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.     

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.     

The modification of the lone tryptophan residue in human serum albumin by 2-hydroxy-5-nitrobenzyl bromide. Characterization of the modified protein and the binding of L-tryptophan and benzodiazepines to the tryptophan-modified albumin.

The possible function of the lone tryptophan residue of human serum albumin in the stereospecific binding site for indole and benzodiazepine compounds was investigated by chemical modification. This residue can be selectively modified with 2-hydroxy-5-nitrobenzyl bromide. The modification alters the conformation of the albumin only slightly, as revealed by circular dichroism, fluorescence, and ultraviolet absorption measurements. A decrease in the association constants of L-tryptophan and diazepam of about 30 - 50% and a decrease in the extrinsic Cotton effects of four benzodiazepine derivatives of about 10 - 15% were found as specific effects of the tryptophan modification. The tryptophan modification itself did not change the number of binding sites of diazepam and L-tryptophan. It is suggested that the lone tryptophan residue of human serum albumin is not directly involved in the specific binding site for indole and benzodiazepine compounds. However, the modification alters the properties of the binding site either by an incomplete refolding of the albumin after urea treatment, or a more selective allosteric effect of the modified tryptophan residue.     

A highly reactive tyrosine residue as part of the indole and benzodiazepine binding site of human serum albumin.

The interaction of L-tryptophan and four benzodiazepine derivatives with tyrosine-modified human serum albumin was investigated by equilibrium dialysis and circular dichroism measurements. Out of the 18 tyrosine residues of the human serum albumin molecule, only 9 could be modified with tetranitromethane. At least up to a degree of modification of 5, the conformation of human serum albumin was not changed and no crosslinking and fractionation has been found, as revealed from circular dichroism measurements in the far ultraviolet range and from SDS polyacrylamide electrophoresis. The modification of only 2 out of the 9 accessible tyrosine residues of human serum albumin strongly affects the binding of L-tryptophan and diazepam to their common, stereospecific bindining site. This was evidently shown by a reduction of the association constants by more than 90% and by a large reduction of the extrinsic Cotton effects of four benzodiazepines bound to human serum albumin. The numbers of binding sites remained unchanged. Strong evidence was presented that only one tyrosine residue, which reacts faster with tetranitromethane than all others, is mainly involved in the specific indole and benzodiazepine binding site of human serum albumin. The location of this highly reactive tyrosine residue and that of the specific indole and benzodiazepine binding site within the human serum albumin primary structure is discussed.     

The interaction of 8-anilino-1-naphthalenesulfonate with polylysine and polyarginine

Comparative studies on the interaction of 8-anilino-1-naphthalenesulfonate (ANS) with polylysine and polyarginine have been made by equilibrium dialysis and fluorescence or circular dichroism measurements, to investigate the structural characteristics of the polypeptides. The results are summarized as follows: (i) ANS binds to either of the polypeptides primarily by electrostatic interaction while hydrophobic interaction partially facilitates the dye binding; both interactions are stronger in the polyarginine-dye binding than the polylysine-dye binding. (ii) The fluorescence of ANS is more intensified when the dye binds to polyarginine than to polylysine regardless of the value of r (number of bound dye per amino-acid residue) of polypeptide-dye complexes, although the intensification depends on the r value and becomes maximum at r = 0.25–0.35 for both cases. (iii) The binding of ANS to each polypeptide is cooperative at r < 0.4. (iv) The circular dichroism is more efficiently induced in the spectral region of ANS by binding to polyarginine than to polylysine. From these results, it was concluded that, compared to polylysine, polyarginine suffers some structural change by ANS binding into a more compact molecular configuration having some regularity with a lower dielectric environment.     

Human serum albumin. Spectroscopic studies of binding and proximity relationships for fatty acids and bilirubin.

