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.     

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.     

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.     

Ligand binding induces a sharp decrease in hydrophobicity of folate binding protein assessed by 1-anilinonaphthalene-8-sulphonate which suppresses self-association of the hydrophobic apo-protein

High affinity folate binding protein (FBP) regulates as a soluble protein and as a cellular receptor intracellular trafficking of folic acid, a vitamin of great importance to cell growth and division. We addressed two issues of potential importance to the biological function of FBP, a possible decrease of the surface hydrophobicity associated with the ligand-induced conformation change of FBP, and protein-inter-protein interactions involved in self-association of hydrophobic apo-FBP. The extrinsic fluorescent apolar dye 1-anilinonaphthalene-8-sulphonate (ANS) exhibited enhanced fluorescence intensity and a blueshift of emission maximum from 510-520 to 460-470nm upon addition of apo-FBP indicating binding to a strongly hydrophobic environment. Neither enhancement of fluorescence nor blueshift of ANS emission maximum occurred when folate-ligated holo-FBP replaced apo-FBP. The drastic decrease in surface hydrophobicity of holo-FBP could have bearings on the biological function of FBP since changes in surface hydrophobicity have critical effects on the biological function of receptors and transport proteins. ANS interacts with exposed hydrophobic surfaces on proteins and may thereby block and prevent aggregation of proteins (chaperone-like effect). Hence, hydrophobic interactions seemed to participate in the concentration-dependent self-association of apo-FBP which was suppressed by high ANS concentrations in light scatter measurements.     

Analysis of heterogeneous fluorescence decays in proteins. Using fluorescence lifetime of 8-anilino-1-naphthalenesulfonate to probe apomyoglobin unfolding at equilibrium

The solvatochromic fluorescent dye 8-anilino-1-naphthalenesulfonate (ANS) is one of the popular probes of protein folding. Folding kinetics is tracked with ANS fluorescence intensity, usually interpreted as a reflection of protein structure-the hydrophobicity of the binding environments. Such simplistic view overlooks the complicated nature of ANS-protein complexes: the fluorescence characteristics are convoluted results of the ground state populational distribution of the probe-protein complex, the structural changes in the protein and the excited state photophysics of the probe. Understanding of the interplay of these aspects is crucial in accurate interpretation of the protein dynamics. In this work, the fluorescence decay of ANS complexed with apomyoglobin at different conformations denatured by pH is modeled. The fluorescence decay of the ANS-apomyoglobin complex contains information on not only apomyoglobin structure but also molecular populational distributions. The challenge in modeling fluorescence decay profiles originates from the convolution of heterogeneous binding and excited-state relaxation of the fluorescent probe. We analyzed frequency-domain fluorescence lifetime data of ANS-apomyoglobin with both maximum entropy methods (MEM) and nonlinear least squares methods (NLLS). MEM recovers a model of two expanding-and-merging lifetime distributions for ANS-apomyoglobin in the equilibrium transition from the native (N) through an intermediate (I-1) to the acid-unfolded state U(A). At pH 6.5 and above, when apomyoglobin is mostly populated at the N-state, ANS-apomyoglobin emits a predominant long-lifetime fluorescence from a relaxed charge transfer state S(1,CT) of ANS, and a short-lifetime fluorescence that is mainly from a nascent excited-state S(1,np) of ANS stabilized by the strong ANS-apomyoglobin interaction. Lowering the pH diminishes the contribution from the S(1,np) state. Meanwhile, more protein molecules become populated at the U(A) state, which exhibits a short lifetime that is not distinguishable from the S(1,np) state. At pH 3.4, when the population of the U(A) becomes significant, the short-lifetime fluorescence comes predominantly from ANS binding to the U(A). Further lowering the pH leads to more exposure of the bound ANS. The long lifetime shifts toward and finally merges with the short lifetime and becomes one broad distribution that stands for ANS binding to the U(A) below pH 2.4. The above expanding-and-merging model is consistent with F-statistic analysis of NLLS models. The consistency of this model with the knowledge from the literature, as well as the continuity of the decay parameters changing upon experimental conditions are also crucial in drawing the conclusions.     

