And 1-anilinonaphthalene-8-sulfonate Research Articles

Distinct stability of recombinant L and H subunits of human ferritin: calorimetric and ANS binding studies.

Thermodynamic and pH stability of recombinant human L- and H-ferritins were probed by differential scanning calorimetry and 8-anilino-1-naphthalenesulfonate (ANS) binding in the pH range 2-7. At pH 2.0-2.8 they were dissociated into subunit monomers and in this pH interval the H-subunit displayed a single calorimetrically-revealed domain with properties of a molten globule-like state: low enthalpy (6.3-8.0 J/g or 169-172 kJ/mol) and Tm of thermal unfolding (approximately 50 degrees C), a wide transition range (approximately 20 degrees C) and high ANS binding. In contrast, at pH 2 the L-ferritin subunit showed two calorimetric domains with Tm of 35 and 40 degrees C with similar unfolding enthalpies and with moderate extent of interactions, as indicated by the ratio of calorimetric enthalpy (293.9 kJ/mol) and van't Hoff enthalpy (174.2 kJ/mol) for the thermal transition. A pH increase from 2.0 to 2.8 determined the coupling of the two domains into a single cooperative folding unit and drastic increase of the transition temperature (from 37 to 80 degrees C). The contacts between the two domains in the L-subunit appeared to contribute to about 30% of the total stabilization free energy. The unfolding enthalpies, heat capacity changes and pronounced ANS binding of the L-subunit at pH 2.0-2.8 indicated that part of the structure lacked 'meltable' tertiary interactions. The results indicate that H- and L-subunits are stabilized by largely different intra-chain interactions with a critical contribution to L-subunit stability of embedded salt bridge(s) absent in the H-subunit.

Extrinsic thermostabilization factors and thermodenaturation mechanisms for phosphoenolpyruvate carboxylase (PEPC) from an extremely thermophilic bacterium Rhodothermus obamensis

The thermodenaturation of phosphoenolpyruvate carboxylase (PEPC) from an extremely thermophilic bacterium Rhodothermus obamensis, capable of growth at temperature up to 85°C, was probed at different denaturation temperatures by UV-visible absorption, fluorescence emission and 1-anilinonaphthalene-8-sulfonate (ANS) binding and renaturation was assessed from different states of denaturation. Under severe denaturation conditions at 100°C, the enzyme was rapidly inactivated and its global structure immediately reached the irreversibly aggregated state by passing through the dissociated and the putative scrambled states as observed by UV-visible absorption spectroscopy. However, under milder conditions of denaturation at 93°C, the enzyme was gradually inactivated, and its global structure shifted sequentially from the dissociated state to the scrambled state. At 80°C, about 50% of the activity was left and no apparent change in the global structure occured even after 30 h. In addition, ANS binding to the enzyme was greatly increased in accordance with the change in global structure. This implies that the hydrophobic regions of the enzyme tend to be exposed to solvent due to thermal dissociation and unfolding. The extrinsic thermostabilization factors that enhance the thermostability of the enzyme successfully suppress the thermodenaturation of the enzyme, especially the dissociation of its tetrameric form. Of these factors, the substrate for the enzyme, phosphoenolpyruvate (PEP), causes the reassociation of the dissociated inactive form of the enzyme to the active form. These results suggest that the global thermodenaturation of the enzyme results from the temperature-dependent shift of three different states and that the extrinsic thermostabilization factors act to a large extent to maintain quaternary structure.

Heat-induced conformational change and increased chaperone activity of lens a-crystallin

PURPOSE. Alpha-crystallin is the major structural protein of the eye lens known to have chaperone-like activity. Our objective is to elucidate the nature of the thermal transition that a-crystallin undergoes at 60°C and the effect of this transition on the chaperone activity. METHODS. FPLC size exclusion chromatography, far- and near-ultraviolet circular dichroism, and tryptophan (Trp) and 1-anilino-8-naphthalenesulfonate (ANS) fluorescence were used to study conformational change. Turbidity of dithiothreitol (DTT)-reduced insulin was used to study chaperone activity. RESULTS. The thermal transition was identified as a conformational change in mainly tertiary (partial unfolding) and quaternary high-molecular-weight (HMW) aggregation structures, along with a loss of 10 percentage points of secondary structure (ß-sheet). Initial partial perturbation in tertiary structure increased chaperone activity, but the increase was less in the HMW aggregate. Similar results were observed in in vivo -formed HMW a-crystallin. CONCLUSIONS. The conformational change and HMW aggregation of a-crystallin observed at 60°C, as well as in vivo -formed HMW aggregates, increased chaperone activity.

