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-GalNAc and for jack fruit agglutinin (JFA) is Me-Gal. 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.Abbreviations SBA Soybean agglutinin - WBA I Winged bean agglutinin (Basic) - JFA Jack fruit agglutinin - PNA Peanut agglutinin - Con A Concanavalin A - Dansyl (Dns) 5-dimethylaminonaphthalene-I-sulphonyl - 2GaINDns N-dansylgalactosamine - dGal 2-deoxygalactose - l-Ara l-arabinose - d-Fuc d-fucose - l-Rha l-rhamnose - N-acetyllactosamine Galß4GlcNAc - melibiose Gal6Glc     

Analysis of saccharide binding to Artocarpus integrifolia lectin reveals specific recognition of T-antigen (beta-D-Gal(1----3)D-GalNAc)

The binding of Artocarpus integrifolia lectin to N-dansylgalactosamine (where dansyl is 5-dimethylaminonaphthalene-1-sulfonyl) leads to a 100% increase in dansyl fluorescence with a concomitant blue shift in the emission maximum by 10 nm. This binding is carbohydrate-specific and has an association constant of 1.74 X 10(4) M-1 at 20 degrees C. The lectin has two binding sites for N-dansylgalactosamine. The values of -delta H and -delta S for the binding of N-dansylgalactosamine are in the range of values reported for several lectin-monosaccharide interactions, indicating an absence of nonpolar interaction of the dansyl moiety of the sugar with the combining region of the protein. Dissociation of the bound N-dansylgalactosamine from its complex with the lectin and the consequent change in its fluorescence on addition of nonfluorescent sugars allowed evaluation of the association constant for competing ligands. The thermodynamic parameters for the binding of monosaccharides suggest that the OH groups at C-2, C-3, C-4, and C-6 in the D-galactose configuration are important loci for interaction with the lectin. The acetamido group at C-2 of 2-acetamido-2-deoxygalactopyranose and a methoxyl group at C-1 of methyl-alpha-D-galactopyranoside are presumably also involved in binding through nonpolar and van der Waals' interactions. The T-antigenic disaccharide Gal beta 1----3GalNAc binds very strongly to the lectin when compared with methyl-beta-D-galactopyranoside, the beta(1----3)-linked disaccharides such as Gal beta 1----3GlcNAc, and the beta(1----4)-linked disaccharides, N-acetyllactosamine and lactose. The major stabilizing force for the avid binding of T-antigenic disaccharide appears to be a favorable enthalpic contribution. The combining site of the lectin is, therefore, extended. These data taken together suggest that the Artocarpus lectin is specific toward the Thomsen-Friedenreich (T) antigen. There are subtle differences in the overall topography of its combining site when compared with that of peanut (Arachis hypogaea) agglutinin. The results of stopped flow spectrometry for the binding of N-dansylgalactosamine tot he Artocarpus lectin are consistent with a simple single-step bimolecular association and unimolecular dissociation rate processes. The value of K+1 and K-1 at 21 degrees C are 8.1 X 10(5) M-1 s-1 and 50 s-1, respectively. The activation parameters indicate an enthalpy-controlled association process.     

Resonance Raman spectroscopic studies in copper reconstituted and hybrid hemoglobins: probe into subunit heterogeneity

Copper reconstituted hemoglobin (CuHb), copper containing T-state hybrid hemoglobins like alpha2(Ni)beta2(Cu), and alpha2(Cu)beta2(Ni), and intermediate R-state hybrids like alpha2(CO-Fe)beta2(Cu) and alpha2(Cu)beta2(Fe-CO) are studied using resonance Raman (RR) spectroscopy at two different excitation wavelengths. The high frequency RR region in CuHb indicates the presence of both 4- and 5-coordinate forms of Cu(II). In hybrid Hbs, the presence of two distinct metal ion environments within one particular subunit is evident. This is also consistent with previous findings using EPR spectroscopy and sulfydryl reactivity studies on these hybrid Hbs. The low frequency RR region on these copper derivatives of HbA further suggests the existence of two different heme moieties within the subunit.     

