The interaction of flavins with egg white riboflavin-binding protein

The properties of the riboflavin-binding site in the riboflavin-binding protein from egg white have been elucidated by determining constants for binding of flavin analogs to the protein and by changes in absorption spectra of free and bound flavins. The spectral changes and unfavorable interaction of the protein with charged species indicate that the overall flavin environment in the holoprotein is hydrophobic. Modification of either ring or side-chain portions of flavin usually results in a decrease of binding energy. Although no one portion of the structure is absolutely essential, both 7- and 8-methyl groups and 2′-hydroxyl group contribute most significantly to binding. The binding site at the region of C-2 and N-3 of the isoalloxazine is rather insensitive to the relative site of a substituent and thus relatively open, whereas considerable steric limitation is imposed at C-8, N-10, especially C-1′, and 4carbonyl positions. The hydroxyl groups of the N-10 side chain contribute in a stereoselective manner by formation of hydrogen bonds. Studies with model compounds that represent only a part of flavin suggest that the dimethylbenzenoid portion of the ring is involved in primary interactions of binding, and relatively buried in the protein. The quenching of protein fluorescence upon binding is mainly due to ground-state stacking interaction between a trytophanyl residue at the binding site and the quinoxaline portion, and not to Förster energy transfer.     

One-electron reduction of flavoproteins: pulse radiolysis of chicken egg white riboflavin binding protein

Reduction of the chicken egg white riboflavin binding protein by the hydrated electron results in competitive formation of both a disulfide-electron adduct and an anionic flavin semiquinone bound to the protein. The former decays to products that cannot be observed under the conditions of our experiments. The latter is rapidly protonated to the stable neutral semiquinone. The pH dependence of the rate constant associated with this protonation suggests that an acid/base group on the protein donates a proton to the anionic semiquinone.     

In-vitro riboflavin binding and endogenous flavins in Phycomyces blakesleeanus

Several types of membrane-localized flavin binding sites were investigated in sporangiophores (spph) and mycelia of Phycomyces blakesleeanus. In-vitro binding of riboflavin, riboflavin-5′-phosphate, and flavin-adenine-dinucleotide was demonstrated with unfractionated membrane preparations by means of competition of [¹⁴C]riboflavin binding. Saturation of binding was only obtained with the highly water-soluble riboflavin-5′-phosphate, but by extrapolation it was shown that riboflavin showed the highest affinity towards the binding sites (K_D about 4·10^-6M). The number of binding sites was estimated to be 0.7 nmol g^-1 fresh-weight equivalent. Analysis of endogenous soluble flavin revealed that only riboflavin, riboflavin-5′-phosphate, and flavin-adenine-dinucleotide occurred in Phycomyces, and at a concentration of at least 1 nmol g^-1 fresh-weight equivalent in entire spph. Thus, the measured binding sites could reach saturation in-vivo. In the apical part of spph to which blue-light sensitivity is restricted, the amount of soluble flavin was three-fold higher. Exclusively in this zone, heat-labile riboflavin proteins were measured at a concentration of about 3 nmol g^-1 fresh-weight equivalent. The amount of covalently bound flavin was higher in spph tips than in intact spph (8 nmol and 3 nmol g^-1 fresh-weight equivalent, respectively). In either case, the concentrations of the flavin-membrane complexes were higher than the theoretical calculated concentration of (anisotropic) blue-light photoreceptor in Phycomyces (Bergman et al. 1969), and their involvement in blue-light photoreception is considered.     

Effects of pH and ionic strength on the binding of L-tri-iodothyronine to the solubilized nuclear receptor.

K'A (apparent association constant) and Bmax. (total receptor concentration) describing the interaction of tri-iodothyronine (T3) and its solubilized rat liver nuclear receptor (R) are found to be moderately consistent in successive preparations, but both quantities diminished after a few days. To achieve comparability in the effects of ionic strength (I) and of pH on K'A and Bmax, appropriate measurements have been made simultaneously on single preparations. K'A and Bmax. were found to be effectively unchanged over the range I0.05-0.60. Both parameters have been measured over the range pH 6.4-9.0 and the values of K'A analysed in terms of the 4'-OH ionization of T3 and that of a cationic acidic group, shown to require pK' = 7.6. This group could be identified either with the terminal alpha-NH3+ of T3 or with a group (RH+) in the receptor site. On the balance of evidence the first possibility is the more likely, in which case the variation of Bmax. with pH is ascribed to conformational changes in the receptor protein.     

