An investigation into the altered binding mode of green tea polyphenols with human serum albumin on complexation with copper

Green tea is rich in several polyphenols, such as (?)-epicatechin-3-gallate (ECG), (?)-epigallocatechin (EGC), and (?)-epigallocatechin-3-gallate (EGCG). The biological importance of these polyphenols led us to study the major polyphenol EGCG with human serum albumin (HSA) in an earlier study. In this report, we have compared the binding of ECG, EGC, and EGCG and the Cu(II) complexes of EGCG and ECG with HSA. We observe that the gallate moiety of the polyphenols plays a crucial role in determining the mode of interaction with HSA. The binding constants obtained for the different systems are 5.86?±?0.72?×?10⁴ M^?1 (K _ECG-HSA), 4.22?±?0.15?×?10⁴ M^?1 (K _{ECG-Cu(II)-HSA}), and 9.51?±?0.31?×?10⁴ M^?1 (K _{EGCG-Cu(II)-HSA}) at 293?K. Thermodynamic parameters thus obtained suggest that apart from an initial hydrophobic association, van der Waals interactions and hydrogen bonding are the major interactions which held together the polyphenols and HSA. However, thermodynamic parameters obtained from the interactions of the copper complexes with HSA are indicative of the involvement of the hydrophobic forces. Circular dichroism and the Fourier transform infrared spectroscopic measurements reveal changes in α-helical content of HSA after binding with the ligands. Data obtained by fluorescence spectroscopy, displacement experiments along with the docking studies suggested that the ligands bind to the residues located in site 1 (subdomains IIA), whereas EGC, that lacks the gallate moiety, binds to the other hydrophobic site 2 (subdomain IIIA) of the protein.     

Properties and characteristics of an anti-human chorionic gonadotropin monoclonal antibody

The product of a hybrid cell clone, P₃W₈₀, obtained as ascites fluid from mouse peritoneal cavity had high titres of anti-human chorionic gonadotropin antibodies e.g. 30 to 40% binding of¹²⁵I-human chorionic gonadotropin at 10⁷ dilution in a radioimmunoassay. The antiserum SB₆ (raised against Β-human chorionic gonadotropin distributed by National Institutes of Health, USA gave similar binding at 5000 dilution in parallel runs. The monoclonal antibody recognized best human chorionic gonadotropin (0.3 mlU of hormone/tube withB/B ₀ 75%), but also bound Β and a subunits of human chorionic gonadotropin, 12 and 800 folds lower than human chorionic gonadotropin respectively No binding was observed with carboxy terminal peptides of Β-human chorionic gonadotropin ranging from 93 to 145 amino acid residues, indicating the lack of recognition of the C-terminal region. No cross-reaction with human leutinizing hormone was obtained at the physiological surge levels, a significant competition (B/B ₀75 %. obtainable only at 60 mlU of LER 960 human leutinizing hormone/ tube. The antibody had heavy chain of IgG₁ and light chain of kappa type. It neutralized the bio-activity of human chorionic gonadotropin bothin vitro and invivo.     

Nucleotides do not modulate rat luteocyte human chorionic gonadotropin responsiveness by inhibiting human chorionic gonadotropin binding

Nucleotide inhibition of ¹²⁵I-labeled human chorionic gonadotropin binding to luteocyte receptor was studied by investigating effects of nucleotides on the apparent equilibrium association constant (K_a) and number of binding sites (B_max), and on rate constants for association (k₊₁) and dissociation (k_?1, k_?2). K_aandB_max were determined by various analyses of equilibrium binding data using washed 2000g pellet of an ovarian homogenate from rats 7 days after pregnant mare's serum gonadotropin-human chorionic gonadotropin priming. Adenyl and guanyl nucleotides, as well as other nucleotides, lowered the K_a of ¹²⁵I-labeled human chorionic gonadotropin binding to luteocyte receptor without affecting B_max. The degree of inhibition was dose related at nucleotide concentrations greater than 10^?3 m. GTP and guanyl-5′-ylimidodiphosphate inhibitions were similar in the presence or absence of EDTA (1.25 × 10^?3 m). ATP and GTP lowered K_a by slowing the rate of association. Inhibition of binding could not be demonstrated at lower nucleotide concentrations even when luteocyte membranes were purified partially by sucrose density gradient ultracentrifugation. In light of the high nucleotide concentrations required to inhibit ¹²⁵I-labeled human chorionic gonadotropin binding and the inhibition by Mg²⁺ and PP₁ at similar concentrations, the effect appears to be a nonspecific ionic effect. Therefore, in contrast to the glucagon-hepatocyte system, luteocyte human chorionic gonadotropin responsiveness does not appear to be modulated by nucleotide inhibition of human chorionic gonadotropin-receptor interaction.     

