Effects of surface hydrophobicity on the catalytic iron ion retention in the active site of two catechol 1,2-dioxygenase isoenzymes

The different behaviour of two isozymes (IsoA and IsoB) of catechol 1,2-dioxygenase (C1,2O) from Acinetobacter radioresistenss13 on a hydrophobic interaction, Phenyl-Sepharose chromatographic column, prompted us to investigate the role of superficial hydrophobicity on structural-functional aspects for such class of enzymes. The interaction of 8-anilino-1-naphtalenesulphonate (ANS), a fluorescent probe known to bind to hydrophobic sites in proteins, revealed that the two isoenzymes have a markedly different hydrophobicity degree although a similar number of hydrophobic superficial sites were estimated (2.65 for IsoA and 2.18 for IsoB). ANS is easily displaced by adding the substrates catechol or 3-methylcatechol to the adduct, suggesting that the binding sites are in the near surroundings of the catalytic clefts. The analysis of the hydropathy profiles and the possible superficial cavities allowed to recognize the most feasible region for ANS binding.The lower hydrophobicity detected in the near surroundings of the catalytic pocket of IsoB supports its peculiarity to lose the catalytic metal ions more easily than IsoA. As previously suggested for other metalloenzymes, the presence of more hydrophilic and/or smaller residues near to the active site of IsoB is expected to increase the metal ligands mobility thus increasing the metal ion dissociation rate constants, estimated to be 0.078 h^–1 and 0.670 h^–1 for IsoA and IsoB respectively. Abbreviations: C1,2O – catechol dioxygenase; ANS – 8-anilino-1-naphthalenesulphonate; PHO – phenol hydroxylase oxygenase     

Binding of 1-anilinonaphthalene-8-sulfonic acid to type I collagen

The existence of a hydrophobic cluster on the COOH-telopeptides of type I collagen has been observed by studies on the binding of 1-anilinonaphthalene-8-sulfonic acid (ANS) to this protein. Collagen contains one binding site for the fluorescent probe. This hydrophobic cluster remains after pepsin digestion thus indicating that it is formed by the undegraded portions of the COOH-extrahelical ends of the protein. Energy transfer from tyrosine to ANS has been observed. The triple helix of collagen does not bind ANS.     

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.     

Structure of Avian Thymic Hormone, a High-Affinity Avian β-Parvalbumin, in the Ca-Free and Ca-Bound States

Originally isolated on the basis of its capacity to stimulate T-cell maturation and proliferation, avian thymic hormone (ATH) is nevertheless a parvalbumin, one of two β-lineage isoforms expressed in birds. We recently learned that addition of Ca²⁺-free ATH to a solution of 8-anilinonaphthalene-1-sulfonate (ANS) markedly increases ANS emission. This behavior, not observed in the presence of Ca²⁺, suggests that apolar surface area buried in the Ca²⁺-bound state becomes solvent accessible upon Ca²⁺ removal. In order to elucidate the conformational alterations that accompany Ca²⁺ binding, we have obtained the solution structure of the Ca²⁺-free protein using NMR spectroscopy and compared it to the Ca²⁺-loaded protein, solved by X-ray crystallography. Although the metal-ion-binding (CD-EF) domains are largely coincident in the superimposed structures, a major difference is observed in the AB domains. The tight association of helix B with the E and F helices in the Ca²⁺-bound state is lost upon removal of Ca²⁺, producing a deep hydrophobic cavity. The B helix also undergoes substantial rotation, exposing the side chains of F24, Y26, F29, and F30 to solvent. Presumably, the increase in ANS emission observed in the presence of unliganded ATH reflects the interaction of these hydrophobic residues with the fluorescent probe. The increased solvent exposure of apolar surface area in the Ca²⁺-free protein is consistent with previously collected scanning calorimetry data, which indicated an unusually low change in heat capacity upon thermal denaturation. The Ca²⁺-free structure also provides added insight into the magnitude of ligation-linked conformational alteration compatible with a high-affinity metal-ion-binding signature. The exposure of substantial apolar surface area suggests the intriguing possibility that ATH could function as a reverse Ca²⁺ sensor.     

