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

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

Effect of the bidistilled modified water on bovine serum albumin conformation. Fluorescent spectroscopy data

It was shown that bidistilled modified water induces a marked decrease in the intensity of intrinsic fluorescence of bovine serum albumin and increases the binding of this protein to the fluorescent probe 1.8 ANS. These effects can be interpreted as a denaturing action of bidistilled modified water on the protein and a change in its conformational state, which is probably caused by changes in the microenvironment of the protein molecule. In addition, a substantial increase in the intrinsic fluorescence of bidistilled modified water, as compared with that of distilled water, was found.     

Reevaluation of ANS binding to human and bovine serum albumins: key role of equilibrium microdialysis in ligand - receptor binding characterization

In this work we return to the problem of the determination of ligand-receptor binding stoichiometry and binding constants. In many cases the ligand is a fluorescent dye which has low fluorescence quantum yield in free state but forms highly fluorescent complex with target receptor. That is why many researchers use dye fluorescence for determination of its binding parameters with receptor, but they leave out of account that fluorescence intensity is proportional to the part of the light absorbed by the solution rather than to the concentration of bound dye. We showed how ligand-receptor binding parameters can be determined by spectrophotometry of the solutions prepared by equilibrium microdialysis. We determined the binding parameters of ANS - human serum albumin (HSA) and ANS - bovine serum albumin (BSA) interaction, absorption spectra, concentration and molar extinction coefficient, as well as fluorescence quantum yield of the bound dye. It was found that HSA and BSA have two binding modes with significantly different affinity to ANS. Correct determination of the binding parameters of ligand-receptor interaction is important for fundamental investigations and practical aspects of molecule medicine and pharmaceutics. The data obtained for albumins are important in connection with their role as drugs transporters.     

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.     

Detection of conformational changes in chloroplast coupling factor 1 by 8-anilino-1-naphthalene-sulphonate fluorescence changes

Chloroplast coupling factor 1 (CF1) contains a high-affinity binding site for 8-anilino-1-napthalene sulphonate (ANS,Kd = 5-6 microM). The binding of ANS to the enzyme is associated with a fluorescence enhancement and a blue-shift in the emission spectrum. ANS only slightly inhibits ATP hydrolysis by CF1. Adenine nucleotides and inorganic phosphate induce a fast ANS fluorescence quenching of about 50% which is due to a decrease in the affinity of the enzyme for ANS (Kd increases from 6 microM to 22 microM) and in the fluorescence quantum yield of the bound probe (by 33%) but not in the number of ANS sites (n = 1). Conversely, Mg and Ca ions induce a fluorescence enhancement of bound ANS. Inactivation of the enzyme enhances ANS fluorescence, eliminates the response to adenine nucleotides and inorganic phosphate but increases the response to divalent metals. The affinity of latent CF1 for ADP (Kd = 12 microM) is considerably higher than for ATP (Kd = 95 microM) in buffer containing EDTA. The Kd for inorganic phosphate is 140 microM. Mg increases the apparent affinity for ATP (Kd = 28 microM) but not for ADP or Pi. Binding of ATP to the tight-sites does not inhibit the ADP or Pi-induced fluorescence quenching but decreases the affinity for ADP (Kd = 34 microM) and for inorganic phosphate (Kd = 320 microM). These results suggest that the ADP and phosphate binding sites are different but not independent from the tight sites. Activation of a Mg-specific ATPase in CF1 by octyl glucoside decreases the affinity for ADP and inorganic phosphate by about threefold but increases the affinity for ATP. ATPase activation of CF1 also increases the Ki for ADP inhibition of ATP hydrolysis. ATPase activation also influences the ANS responses to Ca and Mg. Ca-ATPase activation increases the fluorescence enhancement and the apparent affinity for Ca whereas Mg-ATPase activation specifically increases the Mg-induced fluorescence enhancement. The fluorescence of CF1-bound ANS is enhanced by Dio-9 and quenched by phloridzin, quercetin, Nbf-Cl and FITC. Nbf-Cl and FITC completely inhibit the ADP-induced fluorescence quenching whereas Dio-9 inhibits the Mg-induced fluorescence enhancement. ANS does not relieve the quercetin or phloridzin inhibition of ATP hydrolysis indicating that these inhibitors do not compete with ANS for a common binding site. ANS may be used, therefore, as a sensitive probe to detect conformational changes in CF1 in response to activation or inactivation and to binding of substrates and of inhibitors.     