Calcium-Dependent Solvation of the Myristoyl Group of Recoverin

Recoverin is an N-myristoylated calcium-binding protein present in the photoreceptor cells of the mammalian retina. It is believed to function as a calcium sensor in visual signal transduction by coupling the kinetics of the recovery phase of the photoresponse to changes in the levels of intracellular Ca2+. Upon binding Ca2+, recoverin undergoes a conformational change that allows it to associate with membranes in a manner that requires N-myristoyl modification. It has been proposed that, in the Ca(2+)-free conformation, the myristoyl group is sequestered in a hydrophobic part of the protein, and in the Ca(2+)-bound conformation, the myristoyl group is exposed to solution. The crystal structure of Ca(2+)-bound recoverin reveals an exposed cluster of hydrophobic residues, raising the possibility that residues in this region may function as part of an intramolecular myristoyl binding site. Fluorescence spectroscopy analysis of interactions between recoverin and 1-anilinonaphthalene-8-sulfonate (ANS) shows that an increase in solvent-accessible hydrophobic surface accompanies Ca2+ binding. 1H nuclear magnetic resonance (NMR) spectra of myristoyl protons show dispersed chemical shifts in the Ca(2+)-free conformation that become relatively uniform upon the addition of Ca2+. Two-dimensional nuclear Overhauser effect (NOE) spectra of Ca(2+)-free recoverin show NOE contacts between myristoyl protons and aromatic ring protons. Tryptophan fluorescence quenching by acrylamide indicates that the myristoyl group is in proximity to a tryptophan residue only in the Ca(2+)-free conformation. These results indicate that the myristoyl group is in contact with residues in the hydrophobic cluster in Ca(2+)-free recoverin and that it is exposed to solution in the Ca(2+)-bound conformation.

Alloxan-induced alterations in composition and dynamics of red blood cell membranes: I. Effect of alloxan on intact red blood cells and isolated erythrocyte membranes

Changes of dynamics and chemical composition in membranes of intact red blood cells and isolated erythrocyte membranes treated with alloxan were investigated in order to assess whether alloxan-induced generation of active forms of oxygen may be critical for erythrocyte destroying. In vitro incubation of native red blood cells or prepared erythrocyte membrane ghosts with various concentrations of alloxan gave rise both to levels of membrane TBA-reacting substance and lipid membrane micro-viscosity both in the deeper and surface regions of lipid bilayer, as evidenced by fluorescence polarization technique. The amount of membrane phospholipid decreased upon alloxan action and that of membrane cholesterol remained rather unchangeable, thus resulting in significant elevation of membrane cholesterol : phospholipid (C : PL) ratio. Both time course and concentration effect of alloxan were found to change exponentially with the different rates of the reaction. There was a linear correlation between 1,6-diphenylhexatriene-1,3,5 (DPH) and 1-anilinonaphthalene-8-sulfonate (ANS) anisotropy coefficients and C : PL ratio (respectively r = 0.697 and r = 0.580) as well as TEARS levels ( r = 0.386 for r DPH and r = 0.324 for r ANS), thus implying the possible effect of membrane dialdehydes on bilayer components immobilization. Regression coefficients significance testing showed reaction rates ofTBARS and C:PL changes to be significantly parallel, contrary to those of fluorescence anisotropy coefficients assessing considerably slower dynamics of alloxan-induced changes. The relevance of changes induced by alloxan in isolated erythrocyte ghosts and intact red blood cells and the compatibility of the present results with several previous studies support the widespreading idea pointing the cell membrane as a main target of damage during alloxan action.