Investigations of the inhibitory effect of propranolol,chlorpromazine, quinine,and dicyclohexylcarbodiimide on the swelling of yeast mitochondria in potassium acetate. Evidences for indirect effects mediated by the lipid phase

The mode of action of propranolol, chlorpromazine, and quinine, three cationic drugs inhibiting swelling of yeast mitochondria in potassium acetate, was investigated by looking at their effect on fluorescent probes of the polar heads and of the nonpolar moiety of the membranes, under inhibitory conditions of swelling. As expected, propranolol and chlorpromazine exhibited specificity for anionic phospholipids since they increased the binding of the anionic probe 1-anilino 8-naphthalenesulfonate (ANS). Although propranolol did not release 1,6-diphenyl-1,3,5-hexatriene (DPH) from the hydrophobic moiety of the membrane, it increased the excimer/ monomer fluorescence ratio of 10-(1-pyrene)decanoate, suggesting that it induced a limitation in the movements of the aliphatic chains of phospholipids. Opposite to propranolol, chlorpromazine removed DPH from the membrane, suggesting that it bound essentially to the hydrophobic moiety. However, chloramphenicol, which was also able to remove DPH but did not increase the binding of ANS, did not inhibit swelling. Inhibition by chlorpromazine therefore appeared to be related to its binding to the hydrophobic moiety of anionic phospholipids. Quinine had no effect on membrane properties: at inhibitory concentrations of swelling in potassium acetate, it did not inhibit swelling in ammonium phosphate (mediated by the phosphate/H⁺ cotransporter), whereas propranolol and chlorpromazine did, suggesting a more specific effect of quinine on (a) protein(s) involved in the K⁺/H⁺ exchange. Dicyclohexylcarbodiimide (DCCD), which irreversibly inhibits the swelling in potassium acetate, bound to ethanolamine heads; despite this effect, DCCD had no major consequences on the binding of the probes. Consequently, propranolol and chlorpromazine are of no help for characterizing protein(s) catalyzing the K⁺/H⁺ exchange, although their effect on lipids seems to involve limited zones of the inner mitochondrial membrane. Quinine and DCCD, although they also bind to lipids, may inhibit the activity by acting on a limited number of proteins.     

Ans fluorescence and electrokinetic potential of activated lymphocytes by con A or LPS in vivo

The $\text{in vivo}$ effect of Concanavalin A (Con A), or bacterial lipopolysaccharide (LPS) on mouse spleen cell populations was investigated. The membrane fluorescence changes of activated splenic lymphocytes were studied during two weeks after the injection of polyclonal immunogens. 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 both immunogens. These fluorescence intensity changes would be in direct relation to the electrokinetic surface potential changes of activated lymphocytes, as assessed by the electrophoretic mobility analysis. By comparison with the inverse relationship observed in our previous study, it would be concluded that the relation (direct or inverse) between ANS fluorescence and electrokinetic potential depends on the net electrical charge of the antigens used.     

Influence of carbon source and surface hydrophobicity on the aggregation of the yeast Kluyveromyces bulgaricus

Aggregation of the yeast Kluyveromyces bulgaricus is mediated by the galactose-specific lectin KbCWL1. This lectin contains hydrophobic amino acids and its activity is calcium dependent. A specific fluorescent probe, 1-anilinonaphthalene-8-sulfonic acid in the free acid form (ANS; Sigma Chemical Co., St. Louis, Missouri), was used to study the hydrophobic areas on the cellular surface of K. bulgaricus. Changes in surface hydrophobicity during the growth and aggregation of yeast cells were studied. Surface hydrophobicity increased during growth and depended on the amount of yeast cells in the culture medium. During growth, the size of the hydrophobic areas on the cell surface was measured using ANS and was found to increase with the percentage of flocculating yeasts. Our results strongly suggest that the hydrophobic areas of the cell walls of yeast cells are involved in the aggregation of K. bulgaricus.     

Quantitation of the energetic state in mitochondria and submitochondrial vesicles with 8-anilino-1-naphthalene sulfonic acid

Some of the apparently anomalous findings made with the fluorescent probe 8-anilino-1-naphthalene sulfonic acid (ANS) have been reinvestigated using rat liver mitochondria. The results have been found compatible with current views on energy conservation.The direction of fluorescence and proton flux changes under different conditions have been delineated. The relation of these results to consideration of membrane polarity and organization is discussed.The reliability of ANS fluorescence changes in determining the level of energization of mitochondria and submitochondrial preparations is discussed.Abbreviations used ANS 8-anilino-1-naphthalene sulfonic acid - F _E and H⁺ _E O₂ dependent change in fluorescence and H⁺ in mitochondria and SMP - SMP submitochondrial preparation     

