Proton linkage of complex formation between cytochrome c and cytochrome b5: electrostatic consequences of protein-protein interactions.

Two potentiometric methods have been used to study the pH-dependent changes in proton binding that accompany complex formation between cytochrome c and cytochrome b5. With one method, the number of protons bound or released upon addition of one cytochrome to the other has been measured as a function of pH. The results from these studies are correlated with the complexation-induced difference titration curve calculated from the titration curves of the preformed complex and of the individual proteins. Both methods demonstrate that complex formation at acid pH is accompanied by proton release, that complex formation at basic pH is accompanied by proton uptake, and that the change in proton binding at neutral pH, where stability of complex formation is maximal, is relatively small. Under all conditions studied, the stoichiometry of cytochrome c-cytochrome b5 complex formation is 1:1 with no evidence of higher order complex formation. Although the dependence of complex formation on pH for interaction between different species of cytochrome c and cytochrome b5 are qualitatively similar, they are quantitatively different. In particular, complex formation between yeast iso-1-cytochrome c and lipase-solubilized bovine cytochrome b5 occurs with a stability constant that is 10-fold greater than observed for the other two pairs of proteins under all conditions studied. Interaction between these two proteins is also significantly less dependent on ionic strength than observed for complexes formed by horse heart cytochrome c with either form of cytochrome b5.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of calmodulin with the phosphofructokinase target sequence

Ca4.calmodulin (Ca4.CaM) inhibits the glycolytic enzyme phosphofructokinase, by preventing formation of its active tetramer. Fluorescence titrations show that the affinity of complex formation of Ca4.CaM with the key 21-residue target peptide increases 1000-fold from pH 9.0 to 4.8, suggesting the involvement of histidine and carboxylic acid residues. 1H NMR pH titration indicates a marked increase in pKa of the peptide histidine on complex formation and HSQC spectra show related pH-dependent changes in the conformation of the complex. This unusually strong sensitivity of a CaM-target complex to pH suggests a potential functional role for Ca4.CaM in regulation of the glycolytic pathway.     

Parameters involved in binding of porcine pancreatic alpha-amylase with black bean inhibitor: role of sulfhydryl groups, chloride, calcium, solvent composition and temperature

The amylase inhibitor of black (kidney) beans (Phaseolus vulgaris; MW 53,000) forms a 1:1 stoichiometric complex with porcine pancreatic alpha-amylase (MW 52,000) at pH 5.40. The single sulfhydryl group of the inhibitor and the two sulfhydryl groups of alpha-amylase are not involved in recognition and binding. Chloride ions, required for activity of alpha-amylase at both pH 5.40 and 6.90, are important for inhibitor--enzyme binding at pH 6.90 but not at pH 5.40. Calcium-free alpha-amylase binds with the inhibitor. An increase in the ionic strength of the solvent increases the rate of binding of the inhibitor with alpha-amylase; a decrease in the dielectric constant decreases the rate of binding; and decreasing the temperature increases the dissociation constant, Kd, of the complex. These data support the hypothesis that hydrophobic interaction is of primary importance in complex formation. The activation energy, Ea, for complex formation was found to be 12.4 kcal/mol at pH 5.40 and 24.2 kcal/mol at pH 6.90. In the presence of the poor substrate, p-nitrophenyl-alpha-D-maltoside, the Ea for complex formation was 4.1 kcal/mol at pH 6.90.     

A calorimetric investigation of the binding of indole and phenylethane boronic acid to chymotrypsin

