Saccharide binding to three Gal/GalNAc specific lectins: Fluorescence, spectroscopic and stopped-flow kinetic studies

Fluorescence and stopped-flow spectrophotometric studies on three plant lectins fromPsophocarpus tetragonolobus (winged bean),Glycine max (soybean) andArtocarpus integrifolia (jack fruit) have been studied usingN-dansylgalactosamine as a fluorescent ligand. The best monosaccharide for the winged bean agglutinin I (WBA I) and soybean (SBA) is Me-agrGalNAc and for jack fruit agglutinin (JFA) is Me-agrGal. Examination of the percentage enhancement and association constants (1.51×106, 6.56×106 and 4.17×105 M–1 for SBA, WBA I and JFA, respectively) suggests that the combining regions of the lectins SBA and WBA I are apolar whereas that of JFA is polar. Thermodynamic parameters obtained for the binding of several monosaccharides to these lectins are enthalpically favourable. The binding of monosaccharides to these lectins suggests that the-OH groups at C-1, C-2, C-4 and C-6 in thed-galactose configuration are important loci for interaction with these lectins. An important finding is that the JFA binds specifically to Galß1-3GaINAc with much higher affinity than the other disaccharides which are structurally and topographically similar.The results of stopped-flow spectrometry on the binding ofN-dansylgalactosamine to these lectins are consistent with a bimolecular single step mechanism. The association rate constants (2.4×105, 1.3×104, and 11.7×105 M–1 sec–1 for SBA, WBA I and JFA, respectively) obtained are several orders of magnitude slower than the ones expected for diffusion controlled reactions. The dissociation rate constants (0.2, 3.2×10–2, 83.3 sec–1 for SBA, WBA I and JFA, respectively) obtained for the dissociation ofN-dansylgalactosamine from its lectin complex are slowest for SBA and WBA I when compared with any other lectin-ligand dissociation process.

Photobleaching reduced fluorescence correlation spectroscopy

A dual beam excitation-depletion pulse technique is proposed for photobleaching reduced fluorescence correlation spectroscopy (FCS). Excitation pulse promote the molecules to the excited singlet state (S-1), a fraction of that population goes to energetically favorable metastable triplet state (T-1) due to strong intersystem crossing. The depletion pulse followed by excitation pulse instantaneously depletes the triplet states thereby recycling the bleached molecules back to the ground state (S-0). FCS study on diffusing Fluorescein and Rh6G molecules show more than 95% reduction in triplet state population and the associated photobleaching. (c) 2010 American Institute of Physics.

Studies on nucleotidases in plants: Fluorescence and kinetic properties of nucleotide pyrophosphatase from mung bean (Phaseolus aureus) seedlings

Nucleotide pyrophosphatase of mung bean seedlings has earlier been isolated in our laboratory in a dimeric form (Mr 65,000) and has been shown to be converted to a tetramer by AMP and to a monomer by p-hydroxymercuribenzoate. All the molecular forms were enzymatically active with different kinetic properties. By a modified procedure using blue-Sepharose affinity chromatography, we have now obtained a dimeric form of the enzyme which is desensitized to AMP interaction. The molecular weight of the desensitized form of the enzyme was found to be the same as that of the native dimeric enzyme. However, the desensitized enzyme functioned with a linear time course, contrary to the biphasic time course exhibited by the native enzyme. In addition, it was not converted to a tetramer on the addition of AMP, had only one binding site for adenine nucleotides, and p-hydroxy-mercuribenzoate had no effect on the time course of the reaction or on the molecular weight of the enzyme. The temperature optimum of the desensitized enzyme was found to be 67 °C in contrast to the optimum of 49 °C for the native dimer. Fifty percent of the tryptophan residues of the desensitized enzyme were not accessible for quenching by iodide. Fluorescence studies gave Kd values of 0.34, 2.2, and 0.8 mImage for AMP, ADP, and ATP, which were close to the Ki values of 0.12, 2.2, and 0.9 mImage , respectively, for these nucleotides. The binding and inhibition studies with AMP and its analogs showed that the 6-amino group and the 5′-phosphate group were essential for the inhibition of the enzyme activity.