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

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

Osmotic perturbations induce differential movements in the core and periphery of proteins, membranes and micelles

Polymeric structures, namely, micelles, membranes and globular proteins share the property of two distinct regions: a hydrophobic core and a hydrophilic exterior. The dynamics of these regions of the polymeric structures were probed using selective fluorophores 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-anilinonaphthalene-8-sulfonate (ANS), respectively. Perturbation of the polymers by external osmotic pressure, ionic strength and temperature was monitored in the two regions using steady state measurements of fluorescence intensity and anisotropy. While the fluorescence lifetime of DPH and ANS did not change significantly, parallel change in steady state anisotropy values and the rotational correlation time indicated mobility in the probe/probe-domain. Osmotic perturbation of the polymers in electrolyte media led to decreased DPH mobility. Enhanced ellipticity at 222 nm in bovine serum albumin was observed in 1.5 M NaCl and sucrose media. ANS exhibited a decreased anisotropy with progressive dehydration in proteins in NaCl media, in dimyristoylphosphatidylcholine (DMPC) vesicles in sucrose media, and in neutral laurylmaltoside micelles in both NaCl and sucrose media. Thus, ANS showed responses opposite to that of DPH in these systems. A comparison with several domain selective probes indicated that DPH reported findings common to depth probes while ANS reported data common to interfacial probes used for voltage monitoring.     

Effect of phospholipase A on the structure and functions of membrane vesicles from Mycobacterium phlei.

The phospholipid composition of the electron transport particles and coupling factor-depleted electron transport particles of Mycobacterium phlei are the same, but they differ in contents. The accessibility of partially purified phospholipase A to these membrane phospholipids was found to be different. Treatment of membranes of Mycobacterium phlei with phospholipase A impairs the rate of oxidation as well as phosphorylation. The inhibition of phosphorylation can be reversed by washing the membranes with defatted bovine serum albumin. The reconstitution of membrane-bound coupling factor-latent ATPase activity to phospholipase A-treated depleted electron transport particles and their capacity to couple phosphorylation to oxidation of substrates remained unaffected after phospholipase A treatment. However, the pH gradient as measured by bromthymol blue was not restored after reconstitution of phospholipase A-treated depleted electron transport particles with membrane-bound coupling factor-latent ATPase. These findings show that the phosphorylation coupled to the oxidation of substrates can take place without a pronounced pH gradient in these membrane vesicles. The dye 1-anilino-8-naphthalene sulfonic acid (ANS) exhibited low levels of energized and nonenergized fluorescence in phospholipase A-treated membranes. This decrease in the level of ANS fluorescence in phospholipase A-treated membranes was found to be directly related to the amount of phospholipids cleaved. The decrease in the energy-dependent ANS response in phospholipase A-treated electron transport particles, as compared with untreated electron transport particles, was shown to be a result of a change in the apparent K-d of the dye-membrane complex, and of a decrease in the number of irreversible or slowly reversible binding sites, with no change in the relative quantum efficiency of the dye. The decrease in ANS fluorescence in phospholipase A-treated particles appears to be due to a decrease in the hydrophobicity of the membranes.     

Interaction of a hydrophobic fluorescent probe with mouse embryo fibroblasts

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

Intracellular fluorescence decay time of anilinonaphthalene sulfonate

This paper presents the intracellular fluorescence decay time of the probe anilinonaphthalene sulfonic acid (ANS) and compares the results to certain ANS complexes in vitro. There is relatively constant decay time for intracellular ANS over a range of concentrations in the incubating medium, compared with marked variation in results with the complex of ANS-bovine serum albumin in vitro when concentration of the probe is varied. Calculation of the apparent rotational relaxation time from the Perrin equation, using ANS intracellular decay time and polarization data gave a tentative value of circa 66 ns. By comparison with the results of ANS complexes with cell fractions and with certain lipids these data support the concept that intracellularly the compound may be largely membrane located with a portion of the molecule in the lipid phase. Cells damaged by heating or alcohol show longer decay time than those which have taken up ANS in the living state. Suggestions for refinement of technique are included in the discussion.     