Influence of mass transfer and surface ligand heterogeneity on quantitative BIAcoreTM binding data. Analysis of the interaction of NC10 Fab with an anti-idiotype Fab′

The interaction of monovalent Fab fragments of NC10, an antiviral neuraminidase antibody, and the anti-idiotype antibody 3-2G12 has been used as a model system to demonstrate experimentally the influence of non-ideal binding effects on BIAcore^TM binding data. Because the association rate constant for these two molecules was found to be relatively high (about 5×10⁵ M ⁻¹ s⁻¹), mass transfer was recognised as a potential source of error in the analysis of the interaction kinetics. By manipulation of the flow rate and the surface density of the immobilised ligand, however, the magnitude to this error was minimised. In addition, the application of site-specific immobilisation procedures was found to improve considerably the correlation of experimental binding data to the ideal 1:1 kinetic model such that the discrepancy between experimental and fitted curves was within the noise range of the instrument. Experiments performed to measure the equilibrium constant (K_D) in solution resulted in a value of similar magnitude to those obtained from the ratio of the kinetic rate constants, even those measured with a heterogeneous ligand or with a significant mass transfer component. For this system, the experimental complexities introduced by covalent immobilisation did not lead to large errors in the K_D values obtained using the BIAcore © 1997 John Wiley & Sons, Ltd.     

Thermodynamics of hCG–monoclonal antibody interaction: an analysis of real time kinetics data obtained using radiolabeled hCG probe

A thermodynamic analysis of the interaction of ¹²⁵I-labeled human chorionic gonadotropin (IhCG) with two of its monoclonal antibodies (MAbs) was carried out. The dissociation profile of IhCG–MAb complex conforms to a two-step model. vant Hoff enthalpies were calculated with the K_A (equilibrium constant) values obtained from dissociation at different temperatures. Free energy and entropy changes were calculated using the standard equations. ΔH values for one of the MAbs, viz. VM7 were favorable at temperatures beyond 30 °C. Interestingly, the ΔS values were also favorable at all temperatures. In the case of MAb VM4a, however, the interaction throughout the temperature range was driven by large favorable entropic contributions, indicating the importance of hydrophobic interaction in the binding of this MAb to hCG. The energetics of the interaction of these two monoclonals with hCG is discussed.     

Isolation and characterization of monoclonal antibodies to (Na + K)-ATPase

Four stable hybridoma cell lines secreting antibodies specific to the membrane (Na⁺ + K⁺)-dependent ATPase isolated from lamb kidney medulla have been produced by fusing mouse myeloma cells with spleen cells from immunized mice. These cell lines produce IgG γ₁ heavy chain and κ light chain antibodies which are directed against the catalytic or α-subunit of the (Na⁺ + K⁺)-ATPase enzyme. Binding studies, using antibodies that were produced by growing hybridomas in vivo and purified by affinity column chromatography, suggest a somewhat higher affinity of these antibodies for the isolated α-subunit than for the ‘native’ holoenzyme. In addition, these monoclonal antibodies show no reactivity with either the glycoprotein (β) subunit of the lamb enzyme nor the (Na⁺ + K⁺)-ATPase from rat kidney, an ouabain-insensitive organ. Cotitration binding experiments have shown that the antibodies from two cell lines originally isolated independently from the same culture plate well population of fused cells bind to the same determinant site and are probably the same antibody. Cotitration and competition binding studies with two other antibodies have revealed two additional distinct antibody binding sites which appear to have little overlap with the first site. One of the three different antibodies isolated caused a partial inhibition of the (Na⁺ + K⁺)-ATPase activity. This antibody appears to be directed against a specific functionally important site of the α-subunit and is a competitive inhibitor of ATP binding. Under optimum conditions of ATPase activity, this inhibitory effect is not altered by the presence of the other two antibodies.     

