'-dGMP approximately equal to 5'-GMP.     

The binding of palmitoyl-CoA to bovine serum albumin

Bovine serum albumin (BSA) is routinely utilized in vitro to prevent the adverse detergent effects of long-chain acyl-CoA esters (i.e., palmitoyl-CoA) in enzyme assays. Determination of substrate saturation kinetics in the presence of albumin would only be valid if the relationship between bound and free substrate concentrations was known. To elucidate the relationship between bound and free palmitoyl-CoA concentrations in the presence of BSA, several different techniques including equilibrium dialysis, equilibrium partitioning, fluorescence polarization and direct fluorescence enhancement were investigated. Direct fluorescence enhancement using a custom synthesized fluorescent probe, 16-(9-anthroyloxy)palmitoyl-CoA (AP-CoA), was the best approach to this question. Measurement of the relationship between mol of palmitoyl-CoA bound per mol of BSA (nu) versus -log[free palmitoyl-CoA] revealed that the binding of palmitoyl-CoA to BSA, like palmitate was nonlinear, suggesting the presence of more than one class of acyl-CoA binding sites. Computer analyses of the binding data gave a best fit to the 2,4 two-class Scatchard model, suggesting the presence of two high-affinity primary binding sites (k1 = (1.55 +/- 0.46) x 10(-6) M-1) and four lower affinity secondary binding sites (k2 = (1.90 +/- 0.09) x 10(-8) M-1). Further analyses using the six parameter stoichiometric (stepwise) ligand binding model supports the existence of six binding sites with the higher affinities associated with the binding of the first mole of palmitoyl-CoA and weaker binding occurring after the first two sites are occupied. The association constants from this model of multiple binding diminish sequentially (i.e., K1 greater than K2 greater than K3 greater than...greater than or equal to K6), suggesting that each mol of long-chain acyl-CoA binds to BSA with decreasing affinities.     

Non‐target sites with single nucleotide insertions or deletions are frequently found in 16S rRNA sequences and can lead to false positives in fluorescence in situ hybridization (FISH)

Fluorescence in situ hybridization (FISH) has impacted profoundly on our knowledge of the in situ ecophysiology and biodiversity of bacteria in natural communities. However, it has many technical challenges including the possibility of false positives from the binding of probes to non‐target rRNA sequences. We show here that probe target sites containing single‐base insertions or deletions can lead to false FISH positives, the result of hybridization with a bulge around the missing base. Experimental and in silico data suggest this situation occurs at a surprisingly high frequency. The existence of such sites is not currently considered during most FISH probe design processes. We describe software to identify potential non‐target sites resulting from single‐base insertions or deletions in rRNA sequences. This software also provides an estimate of the FISH probe hybridization efficiency to these sites.     

Quantitative aspects of the development of a hydrophobic binding site on calmodulin by calcium binding

The interactive binding by calmodulin of Ca²⁺ and 1-anilinonaphthalene-8-sulfonate (1,8-ANS) has been examined. In the presence of saturating levels of Ca²⁺, calmodulin develops one moderately strong binding site for 1,8-ANS, plus one or more weaker sites. The binding of 1,8-ANS by unliganded, or singly liganded, calmodulin is slight; the development of a strong binding site, as well as the characteristic fluorescence enhancement and CD spectrum, requires the binding of two Ca²⁺ ions. Little further change occurs on binding additional Ca²⁺ ions.     

Fluorescence energy transfer between heterologous active sites of affinity-labeled aspartokinase of Escherichia coli.

The distance between aspartokinase and homoserine dehydrogenase active sites was determined using fluorescence energy transfer between modified substrates. The fluorescent 1,N(6)-ethenoadenosine 5'-triphosphate was bound at the kinase active site by Co(III) affinity labeling. Reduced thionicotinamide adenine dinucleotide phosphate quenched the fluorescence of bound nucleotide. Fluorescence depolarization measurements led to a delimitation of the value of the dipolar orientation factor to the range 0.3 to 2.8. The distance between the fluorescent probe and the quencher was 29 +/- 4 A. In the presence of threonine, this distance increased to 36 +/- 5 A. Threonine binding either increased the intersite distance by ca. 7 A or caused a reorientation of the probe at the dehydrogenase site.     

Gastric mucin hydrophobicity: effects of associated and covalently bound lipids, proteolysis, and reduction

The hydrophobic properties of gastric mucus glycoprotein were investigated using the fluorescent probe, bis(8-anilino-1-naphthalenesulfonate). The glycoprotein was subjected to removal of associated and covalently bound lipids, peptic degradation, and disulfide bridge reduction. Fluorescence titration data revealed the presence of 55 hydrophobic binding sites in the intact mucin molecule, 71 binding sites in the glycoprotein devoid of associated lipids, and 53 binding sites in the glycoprotein devoid of associated lipids and covalently bound fatty acids. Proteolytic digestion of the glycoprotein with pepsin essentially abolished the probe binding, while reduction of disulfide bridges resulted in glycoprotein subunits whose combined number of binding sites was about 3 times greater than that of the mucin polymer. The binding of the probe to mucus glycoprotein varied with the pH of the medium, being highest at pH 2.0 and lowest at pH 9.0. The results indicate that lipids contribute to the hydrophobic character of gastric mucin and that hydrophobic binding sites reside on the nonglycosylated regions of the glycoprotein polymer buried within its core.