Conformational changes in antigen-stimulated lymphocytes detected with 1-anilino-8-naphthalene sulphonate

The hydrophobic fluorescent probe, 1-anilino-8-naphthalene sulphonate (ANS), has been used to study conformational changes of mouse antigen-stimulated lymphocytes in vivo. Studies revealed that early conformational changes appear in Bovine Serum Albumin-- or Sheep Erythrocytes-- stimulated splenic and thymic lymphocytes. These conformational changes are detected by fluorescence intensity changes, when ANS is bound to lymphocytes. The kinetic studies further indicate that the course of conformational changes may vary considerably depending on antigens.     

Binding of hydrophobic ligands to plant lectins: Titration with arylaminonaphthalenesulfonates

Binding of the Hydrophobic ligands 1,8-anilinonaphthalenesulfonic acid (ANS) and 2,6-toluidinylnaphthalenesulfonic acid (TNS) to a variety of plant lectins was studied by lectin-induced alteration of the fluorescence spectra of the two ligands. With one exception, all legume lectins examined bound ANS, with affinity constants ranging from 10³ to 10⁴ M^?1. Similar ANS binding was noted for some nonlegume lectins. Titration of the five isolectins from Phaseolus vulgaris with ANS indicated positive cooperative binding of ANS to the two isolectins E₄ and E₃L₁. Titrations with TNS revealed high-affinity sites for this ligand in a number of lectins. Addition of haptenic sugars did not inhibit binding of ANS, suggesting that the hydrophobic binding sites of lectins are independent of the carbohydrate binding sites.     

Study of the structure of mammalian serum albumin using fluorescent probes

Some peculiarities of hydrophobic structure of serum albumine of some mammals were studied by NMR-spectroscopy, solubilization and fluorescent probes. It has been shown that the FNA probe is bound to the most hydrophobic cavities in the protein molecules, and the sizes of these regions in mammalian albumines are very close. The data obtained by ANS probe show that there exists a proportional relationship between the fluorescence intensity, the total volume of hydrophobic cavities and the quantity of "bound water". When using the ANS--Mg1/2 probe in all cases an increase of fluorescence intensity was obtained. It was concerned with the stabilizing effect of magnesium ions on the protein molecule.     

Fluorescence studies of a cytochrome C mixed phospholipid complex

The fluorescent probe 8-anilino-1-naphthalene sulphonic acid (ANS) has been used to demonstrate the accessibility of the heme of cytochrome $\text{c}$ — mixed mitochondrial phospholipid complexes to the solvent. Contrary to earlier reports fluorescence techniques using ANS do not detect redox induced conformational changes of these complexes.     

The use of the fluorescent probe 8-anilino-1-naphthalene sulphonic acid (ANS) as a histochemical stain in plant tissues

Abstract. The fluorescent probe 8-anilino-l-naphthalene sulphonic acid (ANS) has been evaluated as a histochemical stain for plant tissues. The wide specificity of the compound for hydrophobic binding sites limits its analytical use, but renders it of considerable value as a general fluorescent stain for use in epi-illumination fluorescence microscopy. Used in this way it is analogous to the light-microscope stain toluidine blue. ANS has also been found to be a sensitive vascular tracer.     

Membrane permeability transition promoted by phosphate enhances 1-anilino-8-naphthalene sulfonate fluoresence in calcium-loaded liver mitochondria