Follitropin receptors in rat testis characterization with enzymatically 125I-labeled human follitropin

The interaction between enzymatically radioiodinated human follitropin and the follitropin receptors in testis homogenate was investigated in immature and adult rats. The ¹²⁵I-labeled human follitropin exhibited high binding activity, with specific binding of up to 17% in the presence of an excess of testis homogenate.Approx. 50% of the bound hormone could be eluted at pH 5, and the receptor purified tracer exhibited a 3.6-fold increase in binding activity when compared with the original tracer preparation. Quantitative analysis of equilibrium binding data was performed with corrections for the measured specific activity and maximum binding activity of the tracer hormone. The equilibrium association constants (K_a) determined at 24°C were not significantly different in immature and adult rat testis, and the mean value for K_a was 3.9 · 10⁹ M^?1. At 37°C, the K_a value obtained using immature rat testis was 1.3 · 10¹⁰ M^?1. The association of ¹²⁵I-labeled human follitropin with immature rat testis homogenate was time and temperature dependent. In the presence of an excess of unlabeled hormone, 30–60% of the preformed hormone · receptor complex was dissociated after 24 h incubation. A specific and sensitive radioligand-receptor assay for follitropin was developed using immature rat testis homogenate. The minimum detectable dose of purified human follitropin was 0.6 ng, and human urinary and pituitary follitropin, ovine follitropin and pregnant mare serum gonadotropin reacted in the assay with equivalent slopes. The potencies of highly purified pregnant mare serum gonadotropin and highly purified human follitropin were similar in the radioligand-receptor assay, consistent with the follitropin bioactivity of the equine gonadotropin.     

Efforts in diagnosing early leprosy using serological techniques

Skin scrapings obtained from the lesions of leprosy patients of all types showed 96 % positivity to the serum antibody competition test using monoclonal antibody (ML04)to 35 kDa antigen of Mycobacterium leprae. Further, in vitro culture of full thickness skin biopsies from lepromatous patients were noted to release IgG antibodies toM. leprae with a peak antibody response at 48 h. The significance of this local antibody response toM. leprae in skin has been discussed for its possible use in diagnosing early leprosy.     

The thermodynamic parameters of the interaction of concanavalin A with glycosyl-free liposomes: a microcalorimetric study

The nonspecific interaction of the mitogenic lectin Concanavalin A (Con A) with glycosyl-free liposomes of various composition has been investigated by microcalorimetric titration measurements. The results obtained show the following features of main interest: (1) the affinity constants (K_a) of the interaction of Con A with liposomal bilayers are in the order of magnitude 10⁵–10⁶M^?1. The reaction enthalpies (ΔH) are positive, and small (approximately 0.1 KJ mol^?1 lipid), compared to the free energy terms (?ΔG = 30–40 KJ mol^?1 lipid). All lectin–lipid interactions are strongly entropy-controlled (ΔH/TΔS < 1.0). These thermodynamic features are characteristic for hydrophobic interaction processes. (2) The liposomal head-group charge does not significantly affect the lipid-affinity of Con A. Electrostatic forces thus appear to play a minor role in lectin–lipid interactions. (3) The lipid affinity of Con A is sensitive to the fluidity of the liposomal bilayers, increasing with increasing fluidity. Below the gel to liquid-crystal phase transition temperature, the lectin binding to liposomal bilayers is inhibited. (4) The binding isotherms, corresponding to the interaction of Con A with liposomes, composed of tightly packed, saturated phospholipids, exhibit pronounced positive cooperativity. This phenomenon is absent in the binding curves, corresponding to the interaction of Con A with more fluid liposomal bilayers. (5) The Con A specific inhibitor α-D -methylmannopyranoside (50 mM) drastically increases the molar reaction enthalpy. The K_a term is significantly reduced in presence of the inhibitor sugar. Urea induces analogous changes in the thermodynamic parameters of the lectin–lipid interaction. The effects of α-D -methylmannopyranoside are thus not Con A specific, but are attributable to solvent effects. (6) It was shown that the binding of one Con A molecule affects a large number (approximately 1000) of phospholipid molecules in the liposomal bilayer. (7) The affinity constants (K_a) of the interaction of Con A with glycosyl-free lipids are smaller by a factor of approximately 10, compared to the K_a terms, reported for Con A binding to biological membranes. The presence of glycosidic receptor groups thus controls the specificity of lectin–membrane interactions, whereas the nonspecific lectin–lipid interactions appear to represent the main driving force for the strong attachment of the lectin to membrane surfaces.     