Effects of pH and ionic strength on the structure of collagen fibrils

The roles of pH and ionic strength on the structure and stability of collagen fibrils have been investigated by means of x-ray and neutron diffraction techniques. High-angle x-ray diffraction shows that a salt concentration of 0.5M KCl is sufficient to reduce the osmotic swelling and related disordering in the pH range 1–3. The relative intensities of the low-angle meridional x-ray and neutron diffraction Bragg reflections vary with pH. Difference Fourier syntheses between pH 7 and 1.6 data indicate, for both x-ray and neutron diffraction, a reduced scattering contribution from the telopeptides at low pH. Lyotropic relaxation is a crucial step in the appearance at low pH of a doubling of the 668-Å axial periodicity (D) of collagen fibrils. These results suggest that electrostatic interactions are essential for the structural stability of the telopeptide regions and of the 1D and 3D intermolecular staggers between collagen molecules.     

Influence of molybdate, ionic strength and pH on ligand binding to the glucocorticoid receptor

The addition of molybdate to rabbit liver cytosol increased significantly the affinity of the glucocorticoid receptor for [3H] dexamethasone without influencing the concentration of binding sites. This effect was concentration dependent. Analysis of the binding data by curve-fitting and Scatchard plot revealed the occurrence of a complex binding process in the presence of molybdate. The pH-dependence curve of the binding was shifted towards alkaline values by the oxyanion. Taken together, these data suggest that molybdate exerts its effects via an interaction with the receptor molecule.     

Resonance Raman spectra of riboflavin and its derivatives in the bound state with egg riboflavin binding proteins

The resonance Raman spectra of riboflavin (RF) and its derivatives, including 3-deuterated (3-D RF), 3-methyl (3-CH3 RF), 3-carboxymethyl (3-CH2COOH RF), and 7,8-dichlororiboflavins (7,8-Cl RF), in H2O and D2O were observed in the 700-1700 cm-1 region. The fluorescence problem of riboflavin was overcome by complex formation of riboflavin with riboflavin binding proteins. The observed frequencies of Raman lines of RF are in good agreement with those of glucose oxidase obtained by Spiro et al. by the resonance CARS method, although the present spectral range is extended to much lower frequency with a higher signal-to-noise ratio than that for glucose oxidase. The observed Raman lines were assigned to the individual ring modes of isoalloxazine on the basis of the Raman spectra of appropriate model compounds such as uracil, pyrazine, and o-xylene. The 1253 cm-1 line of RF was shifted to ca. 1300 cm-1 for 3-D RF, 3-CH3 RF, and 3-CH2COOH RF, and accordingly can be assigned to the CN stretching mode of Ring III. The 1632 cm-1 line of RF was shifted for 7,8-Cl RF and was assigned to a Ring I mode. No Raman line mainly due to C = O stretching mode was observed in the present resonance Raman spectra.     

The interaction of riboflavin with a protein isolated from hen's egg white: a spectrofluorimetric study.

The interaction of riboflavin with a protein isolated from egg white has been studied spectrofluorimetrically at different pH values. In 0.1 M phosphate buffer pH 7.0; 1:1 complex formation occurs with the association constant Ka = 7.7-10(7) M-1. In the presence of 0.033% sodium dodecyl sulphate, the complex dissociated with a rate constant of 4-10(-2) sec-1 at 29 degrees C. The binding was sensitive to pH and to the antibodies produced against the protein. On lowering the pH from 7 to 4 the binding affinity decreased approximately 100-fold and below pH 4, the binding could not be detected at all. These data, together with those obtained by measuring the fluorescence intensities of riboflavin in presence of N-bromosuccinimide oxidized- and disulphide reduced apoprotein, suggest that carboxyl functions, 1-2 tryptophan residues and 2-3 disulphide bridges are essential for binding. The emission spectra of the protein under different conditions upon excitation at 280 and 295 nm were analyzed to calculate the quantum yield (Q) and the efficiency of energy transfer (e) from tyrosine to tryptophan residues. From these data it was concluded that the energy transfer did not occur with equal efficiency under all conditions and that the tryptophan residues responsible for the riboflavin binding are more accessible to N-bromosuccinimide oxidation than others.     