Interaction of prostaglandins and fatty acids with native and acetaldehyde-alkylated proteins

Using intrinsic and probe fluorescence, microcalorimetry and isotopic methods, the interactions of prostaglandins (PG) E2 and F2 alpha and some fatty acids with native and alkylated proteins (human serum albumin (HSA) and rat liver plasma membrane PG receptors), were studied. The fatty acid and PG interactions with human serum albumin (HSA) resulted in effective quenching of fluorescence of the probe, 1.8-anilinonaphthalene sulfonate (ANS), bound to the protein. Fatty acids competed with ANS for the binding sites; the efficiency of this process increased with an increase in the number of double bonds in the fatty acid molecule. PG induced a weaker fluorescence quenching of HSA-bound ANS and stabilized the protein molecule in a lesser degree compared to fatty acids. The sites of PG E2 and F2 alpha binding did not overlap with the sites of fatty acid binding on the HSA molecule. Nonenzymatic alkylation of HSA by acetaldehyde resulted in the abnormalities of binding sites for fatty acids and PG. Modification of the plasma membrane proteins with acetaldehyde sharply diminished the density of PG E2 binding sites without changing the association constants. Alkylation did not interfere with the parameters of PG F2 alpha binding to liver membrane proteins.     

The use of 8-anilino-1-naphthalenesulfonic acid as a reporter group molecule for circular dichroism and fluorescence measurements. The effect of stearic acid and sodium dodecylsulfate on the conformation of bovine and human serum albumin.

The fluorescence probe ANS(8-anilino-1-naphthalenesulfonic acid) was employed as a reporter group molecule for circular dichroism and fluorescence measurements in order to investigate the effects of stearic acid and sodium dodecylsulfate on the conformation of bovine and human serum albumin. Stearate as well as dodecylsulfate displaces ANS from the binding to both albumins. Besides this displacement, stearate and dodecylsulfate influence the fluorescence properties and the extrinsic Cotton effects on ANS bound to both albumins. It is suggested that the origin of these effects is a microdisorganization of the albumin structure, provoked by the binding of stearate and sodium dodecylsulfate. Each of the four extrinsic CD bands of bound ANS was influenced in a different manner by the addition of stearate and dodecylsulfate. Using the data of the fluorescence measurements and of the circular dichroism measurements it was possible to differentiate the effects of one ligand on both albumins and of both ligands on one albumin more efficiently than would have been possible using one of the two methods alone. It is suggested that the use of ANS as a reporter group molecule for fluorescence and circular dichroism measurements is a very good tool to detect small changes in the environment of ligand binding sites on protein molecules.     

Energy transfer from tryptophan residues of proteins to 8-anilinonaphthalene-1-sulfonate

The binding of the apolar fluorescent dye 8-anilinonaphthalene-1-sulfonate (ANS) to bovine serum albumin (BSA), phospholipase A₂ (PLA₂), ovalbumin, lysozyme, cobrotoxin and N-acetyltryptophanamide was used to assess the factors affecting the efficiency of energy transfer from Trp residues to the ANS molecule. We found that the efficiency of energy transfer from Trp residues to ANS was associated with the ability of proteins to enhance the ANS fluorescence. At the same molar concentration of protein, BSA enhanced ANS fluorescence most among these proteins; its Trp fluorescence was drastically quenched by the addition of ANS. Fluorescence enhancement of ANS in PLA₂-ANS complex increased upon addition of Ca²⁺ or change of the buffer to acidicpH, resulting in a higher efficiency of energy transfer from Trp residues to ANS. There was limited ANS fluorescence enhancement with ovalbumin, lysozyme, cobrotoxin, and N-acetyltryptophanamide and a less efficient quenching in Trp fluorescence. The capabilities of proteins for binding with ANS correlated with the decrease in their Trp fluorescence being quenching by ANS. However, the microenvironment surrounding Trp residues of proteins did not affect the energy transfer. Based on these results, the factors that affected the energy transfer from Trp residues to ANS are discussed.     