Allosteric property of the (Na+K)-ATPase β1 subunit

(Na⁺+K⁺)-ATPase (NKA) comprises two basic α and β subunits: The larger α subunit catalyzes the hydrolysis of ATP for active transport of Na⁺ and K⁺ ions across the plasma membrane; the smaller β subunit does not take part in the catalytic process of the enzyme. Little is known about allosteric regulation of the NKA β subunit. Here, we report a surprising finding that extracellular stimuli on the native β₁ subunit can generate a significant impact on the catalytic function of NKA. By using a β₁ subunit-specific monoclonal antibody JY2948, we found that the JY2948–β₁ subunit interaction markedly enhances the catalytic activity of the enzyme and increases the apparent affinity of Na⁺ and K⁺ ions for both ouabain-resistant rat NKA and ouabain-sensitive dog NKA. This study provides the first evidence to identify an allosteric binding site residing on the NKA β₁ subunit and uncovers the latent allosteric property of the β₁ subunit, which remotely controls the NKA catalytic function.     

A comparison of some activated matrices for preparation of immunosorbents

The adsorption characteristics of monoclonal anti-(β-galactosidase) immobilised to a number of commercially available pre-activated matrices have been investigated in a series of small scale experiments. Binding characteristics were determined by batch isotherm techniques and estimates were obtained of the rate constants governing adsorption to the immobilised antibodies. The capacity of the different matrices for binding antibody and the specific activity of immunosorbents were measured.There was little effect of support matrix on the dissociation constant, K_d, for the interaction between β-galactosidase and immobilised anti-(β-galactosidase). However, the maximum amounts of antibody that could be immobilised, rates of adsorption and desorption of the enzyme to the immobilised antibody and the specific activity of immunosorbents were affected by the choice of support matrix. The importance of the relative sizes of the antigen and immobilised antibody and the influence of the nature of the support matrix on the properties of the resulting immunosorbent when used in large scale applications are discussed.     

Spectroscopic investigation on the toxic interaction of melamine with herring sperm DNA

The toxic interaction of melamine with herring sperm DNA (hs‐DNA) was investigated by using fluorescence and UV–vis absorption spectra techniques. The experimental results showed that the toxic interaction between melamine and hs‐DNA occurred. Fluorescence quenching experiments indicated the existence of electrostatic binding between melamine and hs‐DNA. The binding constants K_A and the binding site numbers were calculated by means of the Stern–Volmer equation and were 9.8 × 10⁴ L mol^?1 and 1.3, respectively. Both the results of fluorescence spectra and UV–vis absorption spectra verified that there are electrostatic binding between melamine and hs‐DNA. The possibility in the presence of a classical intercalation binding mode could be ruled out by using DNA unwinding experiments. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:323–329, 2010; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.20341     

Multi-spectroscopic and molecular docking studies on the interaction of neotame with calf thymus DNA