The heat of formation of the chymotrypsin-phenylethane boronic acid complex has been observed calorimetrically from pH 4 to 8 at 25 degrees C and is found to be pH-dependent, changing from near -6 kcal/mol at pH 4 to -13 kcal/mol at pH 8. The heat of formation of the chymotrypsin-indole complex is a nearly constant -6 kcal/mol over most of the same pH range. alpha-Chymotrypsin has been purified by pH gradient elution from an immobilized lima bean inhibitor column. Solutions of the enzyme up to 400 microM, prepared in this manner, have a zero heat of dilution from pH 5 to 8 in 0.1 M KCl, with or without added 0.05 M Tris, N-(tris[hydroxy-methyl]methyl-2-amino) ethanesulfonic acid, 4-morpholineethanesulfonic acid, or acetate buffers. Binding of phenylethane boronic acid causes a pH-dependent decrease in proton binding to chymotrypsin; the decrease in proton binding evoked by formation of the indole complex is much less, with a much smaller pH dependence. The calorimetric and proton-binding results are applied to a model for boronic acid binding (Hanai, K. (1976) J. Biochem. (Tokyo) 79, 107-116). We conclude that the thermodynamics of formation of the trigonal boronic acid complex are quite similar to those for the formation of the noncovalent complex formed by indole and related ligands. The trigonal-tetrahedral tautomerism in the boronic acid-chymotrypsin complex is characterized by thermodynamic changes similar to those accompanying the binding of virtual substrates to chymotrypsin.     

Chitosan-poly(acrylic acid): mechanism of complex formation and potential industrial applications.

The food industry is interested in polyelectrolytic coagulants of natural origin for the clarification of food beverages and the recovery of colloidal and dispersed particles from processing waste streams. This paper discusses potential industrial applications of recent findings on polymer complex formation obtained with a chitosan-poly(acrylic acid) model system. Process recommendations could be made on the basis of the ionic strength, pH, and charged group concentration of the fluid to be treated. Ionic strength does not affect the complex formation process. The amount of chitosan in the complex formed is controlled by the solution pH. The mechanism of complex formation indicates that pH measurements could be used to monitor the coagulation process.     

Influence of pH on the Structure and Oleic Acid Binding Ability of Bovine α-Lactalbumin

The biological function of ??-lactalbumin (??-LA) depends on its conformation. ??-LA can adopt a stable intermediate state induced by heating or pH change. This intermediate state associates with oleic acid (OA) to form an anti-tumor complex. The effect of temperature on the formation the complex has been studied, whereas the effect of pH on complex formation remains unresolved. The effect of pH on tryptophan residues, hydrophobic clusters and secondary structure of Ca²⁺-depleted bovine ??-LA (BLA) was studied by fluorescence spectroscopy and circular dichroism. BLA was found to adopt a more flexible conformation between pH 7.0 and 9.0 with buried hydrophobic clusters. The binding ability of ??-LA towards OA and the anti-tumor activity of the corresponding complex were also studied. BLA was found to bind more OA over the pH range of 7.0?C9.0 and the corresponding complexes showed a higher anti-tumor activity than those complexes formed under acidic conditions. Our study indicates that alkaline pH aided the formation of the complex as well as its anti-tumor activity. We also propose a possible characteristic structure that facilitates binding of OA.     

Rapid-scan stopped-flow studies of the pH dependence of the reaction between mercuric reductase and NADPH

The reaction of NADPH with the flavoenzyme mercuric reductase has been studied by rapid-scan stopped-flow spectrophotometry at 5 degrees C in the pH range 5.1-9.5. An intermediate formed within the dead time of the apparatus, and proposed to be an NADPH complex of oxidized enzyme, has an almost pH-independent spectrum. At pH 5.1 the formation of this species is followed by a rapid bleaching (k = 145 s-1) of the main flavin absorption band at 455 nm concomitantly with an absorbance increase around 395 nm. This process, which has a kinetic hydrogen isotope effect of 2.4, becomes less prominent at higher pH values and is not detectable above pH 7. It is suggested that this process includes the formation of a covalent thiol-flavin C-4a derivative stabilized by protonation of the active site. In the presence of an excess of NADPH, the final product of the reaction is probably an NADPH complex of two-electron-reduced enzyme, but below pH 6 the final spectrum becomes less intense suggesting a partial formation of four-electron-reduced enzyme. The spectral changes observed above pH 7 are nearly independent of pH. The first measurable step (k = 48 s-1 at pH 9.5) is thought to include the formation of an NADP+ complex of two-electron-reduced enzyme, while the final step (k = 6.3 s-1 at pH 9.5) results in the above-mentioned NADPH complex with two-electron-reduced enzyme. A minimal kinetic scheme rationalizing the observed pH dependence of the reaction and the observed isotope effects is presented.     