Microbial identification by detection of ligation probes on DNA microarray

Microbes in natural and artificial environments as well as in the human body are a key part of the functional properties of these complex systems. The presence or absence of certain microbial taxa is a correlate of functional status like risk of disease or course of metabolic processes of a microbial community. As microbes are highly diverse and mostly notcultivable, molecular markers like gene sequences are a potential basis for detection and identification of key types. The goal of this thesis was to study molecular methods for identification of microbial DNA in order to develop a tool for analysis of environmental and clinical DNA samples. Particular emphasis was placed on specificity of detection which is a major challenge when analyzing complex microbial communities. The approach taken in this study was the application and optimization of enzymatic ligation of DNA probes coupled with microarray read-out for high-throughput microbial profiling. The results show that fungal phylotypes and human papillomavirus genotypes could be accurately identified from pools of PCR amplicons generated from purified sample DNA. Approximately 1 ng/μl of sample DNA was needed for representative PCR amplification as measured by comparisons between clone sequencing and microarray. A minimum of 0,25 amol/μl of PCR amplicons was detectable from amongst 5 ng/μl of background DNA, suggesting that the detection limit of the test comprising of ligation reaction followed by microarray read-out was approximately 0,04%. Detection from sample DNA directly was shown to be feasible with probes forming a circular molecule upon ligation followed by PCR amplification of the probe. In this approach, the minimum detectable relative amount of target genome was found to be 1% of all genomes in the sample as estimated from 454 deep sequencing results. Signal-to-noise of contact printed microarrays could be improved by using an internal microarray hybridization control oligonucleotide probe together with a computational algorithm. The algorithm was based on identification of a bias in the microarray data and correction of the bias as shown by simulated and real data. The results further suggest semiquantitative detection to be possible by ligation detection, allowing estimation of target abundance in a sample. However, in practise, comprehensive sequence information of full length rRNA genes is needed to support probe design with complex samples. This study shows that DNA microarray has the potential for an accurate microbial diagnostic platform to take advantage of increasing sequence data and to replace traditional, less efficient methods that still dominate routine testing in laboratories. The data suggests that ligation reaction based microarray assay can be optimized to a degree that allows good signal-tonoise and semiquantitative detection.

A23187—Channel behaviour: Fluorescence study

Pyranine entrapped soylipid liposomes have been used as a model system to study the proton transport across membrane in the presence of A23187, a carboxylic ionophore specific for electroneutral exchange of divalent cations. An apparent rate constant (k(app)) for transport of protons has been determined from the rate of change of fluorescence intensity of pyranine by stopped flow rapid kinetics in the presence of proton gradient. The variation of k(app) has been studied as a function of ionophore concentration and the results have been compared with gramicidin-a well known channel former under the similar experimental conditions. The rates thus obtained showed that A23187 is not only a simple carrier but also shows channel behaviour at high concentration of ionophore.

Fluorescence and topological gap of conjugated phenylene polymers

We propose that strong fluorescence in conjugated polymers requires a dipole-allowed state to be the lowest singlet. Hückel theory for para-conjugated phenyl rings yields an extended, topologically one-dimensional ?-system with increased alternation, states localized on each ring, and charge-transfer excitations between them. Exact Pariser�Parr�Pople results and molecular spectra for oligomers support a topological contribution and a lowest dipole-allowed singlet in phenylene polymers.

Ricin-membrane interaction: membrane penetration depth by fluorescence quenching and resonance energy transfer

The entry of the plant toxin ricin and its A- and B-subunits in model membranes in the presence as well as absence of monosialoganglioside (GM(1)) has been studied. Dioleoylphosphatidylcholine and 5-, 10-, and 12-doxyl- or 9,10-dibromophosphatidylcholines serve as quenchers of intrinsic tryptophan fluorescence of the proteins. The parallax method of Chattopadhyay and London [(1987) Biochemistry 26, 39-45] has been employed to measure the average membrane penetration depth of tryptophans of ricin and its B-chain and the actual depth of the sole Trp 211 in the A-chain. The results indicate that both of the chains as well as intact ricin penetrate the membrane deeply and the C-terminal end of the A-chain is well inside the bilayer, especially at pH 4.5. An extrinsic probe N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl) ethylenediamine (I-AEDANS) has been attached to Cys 259 of the A-chain, and the kinetics of penetration has been followed by monitoring the increase in AEDANS fluorescence at 480 nm. The insertion follows first-order kinetics, and the rate constant is higher at a lower pH. The energy transfer distance analysis between Trp 211 and AEDANS points out that the conformation of the A-chain changes as it inserts into the membrane. CD studies indicate that the helicity of the proteins increases after penetration, which implies that some of the unordered structure in the native protein is converted to the ordered form during this process. Hydrophobic forces seem to be responsible for stabilizing a particular protein conformation inside the membrane.