Conformational states in .beta.-lactamase: molten-globule states at acidic and alkaline pH with high salt

We present evidence that beta-lactamase is close to fully unfolded (i.e., random coil conformation) at low ionic strength at the extremes of pH and that the presence of salt causes a cooperative transition to a conformation with the properties of a molten globule, namely, a compact state with native-like secondary structure but disordered side chains (tertiary structure). The conformation of beta-lactamase I from Bacillus cereus was examined over the pH 1.5-12.5 region by circular dichroism (CD), tryptophan fluorescence, dynamic light scattering, and 1-anilino-8-naphthalenesulfonate (ANS) binding. Under conditions of low ionic strength (I = 0.05) beta-lactamase was unfolded below pH 2.5 and above pH 11.5, on the basis of the far-UV and near-UV CD and tryptophan fluorescence. However, at high ionic strength and low pH an intermediate conformation (state A) was observed, with a secondary structure content similar to that of the native protein but a largely disordered tertiary structure. The transition from the unfolded state (U) to state A induced by KCl was cooperative and had a midpoint at 0.12 M KCl (I = 0.17 M) at pH 1.6. A similar conformation (state B) was observed at high pH and high ionic strength. The transition from the alkaline U state to state B induced by KCl at pH 12.2 was cooperative and had a midpoint at 0.6 M KCl (I = 0.65 M). Light scattering measurements showed that state B was compact although somewhat expanded compared to the N state. The compactness of state A could not be determined due to its strong propensity to aggregate.(ABSTRACT TRUNCATED AT 250 WORDS)

Estimation of critical micelle concentrations of lysolecithins with fluorescent probes.

The estimation of the critical micelle concentration (cmc) of various lysolecithins with 2-p-toluidinylnaphthalene-6-sulfonate (TNS) and 1-anilinonaphthalene-8-sulfonate (ANS) as fluorescent probes was investigated. The fluorescence yield of TNS in palmitoyl lysolecithin micellar phase was found to be somewhat larger than that of ANS. Further, the fluorescence yield of TNS in the aqueous phase is known to be much smaller than that of ANS. Thus, TNS should be useful for estimating very small cmc values of surfactants such as lysolecithins. The cmc values of lysolecithins estimated by using TNS were 0.5mM for lauroyl lysolecithin, 63μM for myristoyl lysolecithin, 8.3μM for palmitoyl lysolecithin and 6.6μM for oleoyl lysolecithin, in 20mM Tris-HCl buffer solution (pH7.0) at 25°C. It was also found that stearoyl lysolecithin does not form micelles at room temperature, and the Krafft point is about 60°C.

Purification and characterization of human serum C-reactive protein.

A simple and rapid purification method for human serum C-reactive protein (CRP) was developed. CRP was strongly adsorbed on a DEAE-cellulose column and was easily separated from other serum proteins. CRP was purified approximately 1,000-fold with a high yield (50%). The final preparation showed a single band as judged by SDS-polyacrylamide gel electrophoresis, polyacrylamide gel disc electrophoresis, and polyacrylamide gel isoelectric focusing. The fluorescence of the complex of CRP and 8-anilino-1-naphthalene sulfonate (ANS) changed with change of the pH, suggesting that CRP may show pH-dependent conformational change. This finding could account for the peculiar behavior of the protein in isoelectric focusing; it shows an isoelectric point of 7.4 when the starting pH is 7.0, whereas it shows two isoelectric points, 5.3 and 7.4, when the starting pH is 5.5. Ca2+-dependent change of the fluorescence of the complex of CRP and ANS was also detected. These results suggest a pH- and Ca2+-dependent conformational change of CRP.

Effect of ionic strength on the membrane fluidity of rabbit intestinal brush-border membranes: A fluorescence probe study

The effect of ionic strength on the fluidity of rabbit intestinal brush-border membranes has been studied using two fluorescence probes, pyrene and 1-anilino-8-naphthalene sulfonate (ANS). The imposition of a potential gradient on the pyrene-probed membrane vesicles ( out > in ) with increasing NaCl concentration in the medium resulted in a marked enhancement of the excimer formation efficiency, accompanied by a decrease in the ratio of fluorescence intensities of the probe at 392 and 375 nm. Fluorescence polarization of the pyrene-membrane complex is independent of temperature in the absence of salts, while it is dependent on temperature from 10 to 47°C in the presence of salts, as shown by the thermal Perrin plots of polarization. It has been demonstrated that there is a linear relationship between the changes in the pyrene excimer formation efficiency in the membranes and of the values of the binding parameters of ANS for the membranes. From these results, it is suggested that the lipid phase of the membranes becomes more fluid by shielding negatively charged groups of the membrane surface and that there is a fairly close correlation between the membrane organization and the membrane surface charge density.

