Analysis of human chorionic gonadotropin-monoclonal antibody interaction in BIAcore

Kinetic studies of macromolecular ligand-ligate interaction have generated ample interest since the advent of plasmon resonance based instruments like BIAcore. Most of the studies reported in literature assume a simple 1 : 1 Langmuir binding and complete reversibility of the system. However we observed that in a high affinity antigen-antibody system [human chorionic gonadotropin-monoclonal antibody (hCG-mAb)] dissociation is insignificant and the sensogram data cannot be used to measure the equilibrium and kinetic parameters. At low concentrations of mAb the complete sensogram could be fitted to a single exponential. Interestingly we found that at higher mAb concentrations, the binding data did not conform to a simple bimolecular model. Instead, the data fitted a two-step model, which may be because of surface heterogeneity of affinity sites. In this paper, we report on the global fit of the sensograms. We have developed a method by which a single two-minute sensogram can be used in high affinity systems to measure the association rate constant of the reaction and the functional capacity of the ligand (hCG) immobilized on the chip. We provide a rational explanation for the discrepancies generally observed in most of the BIAcore sensograms

Sequence Specific Interaction between Promoter DNA and Escherichia coli RNA Polymerase: Comparative Thermodynamic Analysis with One Immobilized Partner

Sequence specific interaction between DNA and protein molecules has been a subject of active investigation for decades now. Here, we have chosen single promoter containing bacteriophage Delta D-III T7 DNA and Escherichia coli RNA polymerase and followed their recognition at the air-water interface by using the surface plasmon resonance (SPR) technique, where the movement of one of the reacting species is restricted by way of arraying them on an immobilized support. For the Langmuir monolayer studies, we used a RNA polymerase with a histidine tag attached to one of its subunits, thus making it an xcellent substrate for Ni(II) ions, while the SPR Studies were done using biotin-labeled DNA immobilized on a streptavidin-coated chip. Detailed analysis of the thermodynamic parameters as a function of concentration and temperature revealed that the interaction of RNA polymerase with T7 DNA is largely entropy driven (83 (+/- 12) kcal mol(-1)) with a positive enthalpy of 13.6 (+/- 3.6) kcal mol(-1), The free energy of reaction determined by SPR and Langmuir-Blodgett technique was -11 (+/- 2) and -15.6 kcal mol(-1), respectively. The ability of these methods to retain the specificity of the recognition process was also established.

Synthetic arabinomannan glycolipids and their effects on growth and motility of the Mycobacterium smegmatis

Arabinomannan-containing glycolipids, relevant to the mycobacterial cell-wall component lipoarabinomannan, were synthesized by chemical methods. The glycolipids were presented with tri- and tetrasaccharide arabinomannans as the sugar portion and a double alkyl chain as the lyophilic portion. Following synthesis, systematic biological and biophysical studies were undertaken in order to identify the effects of the glycolipids during mycobacterium growth. The studies included mycobacterial growth, biofilm formation and motility assays. From the studies, it was observed that the synthetic glycolipid with higher arabinan residues inhibited the mycobacterial growth, lessened the biofilm formation and impaired the motility of mycobacteria. A surface plasmon resonance study involving the immobilized glycan surface and the mycobacterial crude lysates as analytes showed specificities of the interactions. Further, it was found that cell lysates from motile bacteria bound oligosaccharide with higher affinity than non-motile bacteria.

A kinetic analysis of the tumor-associated galactopyranosyl-(1→3)-2-acetamido-2-deoxy-α-d-galactopyranoside antigen—lectin interaction

A kinetic study of the tumor-associated galactopyranosyl-(1→3)-2-acetamido-2-deoxy-α-d-galactopyranoside (T-antigen) with lectin peanut agglutinin is described. The disaccharide antigen was synthesized by chemical methods and was functionalized suitably for immobilization onto a carboxy-methylated sensor chip. The ligand immobilized surface was allowed interaction with the lectin peanut agglutinin, which acted as the analyte and the interaction was studied by the surface plasmon resonance method. The ligand—lectin interaction was characterized by the kinetic on-off rates and a bivalent analyte binding model was found to describe the observed kinetic constants. It was identified that the antigen-lectin interaction had a faster association rate constant (k a1) and a slower dissociation rate constant (k d1) in the initial binding step. The subsequent binding step showed much reduced kinetic rates. The antigen-lectin interaction was compared with the kinetic rates of the interaction of a galactopyranosyl-(1→4)-β-d-galactopyranoside derivative and a mannopyranoside derivative with the lectin.