Mast cell-lysosomal cationic protein interaction: effects of metabolic inhibitors and decay of activated cells on enhanced fluorescence of anilinonaphthalene sulfonate

It has been shown earlier that the interactions of the isolated rat peritoneal mast cells with cationic protein from rabbit neutrophil lysosomes (band 2 protein) can be studied using anilinonaphthalene sulfonate (ANS) as a fluorescent probe. In the present communication, binding of ANS dye to the mast cells interacting of histamine release by metabolic inhibitors was found to have no effect on enhancement of ANS fluorescence. On the other hand, inhibition of histamine release at high concentration of Ca2+ (14.4 mM) was accompanied by the decrease in enhance fluorescence. In the presence of 7.2 mM of Sr2+, the release of histamine was enhanced with small but significant increase in ANS fluorescence. The cells heated to 42 degrees C partially lost their capacity to release histamine without the loss of enhanced fluorescence. The mast cells interacting with B2 at 10 degrees C for various time intervals showed time-dependent loss in histamine releasing capacity with concomitant loss in enhanced fluorescence. These studies suggest that the enhancement of ANS fluorescence is associated with the early events of the cell membrane caused by interaction of B2 with the cells. The extracellular cations significantly influence this early event.     

Interaction of N-phenyl-1-amino-8-sulfonaphthalene (ANS) with albumin in blood in hyperbilirubinemia

The fluorescent intensity of the N-phenyl-1-amino-8-sulfonaphthalene (ANS) probe significantly decreases in hyperbilirubinemic serum. A decrease of the albumin concentration and absorption of ANS fluorescence by bilirubin cannot explain such a considerable reduction of the probe fluorescence intensity. Measurements of the fluorescence decay kinetics has shown two types of sites occupied by ANS in albumin. ANS quantum yields in hyperbilirubinemic and normal serum are practically identical. The coupling parameters for ANS decrease, but the coupling constant increases under hyperbilirubinemia. As a result the coupling of organic anions with serum albumin significantly decreases if there is high anion concentration, and it does not decrease at low anion concentration. Bilirubin is not a main cause of a decrease of the albumin binding capacity.     

Mechanism of inhibition of Ca2+-transport activity of sarcoplasmic reticulum Ca2+-ATPase by anisodamine

The mechanism of inhibition of Ca2+-transport activity of rabbit sarcoplasmic reticulum Ca 2+-ATPase (SERCA) by anisodamine (a drug isolated from a medicinal herb Hyoscyamuns niger L) was investigated by using ANS (1-anilino-8-naphthalenesulfonate) fluorescence probe, intrinsic fluorescence quenching and Ca 2+-transport activity assays. The number of ANS binding sites for apo Ca2+-ATPase was determined as 8, using a multiple-identical binding site model. Both anisodamine and Ca2+ at millimolar level enhanced the ANS binding fluorescence intensities. Only anisodamine increased the number of ANS molecules bound by SERCA from 8 to 14. The dissociation constants of ANS to the enzyme without any ligand, with 30 mM anisodamine and with 15 mM Ca 2 were found to be 53.0 microM, 85.0 microM and 50.1 microM, respectively. Both anisodamine and Ca2+ enhanced the ANS binding fluorescenc with apparent dissociation constants of 7.6 mM and 2.3 mM, respectively, at a constant concentration of the enzyme. Binding of anisodamine significantly decreased the binding capacity of Ca2+ with the dissociation constant of 9.5 mM, but binding of Ca2+ had no obvious effect on binding of anisodamine. Intrinsic fluorescence quenching and Ca2+-transport activity assays gave the dissociation constants of anisodamine to SERCA as 9.7 and 5.4 mM, respectively, which were consistent with those obtained from ANS-binding fluorescence changes during titration of SERCA with anisodamine and anisodamine + 15 mM Ca2+, respectively. The results suggest that anisodamine regulates Ca2+-transport activity of the enzyme, by stabilizing the trans-membrane domain in an expanded, inactive conformation, at least at its annular ring region.     