Bovine brain phosphatidylinositol transfer protein. Effects of pH, ionic strength and lipid composition on transfer activity

Phosphatidylinositol and phosphatidylcholine are transferred between bilayer membranes in the presence of a specific phosphatidylinositol transfer protein isolated from bovine brain. The effects of pH, ionic strength and lipid composition on the rate of transfer of these phospholipids between small unilamellar vesicles have been investigated. At low ionic strength, phosphatidylinositol transfer between vesicles prepared from phosphatidylcholine and 5 mol% phosphatidylinositol was maximal at about pH 5 and moderately dependent on hydrogen ion concentration in more alkaline regions. A similar dependence on pH was noted for phosphatidylcholine transfer between membranes containing phosphatidylcholine or mixtures of phosphatidylcholine and 5 mol% phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, phosphatidylethanolamine or stearylamine. The rate of transfer between anionic vesicles was somewhat higher than that between neutral or cationic vesicles. At higher ionic strength the transfer reactions in neutral and alkaline regions were less sensitive to pH. Phospholipid transfers between vesicles containing 5 mol% of anionic lipid increased sharply as ionic strength decreased below 0.1. In contrast, phosphatidylcholine transfer between membranes which contained only zwitterionic phospholipids or 5 mol% stearylamine was unaffected by variations of ionic strength. Irrespective of the lipid composition of membranes, pH affected both the apparent Km and Vmax, while ionic strength generally affected the apparent Vmax. These results indicate a significant role of electrostatic interactions in the phospholipid transfer catalyzed by phosphatidylinositol transfer protein.     

Studies on the methods for the determination of phosphorylation sites in highly phosphorylated peptides or proteins: phosphorylation sites of hen egg white riboflavin binding protein

To determine the phosphate binding sites in hen egg white riboflavin binding protein (RBP), a highly phosphorylated peptide, which consisted of 23 amino acid residues including eight phosphoserines, was isolated from the tryptic digest of reduced and carboxymethylated RBP. The conditions of the beta-elimination-addition reaction to convert phosphoserine residues in the peptide to cysteic acids, S-methylcysteines, alanines, and beta-methylaminoalanines (DL-alpha-amino-beta-methylamino propionic acid) were examined. These converted peptides were purified by HPLC and subjected to Edman degradation. The results of Edman degradation indicated that the S-methylcysteine derivative of the peptide gave the most satisfactory result for determining the phosphate binding sites in the peptide. The phosphorylation sites of the peptide determined by the method mentioned above are as follows: His182-Leu-Leu-Ser185-Glu-Ser(P)-Ser(P)-Glu-Glu190-Ser (P)-Ser(P)-Ser(P)-Met-Ser195(P)-Ser(P)-Ser(P)-Glu-Glu-. These studies indicated that the conversion of phosphoserines in phosphoproteins to S-methylcysteines followed by Edman analysis was a useful method for the elucidation of the phosphorylation sites in phosphopeptides.     

Interaction of riboflavin binding protein with riboflavin, quinacrine, chlorpromazine and daunomycin

1. circular dichroism and fluorescence measurements were used to study the reversible unfolding of riboflavin-riboflavin binding protein complex. Both methods showed that the complex was unfolded according to the two-state model. 2. The results suggest that riboflavin was strongly bound in the hydrophobic cleft of the protein and that it could not be dislodged by TFE, MeOH or SDS without major unfolding of the unique tertiary structure of the protein. 3. In addition, it has been also shown that quinacrine, chlorpromazine and daunomycin did not form stereospecific complexes with riboflavin binding protein.     