Abstract

In this paper, we have studied the in vitro binding of neotame (NTM), an artificial sweetener, with native calf thymus DNA using different methods including spectrophotometric, spectrofluorometric, competition experiment, circular dichroism (CD), and viscosimetric techniques. From the spectrophotometric studies, the binding constant (K_b) of NTM-DNA was calculated to be 2?×?10³ M^?1. The quenching of the intrinsic fluorescence of NTM in the presence of DNA at different temperatures was also used to calculate binding constants (K_b) as well as corresponding number of binding sites (n). Moreover, the obtained results indicated that the quenching mechanism involves static quenching. By comparing the competitive fluorimetric studies with Hoechst 33258, as a known groove probe, and methylene blue, as a known intercalation probe, and iodide quenching experiments it was revealed that NTM strongly binds in the grooves of the DNA helix, which was further confirmed by CD and viscosimetric studies. In addition, a molecular docking method was employed to further investigate the binding interactions between NTM and DNA, and confirm the obtained results.     

Comparison of the biological activity of fusicoccin in higher plants with its binding to plasma membranes

The concentration dependences of the binding of fusicoccins (FCs) A, B, C, D, J and H to plasma membranes isolated from maize (Zea mays L.) roots have been studied in parallel with the effects of these compounds on elongation and ⁸⁶Rb transport in detached maize roots. The dissociation constants obtained showed a good correlation between the affinity of the FCs for the plasmalemma and their biological activity. However, the range of physiologically active FC concentrations proved to be about two orders of magnitude higher than that calculated from the dissociation constants. It was also shown that Vicia faba L. mesophyll protoplasts, unlike isolated plasma membranes, have two FC-binding sites, one with a K _D similar to that of the isolated plasmalemma while the other has a substantially higher K _D , apparently corresponding to the physiologically active state of the FC-binding proteins.Abbreviation FC fusicoccin     

Dual activity of the H1-H2 domain of the (Na(+)+K+)-ATPase

(Na⁺+K⁺)-ATPase is a target receptor of digitalis (cardiac glycoside) drugs. It has been demonstrated that the H1-H2 domain of the α-subunit of the (Na⁺+K⁺)-ATPase is one of the digitalis drug interaction sites of the enzyme. Despite the extensive studies of the inhibitory effect of digitalis on the (Na⁺+K⁺)-ATPase, the functional property of the H1-H2 domain of the enzyme and its role in regulating enzyme activity is not completely understood. Here we report a surprise finding: instead of inhibiting the enzyme, binding of a specific monoclonal antibody SSA78 to the H1-H2 domain of the (Na⁺+K⁺)-ATPase elevates the catalytic activity of the enzyme. In the presence of low concentration of ouabain, monoclonal antibody SSA78 significantly protects enzyme function against ouabain-induced inhibition. However, higher concentration of ouabain completely inactivates the (Na⁺+K⁺)-ATPase even in the presence of SSA78. These results suggest that the H1-H2 domain of the (Na⁺+K⁺)-ATPase is capable of regulating enzyme function in two distinct ways for both ouabain-sensitive and -resistant forms of the enzyme: it increases the activity of the (Na⁺+K⁺)-ATPase during its interaction with an activator; it also participates in the mechanism of digitalis or ouabain-induced inhibition of the enzyme. Understanding the dual activity of the H1-H2 domain will help better understand the structure-function relationships of the (Na⁺+K⁺)-ATPase and the biological processes mediated by the enzyme.     

Gonadal receptors I. Evidence for irreversibility in the binding of human chorionic gonadotropin and human luteinizing hormone