Proteoglycans of bovine articular cartilage. Studies of the direct interaction of link protein with hyaluronate in the absence of proteoglycan monomer

When link protein binds to hyaluronate in the absence of proteoglycan monomer a high molecular weight complex is formed. Two assay procedures have been developed to examine the formation of the complex and the rate and stoichiometry of binding of link protein to hyaluronate in the complex. In the first, the complex is isolated by differential centrifugation, and the stoichiometry of binding of link protein to hyaluronate in the sedimented complex is determined. In the second assay, which involves turbidimetry, the rate of complex formation (delta A420/min) is determined, and the amount of complex formed is determined in terms of the maximum turbidity (A420,max) attained. The effects of temperature, pH, initial total solute concentration, and the ratio by weight of link protein to hyaluronate on the amount of complex formed and on the rate of complex formation were examined. There is a linear correlation between the amount of complex formed as determined by turbidity and by differential centrifugation. Using these assays, we examined the specificity of the binding of link protein to hyaluronate and the capacity of hyaluronate oligosaccharides to competitively inhibit the binding of link protein to hyaluronate. Hyaluronate decasaccharide is the oligosaccharide of minimum size that strongly inhibits the binding of link protein to hyaluronate. Proteoglycan monomers dissociate from hyaluronate as the pH is decreased from pH 7 to pH 5. Turbidimetric studies show that the rate of binding of link protein to hyaluronate increases with decreasing pH. The binding affinity of proteoglycan monomers for hyaluronate is decreased at pH 5, whereas the binding affinity of link protein for hyaluronate is not. This difference in the effect of pH on the stability of binding of link protein to hyaluronate, compared with proteoglycan monomer, explains in part the capacity of link protein to stabilize the binding of proteoglycan monomer to hyaluronate at pH 5.     

A comparison study of the interaction between β-lactoglobulin and retinol at two different conditions: spectroscopic and molecular modeling approaches

The interaction between bovin β-Lactoglobulin (β-LG) and retinol at two different pH values was investigated by multispectroscopic, zeta potential, molecular modeling, and conductometry measurements. The steady state and polarization fluorescence spectroscopy revealed that complex formation at two different pH values could occur through a remarkable static quenching. According to fluorescence quenching, one set of binding site at pH 2 and two sets of binding sites at pH 7 were introduced for binding of retinol to β-LG that show the enhancement of saturation score of β-LG to retinol in dimmer condition. The polarization fluorescence analysis represented that there is more affinity between β-LG and retinol at pH 7 rather than at pH 2. The effect of retinol on β-LG was studied by UV-visible, circular dichroism (CD), and synchronous fluorescence, which indicated that retinol induced more structural changes on β-LG at pH 7. β-LG–retinol complex formation at two different pH values was recorded via applying resonance light scattering (RLS) and zeta potential. Conductometry and RLS showed two different behaviors of interaction between β-LG and retinol at two different pH values; therefore, dimmer formation played important roles in different behaviors of interaction between β-LG and retinol. The zeta potential was the implied combination of electrostatic and hydrophobic forces which are involved in β-LG–retinol complex at two different pH values, and the hydrophobic interactions play a dominant role in complex formation. Molecular modeling was approved by all experimental results. The acquired results suggested that monomer and dimmer states of β-LG can be induced by retinol with different behaviors.     

Platform design for extraction and isolation of Bromelain: Complex formation and precipitation with carrageenan

The main objective of this work was to investigate for the first time the molecular mechanism of complex formation between bromelain (a positively charged enzyme) and carrageenan (a natural strong polyelectrolyte, negatively charged) using spectroscopy techniques and thermodynamic approaches. The Bromelain-Carrageenan complex showed a maximal non-solubility at pH around 5.1. The solubility was dependent on pH and ionic strength of the medium. To re-dissolve the formed complex, the pH was changed and 500 mM of NaCl was added to the initial solution, proving the columbic mechanism for the formation of non-soluble complex. The formation of the carrageenan-bromelain complex increased in 8 °C the enzyme thermal stability, while its biological activity was not modified. The amount of total enzyme recovered in solution after precipitation with around 0.08% w/v of carrageenan was 85–90%.     