Binding and proximity relationships of hydrophobic ligands on human serum albumin have been studied using absorption, fluorescence, circular dichroism, and electron paramagnetic resonance spectroscopy. The ligands studied were bilirubin, two conjugated linear polyene fatty acids, cis-parinaric acid and cis-eleostearic acid, and three nitroxide derivatives of stearic acid with doxyl groups at positions 5, 10, and 12, respectively. Binding of polyene fatty acids was monitored by absorption peak shifts, induced circular dichroism, enhancement of fluorescence, and energy transfer between albumin's single tryptophanyl residue and the polyene chromophore. Induced circular dichroism studies indicate excitonic ligand-ligand interaction between bound fatty acids. Fluorescence enhancement of cis-parinaric acid was analyzed using a stepwise multiple equilibrium model, and six binding constants in the range 10(8) to 10(6) M-1 were obtained, in agreement with previous measurements for other fatty acids. The temperature dependence of the equilibrium constants indicates that the binding enthalpy is nearly zero. Fluorescence energy transfer was similarly used to quantitate bilirubin binding to albumin. Energy transfer, nitroxide quenching of fluorescence, and electron paramagnetic resonance spectroscopy were used to elucidate binding geometries which support and extend proposed structural models for albumin. It is suggested that the first two fatty acids bind side-by-side in an antiparallel fashion in domain III of human serum albumin.     

A partially folded intermediate conformation is induced in pectate lyase C by the addition of 8-anilino-1-naphthalenesulfonate (ANS)

Addition of 8-anilino-1-naphthalenesulfonate (ANS) to acid-denatured pectate lyase C (pelC) leads to a large increase in the fluorescence quantum yield near 480 nm. The conventional interpretation of such an observation is that the ANS is binding to a partially folded intermediate such as a molten globule. Far-ultraviolet circular dichroism demonstrates that the enhanced fluorescence results from the induction of a partially folded protein species that adopts a large fraction of native-like secondary structure on binding ANS. Thus, ANS does not act as a probe to detect a partially folded species, but induces such a species. Near-ultraviolet circular dichroism suggests that ANS is bound to the protein in a specific conformation. The mechanism of ANS binding and structure induction was probed. The interaction of acid-unfolded pelC with several ANS analogs was investigated. The results strongly indicate that the combined effects of hydrophobic and electrostatic interactions account for the relatively high binding affinity of ANS for acid-unfolded pelC. These results demonstrate the need for caution in interpreting enhancement of ANS fluorescence as evidence for the presence of molten globule or other partially folded protein intermediates.     

Circular dichroic evidence for regulation of enzymatic activity by nonsubstrate hydrophobic ligand on glutathione S-transferase P.

1-Anilinonaphthalene-8-sulfonic acid (ANS) noncompetitively inhibited enzyme activity of glutathione S-transferase P for both glutathione and 1-chloro-2,4-dinitrobenzene (Ki = 30 microM). Dissociation constant for ANS.GST-P complex calculated from the binding study was 15 microM. From the similar values of the inhibition constant and the dissociation constant, it was concluded that specific ANS binding caused the loss of enzyme activity. In the protein structural analysis by circular dichroism, the secondary structures remarkably changed by ANS binding in accordance with the decrease of enzymatic activities. The conformational change of the protein and the decrease in enzymatic activity were reversed by dissociation of ANS. This fact strongly suggested that the enzymatic activity was regulated by a nonsubstrate hydrophobic ligand.     

Thyroglobulin structure-function: the effect of iodination on the structure of human thyroglobulin

Thyroglobulin of very low iodine content has been prepared from a single non-toxic human goitre. The initial iodine content of the protein (0.038%) has been increased to levels of 0.16% and 0.85% by in vitro treatment with thyroid peroxidase and the resulting proteins studied with respect to their intrinsic fluorescence, circular dichroism spectra and binding of the hydrophobic probe 1,8-anilinonaphthalene sulfonic acid (ANS). While significant differences were observed between levels of iodination in both the ANS binding and intrinsic fluorescence of the thyroglobulin, no significant differences in the near and far UV circular dichroism spectra of the protein as a function of iodine content were observed. These data suggest that, the iodination of thyroglobulin effects specific areas of the protein without significant disruption of its overall secondary structure.     