The effect of the medium on the functional and structural properties of serum albumins. III. Relation between the N-F1-, F1-F2- and F2-E transitions of human serum albumin and temperature and ionic strength

Using fluorescence parameters of tryptophanyl and bound ANS, the acid-induced structural transitions of defatted monomeric human serum albumin were measured as pH-dependences from 6 to 2.5 in the wide range of temperature (10 to 45 degrees C) and ionic strength (from 0.001 to 0.2 M NaCl or 0.067 M Na2SO4). Temperature rise and decrease in ionic strength value result in the splitting of the N-F-transition onto two stages, N-F1 and F1-F2. The N-F1-transition is accompanied by the blue shift of tryptophanyl and ANS fluorescence spectra and increase in the ANS emission yield. The F1-F2-stage is manifested in an additional blue spectral shift and a sharp drop of the ANS emission yield, which is shown to be due to the lowering of albumin affinity for the dye. In the acidic-extension stage (F2-E), the spectra undergo a red shift which means that the nanosecond dipole relaxation of protein groups and bound water becomes faster. In the F2 from, the albumin affinity for ANS is significantly lowered; the association constant of the primary binding site is lower by an order of quantity and two secondary sites are practically disappeared. The complex effect of temperature, ionic strength and pH changes on the properties of ANS-binding sites is considered as a model of possible control influences of these factors upon the albumin transport of amphiphilic anions in organism.     

Effect of albumin on the kinetics of ascorbate oxidation

The fluorescence intensity of the fluorophore in dansyl piperidine-nitroxide is intramolecularly quenched by the nitroxyl fragment. Therefore, the oxidation of ascorbic acid by the fluorophore-nitroxide (FN) probe can be monitored by two independent methods: steady-state fluorescence and electron paramagnetic resonance. Bovine serum albumin (BSA) affects the rate of this reaction. The influence of BSA on the rate is attributed to the adsorption of both ascorbate and the probe to BSA. Adsorption of ascorbate to BSA is confirmed by NMR relaxation experiments. The spatial distribution of the molecules on the BSA surface changes the availability of ascorbate and FN to each other. The results also point out that, in the presence of BSA, the autoxidation of ascorbate is significantly slowed down. The effect is studied at different pH values and explained in terms of the electrostatic interaction between the ascorbate anion and the BSA molecule.     

Response of an Escherichia coli-Bound Fluorescent Probe to Colicin E1

The fluorescent probe, 8-anilino-1-napthalenesulfonate (ANS) binds to Escherichia coli, showing an enhanced fluorescence. The interaction of colicin E1 with sensitive cells causes an increase of about 100% in the fluorescence of the bound ANS, and this change at equilibrium has an apparent "all-or-none" nature as a function of E1 multiplicity. Approximately 6 to 8% of the ANS is bound to the cells at equilibrium. The colicin E1-induced fluorescence increase can be attributed partly to an increase in ANS binding and partly to an increase in the fluorescence yield of the bound ANS. The kinetics of the E1-induced fluorescence increase in sensitive cells are very similar to those of the adenosine triphosphate decrease. The phosphorylation uncoupler p-trifluoromethoxy-carbonylcyanidephenylhydrazone also causes a large change in the fluorescence of bound ANS. Colicin E2 or E3 does not cause any fluorescence change, nor does colicin E1 cause fluorescence change with a colicinogenic strain. ANS appears to be a probe of structural or conformational change in the cell envelope that is closely associated with the colicin E1-induced adenosine triphosphate decrease.     

Spectroscopic studies on the complex formation of suramin with bovine and human serum albumin.

The binding of suramin to bovine and human serum albumin was investigated by gel filtration and spectroscopic measurements. Besides some low-affinity binding sites suramin has, on the bovine serum albumin molecule one and on the human serum albumin molecule two, high-affinity binding sites. Spectroscopic measurements reveal that there are large differences between the albumins in the mechanism of binding to the high-affinity binding sites. Further, it is suggested that high concentrations of suramin provoke an unfolding of the albumin moleculse. In order to explain the unusual behaviour of suramin in connection with the displacement of other ligands from the albumin binding the fluorescence probe 1-anilino-8-naphthalenesulfonic acid (ANS) was employed as a reporter group molecule for fluorescence as well as circular dichroism measurements. By these measurements it could be shown that suramin greatly influences the microorganization of both albumin molecules. In the case of these measurements large differences between bovine and human serum albumin were also found.     