Stereochemical Effects of Methylphosphonate in B- and Z-DNA Helices: Variation in Hydrophobicity and Effective Widths of Grooves

Abstract

Stereochemical effects of methylphosphonate (MP) in B-DNA and Z-DNA duplexes are studied through molecular mechanics approach. Duplexes of different lengths, tetramers, hexamers, dodecamers are examined to assess the interstrand and intrastrand electrostatic effects due to MPs vis-a-vis phosphates. A variety of models which include duplexes with alternating S-MP and R-MP, alternating phosphate and MP and, duplexes posessing MPs in only one of the strands, are examined by considering both the S- and R-stereoisomers. Majority of the calculations are performed with CG sequences to delineate factors responsible for the stability of B- and Z-DNA as well as B × Z-DNA transition under nonionic conditions. The results show that both B- and Z-DNA duplexes are energetically favoured in the presence of MP due to overwhelming reduction in intrastrand as well as interstrand electrostatic repulsive interactions. The effect is distinct in oligomers longer than tetramers. Comparison of energetics of MP B- and Z-DNA duplexes suggests that an oligodeoxynucleotide such as d(CG)₆ with all phosphates replaced by MPs may favour equally both B- and Z-DNA conformations. The analysis further provides an estimate of electrostatic interactions, operating at the grooves under a variety of conditions. Several specific and localised effects due to S-MP and R-MP are seen at CG and GC steps in various B-DNA and Z-DNA models. S-MP in B- DNA reduces the effective major groove width by nearly 3 Å hence denying access to the functional groups of endonucleases thereby enhancing the resistance of MP-DNA to enzymatic digestion. Further, methyl groups of MP render the surface of the DNA helix to be significantly hydrophobic which may explain higher permeability of MP-DNA in membranes as well as its less soluble nature in aqueous media.     

Properties of a thermostable 4Fe-ferredoxin from the hyperthermophilic bacterium Thermotoga maritima

Abstract A ferredoxin has been purified from one of the most ancient and the most thermophilic bacteria known, Thermotoga maritima , which grous up to 90°C. The reduced protein ( M _r approx. 6300) contains a single S = 1 2 [4Fe 4S]¹⁺ cluster with complete cysteinyl ligation, and was unaffected after incubation at 95°C for 12 h. It functioned as an electron carrier for T. maritima pyruvate oxidoreductase. Remarkably, the properties and amino acid sequence of this hyperthermophilic bacterial protein are much more similar to those of ferredoxins from hyperthermophilic archaea, rather than ferredoxins from mesophilic and moderately thermophilic bacteria.     

Proteomic profiling of L-cysteine induced selenite resistance in Enterobacter sp. YSU

Background  

Enterobacter sp. YSU is resistant to several different heavy metal salts, including selenite. A previous study using M-9 minimal medium showed that when the selenite concentration was 100,000 times higher than the sulfate concentration, selenite entered Escherichia coli cells using two pathways: a specific and a non-specific pathway. In the specific pathway, selenite entered the cells through a yet to be characterized channel dedicated for selenite. In the non-specific pathway, selenite entered the cells through a sulfate permease channel. Addition of L-cystine, an L-cysteine dimer, appeared to indirectly decrease selenite import into the cell through the non-specific pathway. However, it did not affect the level of selenite transport into the cell through the specific pathway.     

Anaerobic Immobilized Yeast Cell Fermentation and Anaerobic Remediation in Hybrid Reactor for Mineralization of Dicarboxylic Acid Solid Waste

Summary The solid resinous product (SRP) containing unsaturated/saturated dicarboxylic acid residues, phthalic acid and maleic acid is discharged as a solid waste during cracking of benzene over vanadium at temperatures above 500°C in the dicarboxylic acid manufacturing industry. In the present study the solid waste was diluted with water to a concentration of 0.5% w/v for microbial degradation. The waste was fermented in a reactor containing mesoporous activated carbon on which was immobilized Saccharomyces cerevisiae at an optimum residence time of 24 h at pH 6.5. The immobilized-yeast-treated samples were further treated in an upflow anaerobic reactor at an hydraulic retention time (HRT) of 0.1038 days at a hydraulic flow rate of 7.34 × 10⁻³ m³/day and chemical oxygen demand (COD) loading rate of 2.19 kg/m³/day. The pathway followed in the degradation of dicarboxylic acid into end products by anaerobic metabolism in the yeast cell fermentor and in the upflow anaerobic reactor was confirmed through HPLC, Fourier transform infra red spectroscopy and proton and ¹³C NMR spectroscopy.