Time-resolved fluorescence studies on covalently linked porphyrin�nitroarene complexes. Conformational control of photoinduced electron transfer reactions

Time-resolved fluorescence studies were carried out on a series of free-base and zinc(II) derivatives of meso-tetraphenylporphyrins covalently linked to either 1,3-dinitrobenzene (DNB) or 1,3,5-trinitrobenzene (TNB) acceptor units. These acceptor units were linked at different sites (at the ortho, meta or para positions of one of the phenyl groups of meso-tetraphenylporphyrin) to the donor porphyrins such that the resulting isomeric intramolecular donor-acceptor complexes exhibit different centre-to-centre (ctc) distances and relative orientations. Biexponential fluorescence decay profiles observed for several of these covalently linked complexes were rationalized in terms of the presence of ''closed'' and ''extended'' conformers. Detailed analyses of the fluorescence decay data have provided a comprehensive understanding of the photoinduced electron transfer (PET) reactions occurring in systems containing zinc(II) porphyrin donors. It is observed that although DNB-linked zinc(II) complexes follow the trends predicted for the efficiency of PET with respect to donor-acceptor distance, the TNB-linked zinc(II) porphyrins exhibit a behaviour which is dictated by steric effects. Similarly, although the thermodynamic criteria predict a greater efficiency of charge separation in TNB-linked complexes compared with DNB-linked complexes, the reverse trend observed has been attributed to orientational effects. In the complexes containing free-base porphyrin donors, PET is expected to be less efficient from a thermodynamic viewpoint. In a few of these cases, fluorescence quenching seems to occur by parallel mechanisms other than PET.

Analysis of a conformation-specific epitope of the alpha subunit of human chorionic gonadotropin: Study using monoclonal antibody probes

Monoclonal antibodies (MAbs) have been used extensively for identification of sequence-specific epitopes using either the ELISA or/and IRMA methods, However, attempts to use MAbs for identification of conformation-specific epitopes have been very few as they are considered very labile. We have investigated the stability of conformation-specific epitopes of human chorionic gonadotropin (hCG) using a quantitative solid-phase radioimmnunoassay (SPRIA) technique. Several epitopes are stable to mild modification (chemical and proteolytic) conditions, and epitopes show differential stability for these modifications. Based on these observations, a monoclonal antibody (MAb 16) for an a-subunit-specific epitope of hCG has been used to monitor changes at the epitopic site (identified as epitope 16) on modification of hCG, using SPRIA with immobilized MAb 16. Modifications of amino groups, hydroxyl group of tyrosine as well as carboxyl group of Asp/Glu all bring about sufficient changes in the epitope integrity. Peptide bond hydrolysis at lysine residues damages the epitope, but not at arginine residues, Hydrolysis at tyrosine does not affect the epitope, though modification of the side-chain of tyrosine inactivates the epitope. Destruction of the epitope occurs on reduction of the disulphide bonds. Partial retention of the epitope activity is seen on modification of carboxyl or the epsilon-amino groups of lysine. Based on these results four to six amino acids have been identified to be at the epitopic site, and the data suggest that two peptide segments are brought together by the disulphide bond Cys10-Cys60 to form the epitope.

Role of spacer chain length in dimeric micellar organization. Small angle neutron scattering and fluorescence studies

Micelles of different dimeric amphiphiles Br-, n-C(16)H(33)NMe(2)(+) -(CH)(m)-N(+)Me(2)-n-C16H33, Br- (where m = 3, 4, 5, 6, 8, 10, and 12) adapt different morphologies and internal packing arrangements in aqueous media depending on their spacer chain length (m). Detailed measurements of small angle neutron scattering (SANS) cross sections from different bis-cationic, dimeric surfactant micelles in aqueous media (D2O) are reported. The data have been analyzed using the Hayter and Penfold model for macro ion solution to compute the interparticle structure factor S(Q) taking into account the screened Coulomb interactions between the dimeric micelles. The SANS analysis clearly indicated that the extent of aggregate growth and the variations of shapes of the dimeric micelles depend primarily on the spacer chain length. With spacer chain length, m less than or equal to 4, the propensity of micellar growth was particularly pronounced. The effects of the variation of the concentration of dimeric surfactants with m = 5 and 10 on the SANS spectra and the effects of the temperature variation for the micellar system with m = 10 were also examined. The critical micelle concentrations (cmc) and their microenvironmental feature, namely, the microviscosities that the dimeric micellar aggregates offer to a solubilized, extrinsic fluorescence probe, 1,6-diphenyl-1,3,5-hexatriene, were also determined. The changes of cmcs and microviscosities as a function of spacer chain length have been explained in terms of conformational variations and progressive looping of the spacer in micellar core upon increasing m values.