The interaction of the anionic fluorescence probe, 1-anilinonaphthalene-8-sulfonate, with hepatocytes and hepatoma tissue culture cells

The fluorescence probe 1-anilinonaphthalene-8-sulfonate (ANS) has been used to characterize the anion transport properties of normal hepatocytes and hepatoma tissue culture cells. Incubation of hepatocytes in the presence of ANS (20 micron) resulted in a 35-fold enhancement of fluorescence and a 50 nm blue shift. The time course of this process is biphasic. A rapid initial fluorescence enhancement suggests ANS binding to the plasma membrane, and a slower component reflects the uptake of ANS into intracellular compartments. Analysis of ANS uptake showed this latter process to be saturable, with a Km of 10 micron, to be temperature dependent and to occur only in viable cells. The above observations suggest a carrier-mediated anion transport mechanism. Incubation of hepatoma tissue culture cells with ANS (20 micron) gave a fluorescence emission spectrum similar to that obtained from purified plasma membranes. The kinetics of this interaction only exhibited a rapid initial binding of ANS. The second slow component was now absent, suggesting that ANS transport by the malignant cell system was greatly reduced. Transport of ANS could, however, be stimulated in the presence of the local anesthetic tetracaine. The observed transport was now saturable, temperature dependent, and as in normal hepatocytes, required viable cells, again indicating a carrier-mediated transport system. These studies suggest a significant alteration in membrane function in hepatoma tissue culture cells resulting in a major defect in anion transport.     

Modulation in the photosensitivity of albumin-bound bilirubin

Exposure of BR–albumin complexes to visible light at pH 8.0 led to a change in the fluorescence intensity at 525 nm, which was found to be different for different serum albumins. Whereas a complex of BR with human serum albumin (HSA) showed a marked increase in fluorescence upon photoirradiation, BR–sheep serum albumin (SSA) complex failed to produce a marked increase. On the other hand, a complex of pig serum albumin (PSA) with BR produced a remarkable decrease in fluorescence upon photoirradiation. Equilibration of these complexes with 20 mM chloroform for 1 h resulted in alteration in the photoinduced fluorescence. These photoinduced fluorescence modulations were found to be concentration dependent. Photoirradiation of BR–HSA complex led to a significant decrease in the positive CDCEs of the bisignate CD spectra in a time dependent manner that can be reconciled, to a significant extent, in the presence of chloroform. Taken together, all these results suggest that chiroptical properties/stability of albumin-bound BR varies with albumin species, protein concentration and the presence of chloroform.     

Spectroscopic studies on the interaction of riboflavin with bovine serum albumin

The mechanism of interaction of riboflavin (RF) with bovine serum albumin (BSA) using fluorometric and circular dichroism (CD) methods has been reported. The association constant (K) for RF-BSA binding shows that the interaction is non-covalent in nature. Stern-Volmer analysis of fluorescence quenching data shows that the fraction of fluorophore (BSA) accessible to the quencher (RF) is close to unity, indicating that both tryptophan residues of BSA are involved in the interaction. The high magnitude of rate constant for quenching kq (10(13) M(-1) s(-1) indicates that RF binding site is in close proximity to tryptophan residue of BSA. Thermodynamic parameters obtained from data at different temperatures showed that the binding of RF to BSA predominantly involves the formation of hydrophobic bonds. Binding studies in the presence of a hydrophobic probe 8-anilino-1-naphthalene sulphonic acid, sodium salt (ANS) showed that RF and ANS do not share common sites in BSA. The small decrease in critical micellar concentration of anionic surfactant, sodium dodecyl sulphate in the presence of RF shows that ionic character of RF also contributes to binding and is not solubilized inside the micelle. Significant decrease in concentration of free RF has been observed in the presence of paracetamol. The CD spectrum shows the binding of RF leads to a change in the alpha helical structure of BSA.