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.     

Human aglycosyl-IgG exhibits increased hydrophobicity. Binding/fluorescence studies with 8-anilinonaphthalene-1-sulfonic acid (ANS)

Human monoclonal, aglycosyl-IgG produced in vitro in the presence of tunicamycin, was compared with its native and acid pH-altered counterparts for their respective abilities to bind the fluorescent hydrophobicity probe, 8-anilinonaphthalene sulfonate. A novel technique based on continuous-flow dynamic dialysis (Sparrow et al., 1982, Anal. Biochem. 123:255-264) allowed binding studies under non-equilibrium conditions. While the native IgG conformation exhibits two, weak ANS binding sites (ca. 10(3) l/mol), aglycosyl-IgG has one weak and one moderate affinity (least squares average Ka = 2 X 10(4) l/mol) site, and the acid conformer binds yet another two ANS molecules with moderate affinity (4 X 10(4) l/mol). Increases in affinity and in the number of sites correlate roughly with increased relative percent fluorescence by conventional fluorimetry. The fluorescence lifetime of ANS bound to altered IgGs is about 10% longer (T2 = 15 nsec by time-resolved fluorimetry) than that for native IgG. All populations also exhibit a rapid decay component (T1 = 3 nsec) analogous to that seen for ANS in 50% aqueous dioxane. Results are discussed in relation to structural role(s) for IgG-linked heterosaccharides.     

Substrate binding to phosphoglycerate kinase monitored by 1-anilino-8-naphthalenesulfonate

The interaction between 1-anilino-8-naphthalenesulfonate (ANS) and yeast phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3) and the use of ANS as a probe for studying the structure and function of phosphoglycerate kinase has been investigated. The interaction has been studied by kinetic methods, equilibrium dialysis, and fluorometric titrations. ANS inhibits the activity of the enzyme. More than one inhibitor site exists. ANS is competitive with MgATP and noncompetitive with 3-phosphoglycerate at the first detected inhibitor binding site. The Ki value is 1-2 mM. Several ANS molecules bind to the enzyme. By fluorometric titrations the first detected site has a dissociation constant that is in the same range as Ki or bigger. When ANS interacts with phosphoglycerate kinase its fluorescence is increased and a blue shift occurs. ANS appears to bind to a strongly hydrophobic site. The fluorescence is sensitive to the addition of substrates. ADP, ATP, or combinations of Mg2+ and nucleotide decreases the fluorescence as does free Mg2+. 3-Phosphoglycerate, on the other hand, increases the fluorescence giving evidence for conformational changes upon 3-phosphoglycerate binding.     

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.     

Kinetics of interaction of partially folded proteins with a hydrophobic dye: evidence that molten globule character is maximal in early folding intermediates.

Interaction with 8-anilino-1-naphthalenesulfonate (ANS) is widely used to detect molten globule states of proteins. We have found that even with stable partially folded states, the development of the fluorescence enhancements resulting from such interactions can be relatively slow and kinetically complex. This is probably because initial binding of the dye can induce subsequent changes in the protein structure, so that the ultimate resulting fluorescence enhancement is not necessarily a good, nonperturbing probe of the preexisting state of the protein. When ANS is used to study folding mechanisms the problem is compounded by the difficulty of distinguishing effects due to the development of dye interactions from those due to the changing populations of folding intermediates. Many of these complications can be avoided by experiments where the ANS is introduced only after folding has been allowed to proceed for a variable time. The initial fluorescence intensity after mixing, resulting only from rapid and therefore hopefully relatively nonperturbing interactions with the protein, can be monitored at different refolding times to provide a better reflection of the progress of the reaction, uncomplicated by dye interaction effects. Such studies of the folding of carbonic anhydrase and alpha-lactalbumin have been compared with conventional single-mix experiments and large discrepancies observed. When ANS was present throughout refolding, time-dependent changes attributed to the formation or reorganization of protein-ANS complexes were clearly superimposed on those associated with the actual progress of refolding, and the folding kinetics and population of intermediates were also substantially perturbed by the dye. Thus, it is clear that the pulse method, though cumbersome, should be used where refolding reactions are to be probed by dye binding. The results emphasize that fluorescence enhancement tends to be greatest in early intermediates, in contrast to what, for carbonic anhydrase at least, might appear to be the case from the more conventional experiments. Later intermediates in the folding of both of these proteins actually induce little fluorescence enhancement and therefore may be quite different in nature from equilibrium molten globule states.     