Differential Mechanisms of Binding of Anti-Sigma Factors Escherichia coli Rsd and Bacteriophage T4 AsiA to E. coli RNA Polymerase Lead to Diverse Physiological Consequences

Anti-sigma factors Escherichia coli Rsd and bacteriophage T4 AsiA bind to the essential housekeeping sigma factor, sigma(70), of E. coli. Though both factors are known to interact with the C-terminal region of sigma(70), the physiological consequences of these interactions are very different. This study was undertaken for the purpose of deciphering the mechanisms by which E. coli Rsd and bacteriophage T4 AsiA inhibit or modulate the activity of E. coli RNA polymerase, which leads to the inhibition of E. coli cell growth to different amounts. It was found that AsiA is the more potent inhibitor of in vivo transcription and thus causes higher inhibition of E. coli cell growth. Measurements of affinity constants by surface plasmon resonance experiments showed that Rsd and AsiA bind to or 70 with similar affinity. Data obtained from in vivo and in vitro binding experiments clearly demonstrated that the major difference between AsiA and Rsd is the ability of AsiA to form a stable ternary complex with RNA polymerase. The binding patterns of AsiA and Rsd with sigma(70) studied by using the yeast two-hybrid system revealed that region 4 of sigma(70) is involved in binding to both of these anti-sigma factors; however, Rsd interacts with other regions of sigma(70) as well. Taken together, these results suggest that the higher inhibition of E. coli growth by AsiA expression is probably due to the ability of the AsiA protein to trap the holoenzyme RNA polymerase rather than its higher binding affinity to sigma(70).

Kinetics and the Mechanism of Interaction of the Endoplasmic Reticulum Chaperone, Calreticulin, with Monoglucosylated (Glc1Man9GlcNAc2) Substrate

Calreticulin is a lectin-like molecular chaperone of the endoplasmic reticulum in eukaryotes. Its interaction with N-glycosylated polypeptides is mediated by the glycan, Glc(1)Man(9)GlcNAc(2), present on the target glycoproteins. In this work, binding of monoglucosyl IgG (chicken) substrate to calreticulin has been studied using real time association kinetics of the interaction with the biosensor based on surface plasmon resonance (SPR). By SPR, accurate association and dissociation rate constants were determined, and these yielded a micromolar association constant. The nature of reaction was unaffected by immobilization of either of the reactants. The Scatchard analysis values for K-a agreed web crith the one obtained by the ratio k(1)/k(-1). The interaction was completely inhibited by free oligosaccharide, Glc(1)Man(9)GlcNAc(2), whereas Man(9)GlcNAc(2) did not bind to the calreticulin-substrate complex, attesting to the exquisite specificity of this interaction. The binding of calreticulin to IgG was used for the development of immunoassay and the relative affinity of the lectin-substrate association was indirectly measured. The values are in agreement with those obtained with SPR. Although the reactions are several orders of magnitude slower than the diffusion controlled processes, the data are qualitatively and quantitatively consistent with single-step bimolecular association and dissociation reaction. Analyses of the activation parameters indicate that reaction is enthalpically driven and does not involve a highly ordered transition state. Based on these data, the mechanism of its chaperone activity is briefly discussed.

Kinetics and the mechanism of interaction of the endoplasmic reticulum chaperone, calreticulin, with monoglucosylated (Glc(1)Man(9)GlcNAc(2)) substrate