Emulsion-stabilizing properties of chitosan in the presence of whey protein isolate: Effect of the mixture ratio, ionic strength and pH

The emulsion-stabilizing properties of a chitosan preparation were compared as a function of the whey protein isolate/chitosan mixture ratio (WPI/CNI) and the ionic strength (μ), at pH 5.5 and 6.0. At both pH conditions, general decreases in emulsion stability towards charge neutralization flocculation and syneresis were observed at WPI/CNI > 5. This was particularly evident at pH 6.0, due to a lower surface net charge (lower electrostatic stabilization). In counterpart, when μ was increased, the higher load of chitosan at pH 6.0 produced higher stabilities (higher steric stabilization), in spite of comparable decreases of surface net charge at both pH conditions. The transition from soluble to insoluble protein–chitosan complex formation in mixtures at pH 6.0 and WPI/CNI > 5.0 was due to an emulsion destabilization towards syneresis, whereas soluble complex formation at pH 5.5 also produced syneresis. It showed that soluble protein–chitosan adsorbing complex formation prior homogenization is not essentially linked to emulsion stabilization.     

Complex formation of p-boronophenylalanine with some monosaccharides

To increase the solubility of p-boronophenylalanine (p-bpa) in neutral pH solution, the complex formation of p-bpa with some monosaccharides has been studied by 11B-NMR and UV spectroscopy. The complex formation constants (log K) obtained by the UV method in pH 7.4 solution are 2.43 (fructose), 2.19 (mannitol), 1.28 (galactose), 1.10 (mannose), and 0.85 (glucose), respectively. One hundred milligrams of p-bpa is able to dissolve in 3 ml of 0.3 M fructose solution at pH 7.98. Based on the results obtained, the behavior of p-bpa-monosaccharide complexes in vivo after injection of the complex solution is described.     

NADPH binding induced proton ionization as a cause of nonlinear heat capacity changes in glutamate dehydrogenase

Functional group interactions involved in the formation of the glutamate dehydrogenase-NADPH binary complex have been studied by three independent but complementary approaches: the pH dependence of the overall dissociation constant measured by an improved differential spectroscopic technique; the pH dependence of the enthalpy of complex formation measured by flow calorimetry; and the pH dependence of the number of protons released to, or taken up from, the solvent in the complex formation reaction, measured by titration. We conclude that the coenzyme binds to the enzyme through three distinguishable interactions: a pH-independent process involving the binding of the reduced nicotinamide ring; a relatively weak "proton-stabilizing" process, occurring at low pH involving the shift at a pK of 6.3 in the free enzyme to 7.0 in the enzyme-NADPH complex; and a stronger "proton-destabilizing" process, occurring at a higher pH involving a shift of a pK of 8.5 in the enzyme down to 6.9 in the enzyme-NADPH complex. The proton ionization of the free enzyme involved in this third interaction exhibits some unusual thermodynamic parameters, having delta Go = +11.5 +/- 0.1 kcal mol-1, delta Ho = +19 +/- 1 kcal mol-1, and delta So = +23 eu. We show here that this proton ionization step is directly related to and indeed constitutes the "implicit" shift in enzyme macrostates which we have shown to be responsible for the existence of large highly nonlinear delta Cpo effects in the formation of this complex [Fisher, H. F., Colen, A. H., & Medary, R. T. (1981) Nature (London) 292, 271-272].     

Kinetic studies on a 15-hydroxyprostaglandin dehydrogenase from human placenta.