Effects of a series of alcohols on the binding of a fluorescent dye to erythrocyte membranes.

1. The effects of a series of aliphatic alcohols (methanol to octanol) on membrane proteins of erythrocytes were studied by monitoring the flueorescence of a dye (1-anilino-8-naphthalenesulfonic acid (ANS)) that adsorbs to erythrocyte ghost membranes. Low concentrations of all the alcohols reduced the ANS fluorescence of the membrane-ANS suspensions; lent to those which protect against hypotonic hemolysis on intact erythrocytes; higher concentrations markedly increased the fluorescence. Ethanol and methanol decreased ANS fluorescence at all concentrations. 2. Lytic concentrations of saponin did not increase ANS fluorescence and did not modify the membrane action of the alcohols. 3. None of these effects were observed in liposomes prepared from lipid extracts of the erythrocyte membrane. 4. Since the apparent dissociation constant for the ANS-membrane interaction was unchanged in the presence of the alcohols, it was assumed that the fluorescence changes anesthetic concentration of the alcohols alter the conformation of membrane proteins, as indicated by the decreased number of ANS binding sites.     

A role for phospholipids in the binding and metabolism of drugs by hepatic microsomes. Use of the fluorescent hydrophobic probe 1-anilinonaphthalene-8-sulphonate

1. The pretreatment of rat liver microsomes with phospholipase C or D decreased the N-demethylation of (+)-benzphetamine. The hydroxylation of aniline was essentially unchanged by pretreatment of microsomes with phospholipase C. 2. Some components of the microsomal mixed-function oxidase system were impaired by phospholipases. 3. The fluorescence of 1-anilinonaphthalene-8-sulphonate (ANS) was greatly enhanced by microsomes. Phospholipase C or D markedly decreased ANS–microsome fluorescence. Quantum yield of ANS–microsome fluorescence appeared to be related directly to phospholipid content of microsomes. 4. Most of the drugs studied enhanced ANS–microsome fluorescence. Warfarin, however, displaced ANS fluorescence competitively from microsomes. The latter effect was postulated as being due to warfarin competing with ANS for the cationic site on microsomal phosphatidylcholine. 5. ANS fluorescence was also increased by the presence of phospholipid micelles. The fluorescence of ANS–phosphatidylcholine micelles was modified by warfarin and (+)-benzphetamine in a manner similar to that observed with microsomes. Warfarin decrease of fluorescence was absent when ANS was bound to phosphatidic acid, which lacks a cationic site. 6. Trypsin pretreatment of microsomes did not modify ANS–microsome fluorescence, including drug-induced changes. 7. It was postulated that phospholipids have a permissive role in the metabolism of most drugs by hepatic microsomes and that the ANS probe might reflect interactions of compounds with microsomal membrane phospholipids.     

Effect of the hemolytic lectin CEL-III from Holothuroidea Cucumaria echinata on the ANS fluorescence responses in sensitive MDCK and resistant CHO cells