Interaction of Artepillin C with model membranes: Effects of pH and ionic strength

Artepillin C is the major constituent of green propolis, one of the most consumed products in popular medicine owing to its therapeutic effects, including antitumor activity. Artepillin C differs from other cinnamic acid derivatives due to the presence of two prenylated groups in its structure, believed to enhance access to the cell membrane and resulting in pharmacological activity. The membrane outer leaflet of tumor cells is exposed to an acidic extracellular environment, which could modulate the protonation state of antitumor drugs and hence their interaction with the cell membrane. Herein, we investigated the interaction of Artepillin C with Langmuir monolayers and giant unilamellar vesicles (GUVs) of 1,2?dipalmitoyl?sn?glycerol?3?phosphocholine (DPPC) used as model membranes, in physiological and acidic environments. We observed that protonation of the carboxyl group of Artepillin C is essential for the interaction, with larger shifts induced in the surface pressure isotherms of DPPC monolayers in comparison with deprotonated Artepillin C. Also observed was a decrease in lipid packing inferred from the compressibility modulus and Brewster angle microscopy (BAM) images for monolayers on acidic subphases. Results with microscopy techniques on GUVs confirmed that.Artepillin C causes a curvature stress of the lipid bilayer only in its neutral state, causing the GUVs to burst. The stronger effects of neutral Artepillin C on both monolayers and GUVs were maintained when the ionic strength was increased. Taken together, the results indicate that Artepillin C may have preferential attachment to a more acidic environment which might be an important feature for its antitumor activity.     

pH and ionic strength dependence of protein (un)folding and ligand binding to bovine beta-lactoglobulins A and B

Formation of complexes between bovine beta-lactoglobulins (BLG) and long-chain fatty acids (FAs), effect of complex formation on protein stability, and effects of pH and ionic strength on both complex formation and protein stability were investigated as a function of pH and ionic strength by electrophoretic techniques and NMR spectroscopy. The stability of BLG against unfolding is sharply affected by the pH of the medium: both A and B BLG variants are maximally stabilized against urea denaturation at acidic pH and against SDS denaturation at alkaline pH. The complexes of BLGB with oleic (OA) and palmitic acid (PA) appear more stable than the apoprotein at neutral pH whereas no differential behavior is observed in acidic and alkaline media. PA forms with BLG more stable complexes than OA. The difference between the denaturant concentration able to bring about protein unfolding in the holo versus the apo forms is larger for urea than for SDS treatment. This evidence disfavors the hypothesis of strong hydrophobic interactions being involved in complex formation. Conversely, a significant contribution to FA binding by ionic interactions is demonstrated by the effect of pH and of chloride ion concentration on the stoichiometry of FA.BLG complexes. At neutral pH in a low ionic strength buffer, one molecule of FA is bound per BLG monomer; this ratio decreases to ca. 0.5 per monomer in the presence of 200 mM NaCl. The polar heads of bound FA appear to be solvent accessible, and carboxyl resonances exhibit an NMR titration curve with an apparent pK(a) of 4.7(1).     

Effects of ionic substances on the adsorption of egg white proteins to a stainless steel surface

The surface fouling of food processing equipment by proteins was studied by investigating the adsorption of egg white proteins to the surface of stainless steel (SS) at pH 7.4 and 30 °C, and particularly the effects of different types of ionic substances. Ovalbumin and ovomucoid, acidic egg white proteins, were less adsorbed in the presence of phosphate (P(i)), a multivalent anion, than in the presence of HEPES, an amphoteric ion. On the other hand, lysozyme, a basic egg white protein, was more adsorbed in the presence of P(i) than in the presence of HEPES. Citrate as another multivalent anion and taurine as another amphoteric ion affected the respective adsorption of those egg white proteins similarly to P(i) and HEPES. The adsorption of an egg white protein to an SS surface therefore depended on the combination of the type of protein and the effective charge of the coexisting ionic substance. This behaviour can be well explained by assuming that a small ionic substance precedes a protein in attaching to an SS surface, resulting in an alteration to the effective surface charge. Pretreating SS with a P(i) buffer lowered the amount of ovalbumin adsorbed with the HEPES buffer, demonstrating that P(i) can attach to and remain on the SS surface to affect the subsequent protein adsorption.     