Calreticulin is a lectin-like molecular chaperone of the endoplasmic reticulum in eukaryotes. Its interaction with N-glycosylated polypeptides is mediated by the glycan, Glc(1)Man(9)GlcNAc(2), present on the target glycoproteins. In this work, binding of monoglucosyl IgG (chicken) substrate to calreticulin has been studied using real time association kinetics of the interaction with the biosensor based on surface plasmon resonance (SPR). By SPR, accurate association and dissociation rate constants were determined, and these yielded a micromolar association constant. The nature of reaction was unaffected by immobilization of either of the reactants. The Scatchard analysis values for K-a agreed web crith the one obtained by the ratio k(1)/k(-1). The interaction was completely inhibited by free oligosaccharide, Glc(1)Man(9)GlcNAc(2), whereas Man(9)GlcNAc(2) did not bind to the calreticulin-substrate complex, attesting to the exquisite specificity of this interaction. The binding of calreticulin to IgG was used for the development of immunoassay and the relative affinity of the lectin-substrate association was indirectly measured. The values are in agreement with those obtained with SPR. Although the reactions are several orders of magnitude slower than the diffusion controlled processes, the data are qualitatively and quantitatively consistent with single-step bimolecular association and dissociation reaction. Analyses of the activation parameters indicate that reaction is enthalpically driven and does not involve a highly ordered transition state. Based on these data, the mechanism of its chaperone activity is briefly discussed.

Realtime kinetic analysis of antigen-antibody interaction using solid phase binding: Transformation of hCG-monoclonal antibody complex

Kinetic constants of MAb-hCG interactions have been determined using solid phase binding of I-125[hCG] to immobilized MAb. While association has been shown to follow the expected pattern, dissociation consists of at least two reversible steps, one with a rate constant of 0.0025 min(-1), and a second with a rate constant of 0.00023 min(-1). Validity of affinity constant measurements in the light of the complex reaction kinetics is discussed, A comparison between the method of surface plasmon resonance technology (BIAcore) and solid phase binding (SPB) for determination of kinetic parameters shows that SPB provides not only a cost-effective approach for determination of realtime kinetic parameters of macromolecular ligand-ligate interaction but also a method with several advantages over the BIAcore system in investigating the mechanism of antigen-antibody interaction.

Synthesis of inorganic solids using microwaves

Use of microwaves in the synthesis of materials is gaining importance. Microwave-assisted synthesis is generally much faster, cleaner, and more economical than the conventional methods. A variety of materials such as carbides, nitrides, complex oxides, silicides, zeolites, apatite, etc. have been synthesized using microwaves. Many of these are of industrial and technological importance. An understanding of the microwave interaction with materials has been based on concepts of dielectric heating and of the resonance absorption due to rotational excitation. This review presents a summary of recent reports of microwave synthesis of inorganic materials. Various observations regarding microwave interaction with materials are also briefly discussed.

Real-time kinetic analysis of hCG-monoclonal antibody interaction using radiolabeled hCG probe: presence of two forms of Ag-mAb complex as revealed by solid phase dissociation studies

Real-time kinetics of ligand-ligate interaction has predominantly been studied by either fluorescence or surface plasmon resonance based methods. Almost all such studies are based on association between the ligand and the ligate. This paper reports our analysis of dissociation data of monoclonal antibody-antigen (hCG) system using radio-iodinated hCG as a probe and nitrocellulose as a solid support to immobilize mAb. The data was analyzed quantitatively for a one-step and a two-step model. The data fits well into the two-step model. We also found that a fraction of what is bound is non-dissociable (tight-binding portion (TBP)). The TBP was neither an artifact of immobilization nor does it interfere with analysis. It was present when the reaction was carried out in homogeneous solution in liquid phase. The rate constants obtained from the two methods were comparable. The work reported here shows that real-time kinetics of other ligand-ligate interaction can be studied using nitrocellulose as a solid support. (C) 2002 Elsevier Science B.V. All rights reserved.

Molecular insights into the mechanism of phenotypic tolerance to rifampicin conferred on mycobacterial RNA polymerase by MsRbpA

The protein MsRbpA from Mycobacterium smegmatis rescues RNA polymerase (RNAP) from the inhibitory effect of rifampicin (Rif). We have reported previously that MsRbpA interacts with the beta-subunit of RNAP and that the effect of MsRbpA on Rif-resistant (Rif(R)) RNAP is minimal. Here we attempted to gain molecular insights into the mechanism of action of this protein with respect to its role in rescuing RNAP from Rif-mediated transcription inhibition. Our experimental approach comprised multiple-round transcription assays, fluorescence spectroscopy, MS and surface plasmon resonance in order to meet the above objective. Based on our molecular studies we propose here that Rif is released from its binding site in the RNAP-Rif complex in the presence of MsRbpA. Biophysical studies reveal that the location of MsRbpA on RNAP is at the junction of the beta- and beta'-subunits, close to the Rif-binding site and the (i + 1) site on RNAP.