Kinetic studies have shown that the reaction catalyzed by the human placental 15-hydroxyprostaglandin dehydrogenase proceeds by a single displacement mechanism. Addition of the reactants is ordered with NAD⁺ binding first. The lifetime of the ternary complex is affected by the pH of the reaction mixture. At pH 7.0 a kinetically significant ternary complex is formed, while at pH 9.0 the ternary complex is not kinetically significant (Theorell-Chance mechanism). There is evidence for the occurrence of a kinetically significant isomerization of the enzyme · NADH complex at pH 9.0 but not at pH 7.0. At high substrate concentrations there is formation of unreactive complexes between the 15-hydroxyrostaglandin and both the free enzyme and enzyme · NADH complex and between the 15-ketoprostaglandin and both the free enzyme and enzyme · NAD⁺ complex. The inhibition of the 15-hydroxyprostaglandin dehydrogenase by various prostaglandins and prostaglandin analogs may be explained by the formation of similar unreactive complexes. Certain prostaglandin analogs, arachidonic acid, and ethacrynic acid also affect the activity of the enzyme by causing its irreversible inactivation.     

Complex formation between polyadenylic acid and formycin B

Polyadenylic acid forms a 2:1 complex with the C-nucleoside formyein B at both pH 7.0, 0.15 m-Na⁺ and pH 6.0, 0.15 M-Na⁺. The formation of this complex has been followed by equilibrium dialysis, and by optical rotatory dispersion measurements in the range 333 to 450 nm. At pH 7, melting curves for thermal dissociation of the complex (followed by the optical rotation at 345 nm) show a strongly co-operative helix-coil transition. From the variation of T_m with the free formyein B concentration at this temperature, the partial molar enthalpy of formation of the complex, at the mid-point of the transition, has been calculated as -12.8 kcal./mol of formyein B. Viscometry and optical rotatory dispersion measurements indicate that the structure of the complex at pH 6 is the same as at pH 7, and that it may be formed in preference to the double-helical acid form of poly (A). The structure and properties of the complex are discussed.     

Kinetics of iron and copper catalysis of ascorbate oxidation.

The effects of oxidant, pH and ligands on iron- and copper-catalyzed ascorbate oxidation have been examined. The formation of the catalyst-substrate complex is affected by pH, whereas oxidant affects its breakdown. With copper-ion catalysis, ligands inhibit competitively. With iron catalysis, on the other hand, for a series of aminopolycarboxylic ligands at neutral pH, formation of catalyst-substrate is favored by ligands which form more stable iron complexes. Decreased rates caused by changes in metal environment (ligand or pH) may result for competing activities (e.g., catalase activity competing with peroxidase activity). Evidence for a ternary complex (catalyst-substrate-oxidant) is presented.     

Electrostatic properties of protein-protein complexes

Statistical electrostatic analysis of 37 protein-protein complexes extracted from the previously developed database of protein complexes (ProtCom, http://www.ces.clemson.edu/compbio/protcom) is presented. It is shown that small interfaces have a higher content of charged and polar groups compared to large interfaces. In a vast majority of the cases the average pKa shifts for acidic residues induced by the complex formation are negative, indicating that complex formation stabilizes their ionizable states, whereas the histidines are predicted to destabilize the complex. The individual pKa shifts show the same tendency since 80% of the interfacial acidic groups were found to lower their pKas, whereas only 25% of histidines raise their pKa upon the complex formation. The interfacial groups have been divided into three sets according to the mechanism of their pKa shift, and statistical analysis of each set was performed. It was shown that the optimum pH values (pH of maximal stability) of the complex tend to be the same as the optimum pH values of the complex components. This finding can be used in the homology-based prediction of the 3D structures of protein complexes, especially when one needs to evaluate and rank putative models. It is more likely for a model to be correct if both components of the model complex and the entire complex have the same or at least similar values of the optimum pH.     