The addition of CEL-III to sensitive MDCK cells preincubated with 8-anilino-1-naphthalenesulfonate (ANS) caused an increase in the fluorescence intensity of the probe. The increase in the ANS fluorescence caused by CEL-III was Ca2+-dependent and strongly inhibited by 0.1 M lactose, indicating that Ca2+-dependent binding of CEL-III to specific carbohydrate receptors on the plasma membrane is responsible for this phenomenon. In contrast, no significant effect of CEL-III on the ANS fluorescence was observed in CHO cells, which are highly resistant to CEL-III cytotoxicity. In MDCK cells, energy transfer from tryptophan residues to bound ANS molecules was observed in the presence of CEL-III, but not in CHO cells. Furthermore, the amount of ANS bound to MDCK cells increased as the concentration of CEL-III increased. Therefore, a simple interpretation is that the CEL-III-induced increase in ANS fluorescence is attributable to an increase of the hydrophobic region in the plasma membrane where ANS could bind. Immunoblotting analysis of proteins from cells treated with CEL-III indicated that CEL-III oligomers were irreversibly bound to the cells, and the amount of oligomer bound to MDCK cells was much greater than that bound to CHO cells under any conditions tested. The oligomerization may be accompanied by an enhancement of the hydrophobicity of CEL-III molecules, which in turn provides new ANS-binding sites. The difference in susceptibility of MDCK and CHO cells to CEL-III cytotoxicity may be due to a difference in oligomerization of bound CEL-III.     

Relationship between ANS fluorescence and electrokinetic potential of activated lymphocytes.

An immune response was induced in vivo on C3H/He ♂ mouse strain with Bovine Serum Albumin (BSA), or Sheep Red Blood Cells (SRBC). The membrane fluorescence changes of activated splenic lymphocytes were studied two weeks after the injection of antigens. Experiments were performed with the hydrophobic fluorescent probe: 1-anilino-8-naphthalene sulphonate (ANS). The kinetic studies further indicated that the course of fluorescence changes may considerably vary depending on antigens. Their fluorescence intensities were lower than control values. A maximum decrease of fluorescence was recorded on days 1, 6 and 9 after immunization with BSA-stimulated lymphocytes. SRBC-stimulated lymphocytes exhibited a maximum ANS fluorescence decrease on days 4 and 9 after immunization. These fluorescence phenomena would be in an inverse relationship with the electrokinetic surface potential of activated lymphocytes, as assessed by the electrophoretic mobility analysis (EPM). Some parameters affecting the ANS fluorescence in T and B cells are discussed. Quantification of hydrophobic sites in splenic cells would indicate that forces other than the hydrophobic ones may also be involved in the dye-binding changes following immune activation.     

Changes in E. coli cell envelope structure caused by uncouplers of active transport and colicin E1

It is of interest to inquire whether agents that uncouple or deenergize membranes cause concomitant structural changes. The agents considered here are the uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone and the bacteriocidal protein colicin E1, agents for which there is some precedent for believing that they interact with membranes. In intact E. coli ML 308-225 cells the inhibition of [¹⁴C]-proline active transport by FCCP increases with uncoupler concentration from ～ 20% at 2 μM to ～100% at 5 μM. The increase in the rotational relaxation time (ρ) of the cell-bound fluorescent probe N-phenyl-1-naphthylamine (PhNap)

1 Abbreviations: FCCP – carbonyl cyanide p-trifluoromethoxyphenylhydrazone; ANS – 8-anilino-1-naphthalenesulfonate; PhNap, N-phenyl-1-naphthylamine; EDTA – ethylenediaminetetraacetate.