Probe effects on micellar size

The effect of several probes on the average hydrodynamic radius of micelles, as measured by photon correlation spectroscopy, is reported for several detergent-salt solutions. The micelles of myristyltrimethylammonium bromide, which grow in the presence of electrolyte, show further growth upon addition of pyrene and 1-anilino-8-naphthalenesulfonate (ANS) but shrink upon addition of hexane. In the detergent-salt concentration regime, where minimum sphere values ( R ̄ ⋍ 23 A ̊ ) are observed, hexane does not perturb the apparent size up to its solubility limit. Probe effects on micellar size are also reported for cetyltrimethylammonium chloride, dodecyltrimethylammonium bromide, and sodium dodecyl sulfate at high counterion concentrations.

High pressure study of luminescence from intramolecular CT compounds

The effects of high pressure on the luminescence properties of four organic intramolecular charge transfer compounds were investigated. The compounds were studied in a wide range of liquid and polymeric environments, and for one of the compounds, the effect of chemical substitution was also examined. In general, an increase of pressure affects the luminescence in a way which is in some degree similar to an increase of solvent polarity. A single configuration coordinate model was used to interpret the luminescence in terms of excited state interactions. Three of the compounds exhibited only fluorescence: p-(9-anthryl)-dimethylaniline (ADMA), 6-propionyl-2-dimethylaminonaphthalene (PRODAN), and 1-anilino-8-naphthalenesulfonate (ANS). Fluorescence from the first two compounds in nonpolar media is attributed to an excited state relatively unaffected by the charge transfer process. In polar media, fluorescence is believed to originate from another excited state that possesses considerable charge transfer character, and whose energy is strongly influenced by solvent polarity. At high pressure, an apparent fluorescence shift to higher energy was observed in viscous alcohols apparently due to the introduction of fluorescence from the energetically higher lying non-charge transfer excited state. This explanation is supported by the dual fluorescence decay observed from ADMA coincident with the sign reversal in fluorescence peak shift. Three excited states are believed to affect ANS fluorescence, two of them with charge transfer character. The model describing the excited states of ADMA and PRODAN is consistent with ANS fluorescence except in glycerol, whose anomalous behavior is discussed in the text. The fourth compound, nitroaniline, was studied in its three isomeric forms, with and without N-methylation. In addition to affecting the phosphorescence and fluorescence of p-nitroaniline, pressure studies indicate that nπ* singlet and triplet states figure prominently in the radiative and nonradiative processes of o-nitroaniline and m-nitroaniline as well.

Lipid domains in membranes. Evidence derived from structural perturbations induced by free fatty acids and lifetime heterogeneity analysis.

The interaction of free fatty acids with cell membranes and lipid bilayers was studied by monitoring the emission polarization changes of the fluorescent probes 1,6-diphenyl-1,3,5-hexatriene (DPH) and 8-anilino-1-naphthalene sulfonate (ANS). It was found that cis unsaturated fatty acids incorporated into plasma membranes reduced DPH and increased ANS polarization, while trans-unsaturated and saturated fatty acids had no effect on DPH, but did increase ANS polarization. Although in fluid lipid vesicles, both types of fatty acids decreased DPH polarization, in mixed phase vesicles (dilauroyl phosphatidylcho1ine:dipalmitoy1 phosphatidylcholine) polarization changes of ANS and DPH were similar to that observed in plasma membranes. Alteration in the dimyristoyl phosphatidylcholine phase transition as a function of fatty acid type was used to study the fluid-solid partition behavior in lipid bilayers. Using solution theory, the shift in the transition midpoint temperature was used to infer that the el-unsaturated fatty acids partition into fluid domains while the tmns-unsaturated and saturated fatty acids preferentially partition into solid-like domains. The fluorescent lifetime of DPH in membranes and vesicles was measured by the phase-modulation technique. The results were analyzed in terms of site heterogeneity and indicate that while DPH decay in single phase vesicles is monoexponential, a considerable degree of heterogeneity is observed in mixed phase vesicles and plasma membranes. In particular, evidence is obtained for a DPH binding site which corresponds to the interface between lipid domains. These results, together with the free fatty acid effects, are interpreted in terms of lipid domains in membranes.