The minimal structural requirement of concanavalin A that retains its functional aspects

A systematic investigation of the effects of several commonly used detergents on the conformation and function of concanavalin A at pH 7 in solution form was made by using circular dichroism (CD), intrinsic fluorescence, 1-anilino 8-sulphonic acid (ANS) binding, dynamic light scattering (DLS) and sugar inhibition assay. In the presence of 6.0 mM sodium dodecyl sulphate (SDS), an anionic detergent, and 0.8 mM cetyl tri methyl ammonium bromide (CTAB), a cationic detergent, intermediate states of concanavalin A were obtained having a negative CD peaks at 222 and 208 nm respectively, a characteristic of alpha-helix. These states also retained tertiary contacts with altered tryptophan environment and high ANS binding (exposed hydrophobic area) which can be characterized as molten globule states. Concanavalin A in the presence of 5.0 mM 3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propanesulphonate (CHAPS), a zwitterionic detergent, and 0.07 mM brij-35, a non-ionic detergent, also exists in intermediate states. These intermediates (molten globules) had high ANS binding with native-like secondary (inherent beta-sheet) and tertiary structure. The intermediate states were characterized further by means of dynamic light-scattering measurements and kinetic data. To study the possible functional requirement of the minimum structure, the intermediate states characterized in the presence of detergents were shown to retain the activity with polysaccharide (dextran). The pattern of activity observed was brij-35 > CHAPS > CTAB > SDS. The specific binding and activity of concanavalin A with ovalbumin was investigated as a function of time by turbidity measurements. Cationic and anionic detergents showed significant effects on the structure of concanavalin A as compared with zwitterionic and non-ionic detergents.     

Quenching of the emission of tryptophan, tyrosine, and serum albumins by cupric ion

The effect of cupric ion on the emission of tryptophan, tyrosine, and serum albumins is studied by emission spectroscopy and lifetime measurements. It is found that whenever cupric ion is bound to tryptophan or tyrosine, their emissions are quenched completely. The quenching may be due to an electron transfer mechanism. The fluorescence of complexes of cupric ions with serum albumins is partially quenched; this is because energy is transferred from tryptophan to the complexed cupric ions by a dipolar energy transfer mechanism. It is deduced from the present study that the tryptophan in the human serum albumin molecule is between 11 and 16 Å from the nearest eupric ion binding sites (assumed to be at the surface of the protein) and that one of the tryptophan in the bovine serum albumin molecule is very close to the cupric ion binding sites and the other is near the center of the bovine serum albumin molecule. It is also found that the deuterium solvent effect on serum albumin fluorescence is very small, and that the quenching of bovine serum albumin fluorescence at the N-F transition is the result of quenching of the fluorescence of both tryptophans. The phosphorescence lifetime apparatus, capable of measuring decay times of signals with intensities changing over a few orders of magnitude, and the ratio spectrofluorometer, both of which were constructed in this laboratory, are also described.     

Partial characterization of the 1-anilino-8-naphthalene sulfonate-adrenochrome semicarbazide interaction site in erythrocyte ghost membrane fragments.

The effect of adrenochrome semicarbazide on the conformation of erythrocyte ghost membranes has been studied by ANS fluorescence, lipid and sulfhydryl spin labels and circular dichroism. No large conformational alterations in the membrane were detected by these techniques. Noncompetitive quenching of ANS fluorescence by ADCS suggests ADCS to interact with the membrane at sites close to the ANS binding domain.     

Exploring structural change of protein bovine serum albumin by external perturbation using extrinsic fluorescence probe: spectroscopic measurement, molecular docking and molecular dynamics simulation

Structural modification through binding interaction of plasma protein bovine serum albumin (BSA) with an extrinsic charge transfer fluorophore 5-(4-dimethylamino-phenyl)-penta-2,4-dienoic acid (DMAPPDA) and its response to external perturbation due to interactions with surfactant sodium dodecyl sulphate (SDS) have been explored at physiological pH by steady state absorption, emission, fluorescence anisotropy, red edge excitation shift, far-UV circular dichroism and time resolved spectral measurements in combination with Molecular Docking and Molecular Dynamics (MD) simulation. Interaction of the probe with BSA is reflected by a small change in protein secondary structure with fluorescence enhancement and blue shift of probe emission. Molecular docking studies revealed that the probe binds to the hydrophobic cavity of sub-domain IIA of BSA. The distance for energy transfer from the tryptophan of BSA to the bound DMAPPDA measured by Fluorescence Resonance Energy Transfer is in good agreement with the molecular docking results. MD simulation predicts stabilization of the complex with respect to the bare molecule. Interaction of BSA and SDS with DMAPPDA supports the movement of the probe from hydrophilic free water region to a more restricted hydrophobic zone inside the protein.     