Phosphate and a number of other compounds induce membrane permeability transition (MBT) in Ca²⁺-loaded mitochondria. 1-Anilino-8-naphthalene sulfonate (ANS) was used as a fluorescent probe to investigate perturbations on the inner membrane during MBT. Induction of MBT caused ANS fluoresence enhancement with a biphasic rate that reached a plateau. The enhancement is analogous to that reported for de-energization of mitochondria. The fluoresence level was independent of whether ANS was added before or at different times after phosphate. In the absence of ANS, fluorescence was low and remained unchanged. The initial time course of MBT, as followed by large-amplitude swelling, was similar to that of fluorescence enhancement. Ruthenium red, EGTA, ADP, and cyclosporin A inhibited the enhancement. Only EGTA + ADP (or ATP) reversed the enhancement when added after phosphate. Efflux of matrix Ca²⁺ by sodium acetate or A23187 did not alter ANS fluoresence. The binding parameters (K _d and number of binding sites) were not significantly different, but the fluorescence maximum was more than doubled after MBT. Although the flourescence of bound ANS showed a nonlinear relationship, it was always higher (73.0 +/- 19.0%) after reaching the plateau. Since ANS binding to membranes is nonspecific, the exact mechanism of the enhanced fluorescence is not apparent. The dependence of the initial rate of fluorescence enhancement on Ca²⁺ concentration was nonlinear, with 45 µM at half-maximal rate. The dependence on phosphate was hyperbolic with 0.7 mM at half-maximal rate, which is close to theK _m value of phosphate carrier. The kinetics is compatible with Ca²⁺ binding to some membrane component(s) during MBT and cause ANS fluorescence enhancement. It is suggested that the bilayer-nonbilayer (hexagonal₁₁) transition consequent to Ca²⁺ binding to proteinphospholipid domains containing cardiolipin may play a role in fluorescence enhancement and MBT.     

Lipid-protein interactions of erythrocyte membranes: comparison of normal O, Rh (D) positive with the rare O, Rh null

Phospholipase A₂ modification of lipid-protein interactions of normal O,Rh(D) positive erythrocyte membranes increased the fluorescence intensity of the membrane bound probe, 1-anilinonaphthalene-8-sulfonate (ANS) and increased the N-1-[¹⁴C]-ethyl maleimide ([¹⁴C]-NEM) labeling of sulfhydryl groups in two proteins of molecular weight >200,000. In marked contrast, phospholipase A₂ modification of the rare phenotype O,Rh_null membranes resulted in no significant increase in ANS fluorescence or labeling of sulfhydryl groups by [¹⁴C] NEM. Since the O,Rh_null erythrocytes demonstrated an increased osmotic fragility and decreased survival time, the fluorescence and sulfhydryl labeling data support the conclusion that hydrophobic bonding between β-fatty acid side chains and non-polar regions of asymmetric proteins is necessary for maintaining the native structure of the O,Rh(D) positive membrane. Comparative studies with phospholipase C or D implied that ionic bonding played a similar though less important structural role in both membranes.     

Effect of 1-Anilino-8-naphthalenesulfonate on Properties and Associations of αs-, β- and κ-Caseins

It was indicated from fluorescence spectra and fluorescence titration that a hydrophobic probe, 1-anilino-8-naphthalenesulfonate (ANS), binds to casein components (α_s-, β- and κ-caseins). Fluorescence intensity and affinity of ANS-κ-casein complex were larger than that of ANS-α_s- and ANS-β-casein complexes. Enhancements of fluorescence intensity of complexes of casein components were observed by the addition of KCI or CaCl₂. Reason for the enhancement was postulated to be the increase of the quantum yield of the ANS fluorescence caused by the environmental change of ANS binding region of the casein components.

Marked increase of sedimentation coefficient of β-casein in the presence of KCl or CaCl₂ at 10°C was caused by the addition of ANS. This may be responsible for the stimulation of the Ca-dependent precipitation of β-casein by the addition of ANS.

It was found that α_s · κ-association was prevented by ANS and that hydrophobic interaction have an important role for α_s · κ-association.     

Interaction of native and modified Naja melanoleuca phospholipases A2 with the fluorescent probe 8-anilinonaphtalene-1-sulfonate

The fluorescent probe 8-anilinonaphtalene-1-sulfonate (ANS) binds at the active site of the Naja melanoleuca snake venom phospholipase A2, thus protecting the enzyme against active-site-directed chemical modification. Both hydrophobic and electrostatic interactions are involved in the binding. At pH 7.5, a binding constant of 100 microM was determined, which improved twofold upon addition of the enzymatic cofactor Ca2+. The pH dependence of the ANS binding in the absence and presence of Ca2+ ions showed a perturbation of a group with a pKa value of 5.2, which could be assigned to the carboxylate group of the Ca2+-binding ligand Asp49 at the active site of the protein. Monomeric concentrations of the substrate analog n-decylphosphocholine displace ANS from the protein, indicating again that both ligands bind at the active site. Binding studies with several modified N. melanoleuca enzymes showed that a loss of enzymatic activity on aggregated substrates was correlated with a loss of affinity for the active site bound ANS molecule. It is suggested therefore, that the fluorescent ANS probe can detect structural rearrangements at the active site, which are important for enzymatic activity.     

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.     

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.