Influence of ionic strength on Hoechst 33258 binding with DNA

The binding of Hoechst 33258 with DNA at various ionic strengths of solution and different ligand concentrations has been investigated. Existence of more than one type of interactions of Hoechst 33258 with DNA has been revealed, which were very sensitive to the ionic strength. Hoechst 33258 doesn't show specificity to AT sequences of DNA at low ionic strength. High affinity binding mode becomes obvious at high ionic strength. The values of binding constants and binding site sizes for revealed strong and weak interactions have been determined.     

Influence of the ionic strength on the heat-induced aggregation of the globular protein beta-lactoglobulin at pH 7

The influence of the ionic strength on the structure of beta-lactoglobulin aggregates formed after heating at pH 7 has been studied using static and dynamic light scattering. The native protein depletion has been monitored using size exclusion chromatography. Above a critical association concentration (CAC) well-defined clusters are formed containing about 100 monomers. The CAC increases with decreasing ionic strength. The so-called primary aggregates associate to form self similar semi-flexible aggregates with a large scale structure that is only weakly dependent on the ionic strength. The local density of the aggregates increases with increasing ionic strength. At a critical gel concentration, Cg, the size of the aggregates diverges. Cg decreases from 100 g/l without added salt to 1 g/l at 0.4M NaCl. For C > Cg the system gels except at high ionic strength close to Cg where the gels collapse under gravity and a precipitate is formed.     

Influence of pH and ionic strength on the steric mass-action model parameters around the isoelectric point of protein

The ion-exchange equilibrium and the dependence of the parameters in the steric mass-action (SMA) model on salt concentration and buffer pH around the isoelectric point of protein were studied. Bovine serum albumin (BSA, isoelectric point = 5.4) was used as a model protein and DEAE Sepharose FF as an ion exchanger. Finite batch adsorption experiments and isocratic elution chromatography were performed for the determination of the model parameters (i.e., characteristic charge, equilibrium constant, and steric factor). The results showed that pH had significant effects on the parameters. With an increase of pH from 4.5 to 6.5, the characteristic charge increased from 0.9 to 3.0 and leveled off as a plateau at pH above 5.5. The charge groups in the contact region of protein surface were considered to play a crucial role on the characteristic charge. The decrease of pH and increase of salt concentration lowered the absolute value of the zeta potential of the protein surface and led to a decrease of the equilibrium constant. The steric factor remained unchanged at about 31 at pH 5.5 and 6.0 and increased to 44.5 at pH 5.0 and 96.8 at pH 4.5, mainly as a result of the lower adsorption capacity of BSA at pH <5.5. Furthermore, the increase of the molecular volume of BSA at pH 4.5 would be an additional reason for the increase of the steric factor. Taking into account the effect of the pH and salt concentration on these parameters, the SMA model described the ion exchange equilibrium of protein more accurately.     

Interactions of flavins with melanin. Studies on equilibrium binding of riboflavin to dopa-melanin and some spectroscopic characteristics of flavin-melanin complex

Natural melanins are photoprotective pigments that in mammals are principally found in the skin, hair, and eyes. Although the molecular mechanism of photoprotection of pigmented cells has not yet been established, several hypotheses have been proposed with melanin acting as a light filter, free radical scavenger, and quencher of electronically excited states of reactive intermediates. It can be expected that the detoxicating efficiency of melanin should be enhanced if the melanin and potentially cytotoxic species are brought close together. Such a situation may occur for a number of photosensitizing dyes that have the ability to bind to melanin. The interaction of melanin with flavins has been studied under strictly controlled experimental conditions. The equilibrium dialysis method has been employed to determine dissociation constants and the number of binding sites in melanin at pH 5-9. The data reveal that synthetic DOPA-melanin has two different classes of binding sites with dissociation constants of 10(-6) and 10(-5) M, respectively. The overall binding capacity of melanin, at pH 7, is 250 nmol RF/mg melanin. The amount of bound-to-melanin RF increases with pH. The absorption spectra of melanin complexes with RF and lumiflavin indicate that hydrophobic interaction may be involved in the binding of these flavins by melanin. No changes in flavin fluorescence have been detected after binding of flavin to melanin. It appears that, contrary to cationic photosensitizing dyes, the singlet excited state of flavin molecules is not quenched by melanin.