Development of specific DNA probes and their usage in the detection of Plasmodium vivax infection in blood

The application of nucleic acid probes, in the detection of pathogenic micro-organisms, has become an integral part of diagnostic technologies. In this study, Plasmodium vivax-specific DNA probes have been identified by carrying out genomic subtractive hybridization. In this approach, the recombinant clones from a P. vivax genomic library are screened with radiolabelled human and P. falciparum DNA. The colonies which react with labelled P. falciparum and human DNA are eliminated and those which do not produce any autoradiographic signal have been subjected to further screening procedures. Three Fl vivax specific DNA probes have been obtained by these repeated screenings. Further analyses indicate that these probes are specific and sensitive enough to detect P. vivax infection in clinical blood samples when used in a non-radioactive DNA hybridization assay. (C) 1995 Academic Press Limited

Long-Range Visible Fluorescence Tunability Using Component-Modulated Coupled Quantum Dots

A simple route for tailoring emissions in the visible wavelength region by chemically coupling quantum dots composed of ZnSe and CdS is reported. coupled quantum dots offer a novel route for tuning electronic transitions via band-offset engineering at the material interface. This novel class of asymmetric. coupled quantum structures may offer a basis for a diverse set of building blocks for optoelectronic devices, ultrahigh density memories, and quantum information processing.

Photophysical and electrochemical redox properties of fixed distance porphyrin-quinone systems

A few fixed distance covalently linked porphyrin-quinone molecules have been synthesized in which a benzoquinone is directly attached to a meso/beta-pyrrole position of tri(phenyl/pentafluorophenyl)/tetraphenylporphyrins. The choice of fluoroarylporphyrins permit modulation of Delta G(ET) values for photoinduced electron-transfer reactions in these systems. All short distance porphyrin-quinone molecules showed efficient quenching of the porphyrin singlet excited state. The electrochemical redox data coupled with the steady-state and time-resolved singlet emission data are analysed to evaluate the dependence of Delta G(ET) values on the rate of electron transfer (k(ET)) in these systems. The meso-trifluoroarylporphyrin-quinones are found to be sensitive probes of the surrounding dielectric environment. Varying solvent polarity on the mechanism of fluorescence quenching and k(ET) values revealed that short donor-acceptor distance and the solvent dielectric relaxation properties play a dominant role. (C) 1999 Elsevier Science S.A. All rights reserved.

Fluorescence and visual sensing of nitroaromatic explosives using electron rich discrete fluorophores

Several pi-electron rich fluorescent aromatic compounds containing trimethylsilylethynyl functionality have been synthesized by employing Sonogashira coupling reaction and they were characterized fully by NMR (H-1, C-13)/IR spectroscopy. Incorporation of bulky trimethylsilylethynyl groups on the peripheral of the fluorophores prevents self-quenching of the initial intensity through pi-pi interaction and thereby maintains the spectroscopic stability in solution. These compounds showed fluorescence behavior in chloroform solution and were used as selective fluorescence sensors for the detection of electron deficient nitroaromatics. All these fluorophores showed the largest quenching response with high selectivity for nitroaromatics among the various electron deficient aromatic compounds tested. Quantitative analysis of the fluorescence titration profile of 9,10-bis(trimethylsilylethynyl) anthracene with picric acid provided evidence that this particular fluorophore detects picric acid even at ppb level. A sharp visual detection of 2,4,6-trinitrotoluene was observed upon subjecting 1,3,6,8-tetrakis (trimethylsilylethynyl) pyrene fluorophore to increasing quantities of 2,4,6-trinitrotoluene in chloroform. Furthermore, thin film of the fluorophores was made by spin coating of a solution of 1.0 x 10(-3) M in chloroform or dichloromethane on a quartz plate and was used for the detection of vapors of nitroaromatics at room temperature. The vapor-phase sensing experiments suggested that the sensing process is reproducible and quite selective for nitroaromatic compounds. Selective fluorescence quenching response including a sharp visual color change for nitroaromatics makes these fluorophores as promising fluorescence sensory materials for nitroaromatic compounds (NAC) with a detection limit of even ppb level as judged with picric acid.