The binding of human chorionic gonadotropin and human luteinizing hormone to particulate receptors of rat testes has generally been assumed to follow an equilibrium model similar to that proposed for many enzyme systems. Our work shows that equilibrium dissociation constant (K_d) and number of hormone binding sites (B_max) are highly sensitive to changes in hormone and/ or receptor concentration and to treatment received by tissue or receptor preparation prior to the assay. The results of binding assays obtained using receptor preparation pretreated with hormone (labeled as well as unlabeled) indicated that the binding reaction between hormone and receptor was irreversible and that pretreatment of the tissue with hormone greatly alters the number of high affinity gonadotropin binding sites in the testicular homogenate. Data from studies involving increasing receptor concentrations revealed that increasing the mass of particulate receptors in the binding assays leads to higher K_d as well as B_max values. These findings are incompatible with a binding model based upon occupancy of receptor sites and the state of equilibrium implied. The incompatibilities are analyzed and an alternate model advanced (Bhalla, V.K., Trowbridge, C.G., Chen, C.J.H., Lindeman, J.G. and Rojas, F.J. (1979) Biochim. Biophys. Acta 584, 436–453).     

Interaction Mode between Inclusion Complex of Vitamin K3 with γ- Cyclodextrin and Herring-Sperm DNA

Methods including spectroscopy, electronic chemistry and thermodynamics were used to study the inclusion effect between γ-cyclodextrin (CD) and vitamin K₃(K₃), as well as the interaction mode between herring-sperm DNA (hsDNA) and γ-CD-K₃ inclusion complex. The results from ultraviolet spectroscopic method indicated that VK₃ and γ-CD formed 1:1 inclusion complex, with the inclusion constant K_f = 1.02 × 10⁴ L/mol, which is based on Benesi–Hildebrand's viewpoint. The outcomes from the probe method and Scatchard methods suggested that the interaction mode between γ-CD-K₃ and DNA was a mixture mode, which included intercalation and electrostatic binding effects. The binding constants were K ^θ_25°C = 2.16 × 10⁴ L/mol, and K^θ_37°C = 1.06 × 10⁴ L/mol. The thermodynamic functions of the interaction between γ-CD-K₃ and DNA were Δ_rH_m^θ = ?2.74 × 10⁴ J/mol, Δ_rS_m^θ = 174.74 J·mol^?1K^?1, therefore, both Δ_rH_m^θ (enthalpy) and Δ_rS_m^θ (entropy) worked as driven forces in this action.     

Studies on regulation of chorionic gonadotropin secretion in primates

The regulation of secretion of chorionic gonadotropin in primates has been studied using bothin vivo andin vitro models.In vivo studies using the pregnant bonnet monkey revealed that at the doses tested, the administration of progesterone or estradiol 17Β in combination or alone did not result in any appreciable change in the duration or magnitude of serum chorionic gonadotropin levels. However, administration of lutropin-releasing hormone by intravenous route resulted in significant increase in chorionic gonadotropin levels within 30–60 min and the extent of stimulation seemed to depend on the state of pregnancy. Forin vitro studies, explants or cells prepared from first trimester human placenta has been used. The functional integrity of these cells has been established by demonstrating the binding of [¹²⁵I]-labelled human chorionic gonadotropin antibody to the cells as well as the synthesis of [³H]-labelled human chorionic gonadotropin.In vitro studies using the cells revealed that addition of lutropin-releasing hormone caused a significant increase in chorionic gonadotropin and estradiol 17Β secreted into the medium. Thus bothin vivo andin vitro results suggest that lutropin-releasing hormone could be one of the factors involved in regulation of chorionic gonadotropin secretion in primates.     

Detection and characterization of weak affinity antibody antigen recognition with biomolecular interaction analysis

In biological systems, weak-affinity interactions (association constant, K_a, of less than approximately 10⁴ M ⁻¹) between biomolecules are common and essential to the integrity of such units. However, studies of weak biological interactions are difficult due to the scarcity of analytical methods available for the bioscientist. In this communication, we report on the use of biosensors based on surface plasmon resonance to detect and characterize weak affinity antibody–antigen interactions. Monoclonal antibodies towards carbohydrate antigens were immobilized on sensor surfaces and were used to detect weak binding of the carbohydrate tetraglucose of dissociation constant, K_d, in the millimolar range. Sensorgrams were received in the form of square pulses where the kinetic rate constants were difficult to assess due to the rapid association and dissociation of the antigen to/from the immobilized antibody. © 1997 John Wiley & Sons, Ltd.