Isolation and characterization of denatured serum albumin from rats with endotoxicosis

Due to its rapid breakdown in the body, denatured serum albumin has not been identified in biological samples. In this study we attempted to determine whether denatured albumin could be identified in rats with endotoxicosis. Male Wistar rats were injected with lipopolysaccharide (LPS; 5 mg/kg body weight). Plasma albumin concentration decreased to one-third the normal level at 2 days after the injection. By using the purified IgG against the specific epitope of chemically denatured albumin, two immunoreactive plasma proteins (bands D2 and D3) were identified by native PAGE followed by Western blot analysis. The plasma concentration of these two proteins increased significantly at 1 and 1.5 days after LPS injection. Peptide mass fingerprinting using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI/TOF-MS) identified these two proteins as serum albumin. In order to characterize their conformational nature, ion-exchange chromatography was used to isolate D2 and D3 albumins from rats injected with LPS. Far- and near-UV circular dichroism (CD), tryptophan and 1-anilino-8-naphthalenesulfonate (ANS) fluorescence, and proteolytic susceptibility showed conformational alterations in the D2 and D3 albumins as compared with native albumin. These data indicate the presence of denatured albumin in circulating rat plasma, and this fact may contribute to a further understanding of the molecular mechanisms of albumin breakdown in physiological and pathophysiological conditions.     

Octyl glucoside induced formation of the molten globule-like state of glutamate dehydrogenase.

The interaction between n-octyl-beta-D-glucopyranoside (octyl glucoside) and bovine liver glutamate dehydrogenase (GDH) was studied using techniques including equilibrium dialysis, UV-spectrophotometry, circular dichroism (CD), fluorescence energy transfer and extrinsic spectrofluorometry in 50 mM sodium phosphate buffer solution (pH 7.6). The equilibrium dialysis experiment showed a higher binding of octyl glucoside to GDH that induces up to 80% enzyme inhibition in 20 mM octyl glucoside solution. The CD study indicated that GDH retains its secondary structure in the presence of octyl glucoside, but loses a degree of its tertiary structure by acquiring a more extended tertiary structure. Measurement of the binding of a hydrophobic fluorescent probe, 1-anilino-naphthalene-8-sulfonate (ANS), to GDH revealed that the binding of ANS to GDH is increased in the presence of octyl glucoside, a finding that may be interpreted in terms of the increment of surface hydrophobic patch(es) of GDH because of its binding to octyl glucoside. Fluorescence energy transfer studies also showed more binding of the reduced coenzyme (NADH) to GDH and the Lineweaver-Burk plots (with respect to NADH) indicate the existence of substrate inhibition in the presence of octyl glucoside. These observations are aimed at explaining the formation of the molten globule-like structure of GDH, which is induced by a non-ionic detergent such as octyl glucoside.     

Salt-induced folding of a rabbit muscle pyruvate kinase intermediate at alkaline pH

The effect of alkaline denaturation on the structural and functional characteristics of rabbit muscle pyruvate kinase (PK) was investigated using enzymatic activity measurements and a combination of optical methods such as circular dichroism, fluorescence, and ANS binding. At a critical pH, 10.5, PK exists in an intermediate state (alkaline unfolded state) with predominant secondary structure along with some of the tertiary interactions and a strong binding to the hydrophobic dye ANS. This intermediate retains the enzymatic activity and corresponds to a dimeric state of the molecule. Above pH 10.5, a sudden fall in the spectral properties and enzymatic activity occurs suggesting the dissociation of the molecule followed by unfolding at very high pH. Addition of salts such as NaCl, KCl, and Na₂SO₄ to the alkali-induced state induces both secondary and tertiary structure to a level equivalent to that of native tetramer (salt-induced state). Chemical- and temperature-induced unfolding of the alkali-induced state as well as the salt-induced refolded state of PK reveal the presence of intermediate conformations in the unfolding pathway. The unfolding transition curves are noncoinciding and noncooperative along with ANS binding at intermediate concentrations of denaturants during unfolding. The observations presented in this paper suggest that the native pyruvate kinase tetramer dissociates to an active dimer around pH 10.5 and further to inactive monomer before attaining a completely unfolded monomeric conformation.     