Drug interactions. V. Binding of basic compounds to bovine serum albumin by fluorescent probe technique.

The binding of basic compounds to bovine serum albumin (BSA) was studied by the fluorescence method, using N-phenyl-1-naphthylamine (NPN) and 1-anilinonaphthalene-8-sulfonate (ANS) as fluorescent probes. The spectral property of these probes was used for an indirect measurement of the binding of basic compounds to BSA. The compounds used in the present investigation were chlorpromazine, diazepam, chlordiazepoxide, nitrazepam, aniline, and acetanilide. Its results demonstrated that these compounds bind to the hydrophobic regions on the albumin molecule. It appeared that these compounds may be located at least near a tryptophan residue of albumin and two probes may bind at the same site. In the case of chlorpromazine, this drug competed with NPN but not with ANS in binding to BSA. Competitive binding between these compounds, except chlorpromazine, and the probes was observed. The significance of these results was discussed in relation to various types of binding forces. NPN is a useful probe in the evaluation of interactions of basic compounds to serum albumin, but the usefulness of the probe technique may be limited in that binding studies are related only to the site to which the probe binds.

A conformational study of N-phenyl-1-naphthylamine and 1-anilino-8-naphthalene sulfonate by the empirical method

Conformational analysis of N-phenyl-1-naphthylamine and 1-anilinonaphthalene-8-sulfonate (ANS) was carried out using the empirical method. Properties such as conformational energies and dipole moments were considered. Furthermore, the effect of solvent medium was examined through the effective dielectic constant. The N-phenyl-1-naphthylamine molecule showed two energy minima which were independent of dielectic constant. The ANS molecule also showed two energy minima but the minima changed positions when the dielectic constant increased from 1.0 (vacuum) to 80.0 (highly polar medium). Hydrogen bonding appeared to play an important role in stabilizing these conformations. The minimum energy conformations may have relevance to the binding of ANS to lipid bilayers and bimembranes. The dipole moment, in contrast to the energy minimum, was found to depend on orientation of the sulfonate group rather than of the benzene ring with respect to the naphthalene ring. Thus binding and fluorescence enhancement of ANS may be attributed to the orientation of the sulfonate group, which to a large extent may determine the magnitude of the dipole moment and the degree of electrostatic interactions between the probe and binding domains. Various dimensions like intra-atomic distances, volume and area of the ANS molecule were calculated.

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 approximately 20% at 2 muM to approximately 100% at 5 muM. The increase in the rotational relaxation time (rho) of the cell-bound fluorescent probe N-phenyl-1-naphthylamine (PhNap)1 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 rho value of ANS show the same dependence on FCCP concentration with saturation at 0.3 muM. 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 5muM FCCP...

Studies on DNA-recipient interaction I. Investigation of the interaction between recipients and transfection stimulators with fluorescent probes

The interaction of some basic proteins (protamine, thymus histone and the internal protein of T2H bacteriophage) with Escherichia coli spheroplasts and its membranes were investigated with the aid of two fluorescent probes (5-dimethylamino-1-naphthalenesulfonylchloride (DNS)) and 1-anilino-naphthalene-8-sulfonate (ANS). It was found that not less than 40% (and presumably more) of the total (15 μg · ml −1) DNS-protamine was bound to spheroplasts. Some protein was found in isolated membranes and inside the cell. The double-reciprocal plot analysis showed that the binding of protamine resulted in the reduction of the binding sites for ANS and a great enhancement of the fluorescence yields. Addition of internal protein did not affect the number of binding sites and only slightly influenced the quantum yields. It was found that protamine and internal protein alone, in contrast to histone, did not interact with ANS. The dependence of fluorescence intensity upon protein concentration exhibited saturation kinetics for protamine and internal protein in contrast to histone where no saturation was observed. The values of saturating protamine concentration agreed well with the optimal concentration of the protein in transfection assays. Protamine treatment of spheroplasts at 2°C caused their substantial aggregation. The results were interpreted in terms of the structural and charge changes of the surface of the spheroplasts. The interaction of protamine with spheroplasts was directly related to the enhancement of their transfection by various DNAs.