How does dextran sulfate prevent heat induced aggregation of protein? The mechanism and its limitation as aggregation inhibitor

The effect of dextran sulfate on protein aggregation was investigated to provide the clues of its biochemical mechanism. The interaction between dextran sulfate and BSA varied with the pH values of the solution, which led to the different extent of aggregation prevention by dextran sulfate. Light scattering data with thermal scan showed that dextran sulfate suppressed BSA aggregation at pH 5.1 and pH 6.2, while it had no effect at pH 7.5. Isothermal titration calorimetric analysis suggested that the pH dependency of the role of dextran sulfate on BSA aggregation would be related to the difference in the mode of BSA-dextran sulfate complex formation. Isothermal titration calorimetric analysis at pH 6.2 indicated that dextran sulfate did not bind to native BSA at this pH, but interacted with partially unfolded BSA. While stabilizing native form of protein by the complex formation has been suggested as the suitable mechanism of preventing aggregation, our observation of conformational changes by circular dichroism spectroscopy showed that strong electrostatic interaction between dextran sulfate and BSA rather facilitated the denaturation of BSA. Combining the data from isothermal titration calorimetry, circular dichroism, and dynamic light scattering, we found that the complex formation of the intermediate state of denatured BSA with dextran sulfate is a prerequisite to suppress the aggregation by preventing further oligomerization/aggregation process of denatured protein.     

Effect of pH and the binding anion on In3+-transferrin interaction: spectroscopic study of the perturbed angular correlation of 172-245 keV gamma rays of indium 111

The formation of ternary complexes, transferrin-anion-In111 has been investigated by means of gamma-gamma coincidence spectrometry of the 172-245 keV rays. The angular correlation between the two gamma-rays emitted in cascade depends on the magnetic and electric fields gradients, consequently the chemical structure of metal holder. Any modification of this structure causes the variation of angular correlation. The study of G22 (infinity) as function of pH (G22(infinity): integrated perturbed angular correlation coefficient) has been performed to turn out the hydrolysis of In111 in aqueous solution, metal complex formation in presence of chelating agents (citric acid and sodium bicarbonate) and the formation of protein-metal complexes. The presence of complexing agents limits the domain of In111 colloid existence and allows fast transfer of ionised indium on the transferrin. Two types of metal-protein interactions has been turn out. The first in the weakly acidic range of pH is characterized by an affinity constant near to this of citric acid. The second lying in neutral and basic range of pH, where the formation rate of transferrin-In111 complex is fast (t less than 500 s). In citrate medium, for pH 6-7,5 the rate of metal transfer on the protein, studied by means of G22 (infinity) = f(t), is function of pH. The binding anion appears as an indispensable element for the formation of protein-metal complexes. The In111 previously chelated by 8-Hydroxyquinoline is fixed by the protein if only exits a binding anion in the solution. This mays bring in the formation of an intermediate active state, indispensable step for the ternary complex formation transferrin-anion-In111.     

Transient removal of proflavine inhibition of bovine beta-trypsin by the bovine basic pancreatic trypsin inhibitor (Kunitz). A case for "chronosteric effects"

The formation of the bovine beta-trypsin-bovine basic pancreatic trypsin inhibitor (Kunitz) (BPTI) complex was monitored, making use of three different signals: proflavine displacement, optical density changes in the ultraviolet region, and the loss of the catalytic activity. The rates of the reactions indicated by the three different signals were similar at neutral pH, but diverged at low pH. At pH 3.50, proflavine displacement precedes the optical density changes in the ultraviolet and the loss of enzyme activity by several orders of magnitude in time (Antonini, E., Ascenzi, P., Menegatti, E., and Guarneri, M. (1983) Biopolymers 22, 363-375). These data indicated that the bovine beta-trypsin-BPTI complex formation is a multistage process and led to the prediction that, at pH 3.50, BPTI addition to the bovine beta-trypsin-proflavine complex would remove proflavine inhibition and the enzyme would recover transiently its catalytic activity before being irreversibly inhibited by completion of BPTI binding. The kinetic evidences, by completion of BPTI binding. The kinetic evidences, here shown, verified this prediction, indicating that during the bovine beta-trypsin-BPTI complex formation one transient intermediate occurs, which is not able to bind proflavine but may bind and hydrolyze the substrate. Thus, the observed peculiar catalytic behavior is in line with the proposed reaction mechanism for the bovine beta-trypsin-BPTI complex formation, which postulates a sequence of distinct polar and apolar interactions at the contact area.