and 8-anilino-1-naphthalene-sulfonate (ANS) under these conditions shows the same dependence on FCCP concentration. For cells treated with EDTA to remove part of the outer lipopolysaccharide layer, inhibition of proline transport and the increase in ρ value of ANS show the same dependence on FCCP concentration with saturation at 0.3 μM. EDTA treatment causes a large increase in the binding and rotational relaxation time of PhNap, the latter quantity approaching a value obtained with purified inner membrane. Similar effects are produced in untreated cells by 5 μM FCCP. It is concluded that (a) EDTA treatment removes a permeability barrier t o FCCP and PhNap in the outer membrane; (b) uncoupling by FCCP removes a similar permeability barrier to PhNap; (c) binding of amphiphilic ANS, assumed to be located in the outer membrane, is hardly changed by these treatments; (d) deenergization of the inner membrane by FCCP thus causes a structural change in the outer membrane as measured by the permeability change to hydrophobic PhNap and the increase in ρ values of the amphiphilic ANS; (e) The binding sites reached by PhNap within the permeability barrier at or near the inner membrane are changed by FCCP from their initial state. This is inferred from an increase in PhNap quantum yield extrapolated to infinite cell concentration, and from removal by FCCP of an apparent phase transition sensed by the PhNap rotational relaxation time. Thus, uncoupling and deenergization by FCCP appears to cause structural change both in the outer membrane and inside the permeability barrier of the outer membrane. Transmission of the colicin E1 response in the envelope of intact and EDTA-treated cells can also be monitored by an increase in ANS and PhNap fluorescence intensity, a smaller fractional increase in dye binding, and a large increase in probe rotational relaxation time. The fluorescence changes of ANS again imply structural effects in the outer membrane caused by colicin. The binding and fluorescence changes of PhNap caused by colicin E1 acting on intact cells again imply an effect of deenergization on the permeability barrier of the outer membrane. Fluorescence changes with PhNap in intact and EDTA-treated cells show that the dye binding sites are altered in the presence of colicin E1. It is also shown that the PhNap intensity change can be blocked by low concentrations of vitamin B12, which competes for the colicin E1 receptor. Some properties are presented of the probe chlorotetracycline, which has been proposed by others to be an indicator of magnesium. The probe appears to reside in an environment somewhat similar to that of ANS, but the colicin-induced changes in its fluorescence parameters appear to be small under our conditions.     

Use of fluorescence decay times of 8-ANS-protein complexes to study the conformational transitions in proteins which unfold through the molten globule state

The conformational transitions starting with the native protein, passing the molten globule state and finally approaching the unfolded state of proteins was investigated for bovine carbonic anhydrase B (BCAB) and human -lactalbumin (-HLA) by means of fluorescence decay time measurements of the dye 8-anilinonaphthalene-1-sulphonic acid (8-ANS). Stepwise denaturation was realized by using the denaturant guanidinium chloride (GdmCl). It was shown that 8-ANS bound with protein yields a double-exponential fluorescence decay, where both decay times considerably exceed the decay time of free 8-ANS in water. This finding reflects the hydrophobic environment of the dye molecules attached to the proteins.

The fluorescence lifetime of the short-time component is affected by protein association and can be effectively quenched by acrylamide, indicating that 8-ANS molecules preferentially bind at the protein surface. The fluorescence lifetime of the long-time component is independent of the protein and acrylamide concentration and may be related to protein-embedded dye molecules.

Changes of the long lifetime component upon GdmCl-induced denaturation and unfolding of BCAB and -HLA correlate well with overall changes of the protein conformation. The transition from native protein to the molten globule state is accompanied by an increase of the number of protein-embedded 8-ANS molecules, while the number of dye molecules located at the protein surface decreases. For the transition from the molten globule to the unfolded state was the opposite behaviour observed.     

Binding of naphthalene dyes to the N and A conformers of bovine α-lactalbumin

Contrary to earlier findings, monomeric native α-lactalbumin does bind naphthalene dyes such as ANS and TNS with marked enhancement of their fluorescence. Nanosecond decay measurements indicate there to be two dye binding sites per protein molecule with lifetimes of ca. 2 and 15 ns for ANS and 5 and 11 ns for TNS. The fluorescence titrations curves of α-lactalbumin with ANS and TNS reflect this site multiplicity, i.e., it was not possible to analyze such curves with a single K_diss. The apparent dissociation constants for binding of ANS and TNS to native bovine α-lactalbumin, as determined by an ultracentrifugal technique, ca. 950 and 900 μm, respectively, indicate that such binding is considerably weaker than previously supposed. The A conformer (metal ion-free form) of α-lactalbumin binds ANS and TNS more tightly than the N (native) form of the protein with marked fluorescence enhancement. The A conformer has two dye binding sites with lifetimes for ANS and TNS comparable with those seen with native protein.