Synthesis, biological studies of linear and branched arabinofuranoside-containing glycolipids and their interaction with surfactant protein A

Oligoarabinofuranoside-containing glycolipids relevant to mycobacterial cell wall components were synthesized in order to understand the functional roles of such glycolipids. A series of linear tetra-, hexa-, octa-and a branched heptasaccharide oligoarabinofuranosides, with 1 -> 2 and 1 -> 5 a-linkages between the furanoside residues, were synthesized by chemical methods from readily available monomer building blocks. Upon the synthesis of glycolipids, constituted with a double alkyl chain-substituted sn-glycerol core and oligosaccharide fragments, biological studies were performed to identify the effect of synthetic glycolipids on the biofilm formation and sliding motilities of Mycobacterium smegmatis. Synthetic glycolipids and arabinofuranosides displayed an inhibitory effect on the growth profile, but mostly on the biofilm formation and maturation. Similarly, synthetic compounds also influenced the sliding motility of the bacteria. Further, biophysical studies were undertaken, so as to identify the interactions of the glycolipids with a pulmonary surfactant protein, namely surfactant protein A (SP-A), with the aid of the surface plasmon resonance technique. Specificities of each glycolipid interacting with SP-A were thus evaluated. From this study, glycolipids were found to exhibit higher apparent association constants than the corresponding oligosaccharide portion alone, without the double alkyl group-substituted glycerol core.

Self assembly of bivalent glycolipids on single walled carbon nanotubes and their specific molecular recognition properties

Solubilization of single walled carbon nanotubes (SWNTs) in aqueous milieu by self assembly of bivalent glycolipids is described. Thorough analysis of the resulting composites involving Vis/near-IR spectroscopy, surface plasmon resonance, confocal Raman and atomic force microscopy reveals that glycolipid-coated SWNTs possess specific molecular recognition properties towards lectins.

Synthesis of beta-arabinofuranoside glycolipids, studies of their binding to surfactant protein-A and effect on sliding motilities of M. smegmatis

Surfactant protein A (SP-A), which is a lung innate immune system component, is known to bind glycolipids present at the cell surface of a mycobacterial pathogen. Lipoarabinomannan (LAM), a component of mycobacterial thick, waxy cell wall, is one of the glycolipid ligands for SP-A. In order to assess binding of synthetic glycolipids with SP-A and the glycosidic linkage preferences for the interaction, beta-arabinofuranoside trisaccharide glycolipids constituted with beta-(1 -> 2), beta-(1 -> 3) and beta-(1 -> 2), beta-(1 -> 5) linkages relevant to LAM were synthesized through chemical glycosylations. The efficacies of synthetic glycolipids to interact with SP-A were assessed by using the surface plasmon resonance (SPR) technique, from which association-dissociation rate constants and equilibrium binding constants were derived. The equilibrium binding constants of the interaction of two constitutionally varying beta-arabinofuranoside glycolipids with SP-A were found to be in the millimolar range. A comparison of the results with few alpha-anomeric arabinofuranoside glycolipids showed that glycolipids with beta-anomeric linkages were having relatively lower equilibrium binding constants than those with alpha-anomeric linkages in binding to the protein, whereas oligosaccharides alone, without lipidic chains, exhibited higher equilibrium binding constants. Further, the synthetic compounds inhibited the growth of mycobacteria and affected sliding motilities of the bacteria, although to an extent relatively lesser than that of synthetic compounds constituted with alpha-anomeric linkages.

Nanosphere Templated Metallic Grating Assisted Enhanced Fluorescence

In this paper, enhanced fluorescence from a silver film coated nanosphere templated grating is presented. Initially, numerical simulation was performed to determine the plasmon resonance wavelength by varying the thickness of the silver film on top of a monolayer of 400 nm nanospheres. The simulation results are verified experimentally and tested for enhancing fluorescence from fluorescein isothiocyanate whose excitation wavelength closely matches with the plasmon resonance wavelength of the substrate with 100 nm silver film over nanosphere. The 12 times enhancement in the intensity is attributed to the local field enhancement in addition to the excitation of surface plasmon polaritons along the surface.