Temperature-induced conformational switch in intestinal fatty acid binding protein (IFABP) revealing an alternative mode for ligand binding

IFABP is a small beta-barrel protein with a short helix-turn-helix motif near the N-terminus that is thought to participate in the regulation of the uptake and delivery of fatty acids. In a previous work, we detected by near UV circular dichroism a reversible conformational transition of this protein occurring between 35 and 50 degrees C in the absence of fatty acids. The addition of the natural ligand oleic acid prevents this phenomenon. In both cases, the overall structure of the beta-barrel is maintained. This thermal transition is also detected by the fluorescent probe bis-anilino naphthalene sulfonic acid (bisANS) but not by its monomer ANS. In the present work, we studied in detail the interaction of each compound with IFABP as a function of temperature and in the absence or in the presence of oleic acid. A contrasting behavior was observed for these probes: (i) IFABP is able to bind two molecules of bisANS but only one molecule of ANS and (ii) oleic acid can fully displace ANS but only partially bisANS. Three independent lines of evidence, namely, fluorescence spectroscopy, circular dichroism, and limited proteolysis, indicate that there is an equilibrium among different conformations of IFABP, which differ in the extent of flexibility of the helical domain. This equilibrium can be shifted by raising temperature. bisANS is able to probe a population of IFABP in an altered state, which is more susceptible to cleavage by clostripain as compared to the apo-form, whereas the conformation of IFABP bound to oleic acid is characteristically more ordered. These results highlight the idea of an enhanced flexibility exhibited by IFABP that bears importance on its transport function, supporting the role of a dynamic entry portal region for the fatty acid ligand.     

On the mechanism of human stefin B folding: I. Comparison to homologous stefin A. Influence of pH and trifluoroethanol on the fast and slow folding phases

The folding of human stefin B has been studied by several spectroscopic probes. Stopped-flow traces obtained by circular dichroism in the near and far UV, by tyrosine fluorescence, and by extrinsic probe ANS fluorescence are compared. Most (60 ± 5%) of the native signal in the far UV circular dichroism (CD) appeared within 10 ms in an unresolved “burst” phase, which was followed by a fast phase (t = 83 ms) and a slow phase (t = 25 s) with amplitudes of 30% and 10%, respectively. Similar fast and slow phases were also evident in the near UV CD, ANS fluorescence, and tyrosine fluorescence. By contrast, human stefin A, which has a very similar structure, exhibited only one kinetic phase of folding (t = 6 s) detected by all the spectroscopic probes, which occurred subsequent to an initial “burst” phase observed by far UV CD. It is interesting that despite close structural similarity of both homologues they fold differently, and that the less stable human stefin B folds faster by an order of magnitude (comparing the non-proline limited phase). To gain more information on the stefin B folding mechanism, effects of pH and trifluoroethanol (TFE) on the fast and slow phases were investigated by several spectroscopic probes. If folding was performed in the presence of 7% of TFE, rate acceleration and difference in the mechanism were observed. Protein 32:296–303, 1998. © 1998 Wiley-Liss, Inc.     

Interaction studies of aristolochic acid I with human serum albumin and the binding site of aristolochic acid I in subdomain IIA

Optical spectroscopy and molecular docking methods were used to examine the binding of aristolochic acid I (AAI) to human serum albumin (HSA) in this paper. By monitoring the intrinsic fluorescence of single Trp214 residue and performing displacement measurements, the specific binding of AAI in the vicinity of Sudlow's Site I of HSA has been clarified. An apparent distance of 2.53 nm between the Trp214 and AAI was obtained via fluorescence resonance energy transfer (FRET) method. In addition, the changes in the secondary structure of HSA after its complexation with the ligand were studied with circular dichroism (CD) spectroscopy, which indicated that AAI does not has remarkable effect on the structure of the protein. Moreover, thermal denaturation experiments clearly indicated that the HSA−AAI complexes are conformationally more stable. Finally, the binding details between AAI and HSA were further confirmed by molecular docking studies, which revealed that AAI was bound at subdomain IIA through multiple interactions, such as hydrophobic effect, van der Waals forces and hydrogen bonding.     

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.