Sequencing of T-superfamily conotoxins from Conus virgo: Pyroglutamic acid identification and disulfide arrangement by MALDI mass spectrometry

De novo mass spectrometric sequencing of two Conus peptides, Vi1359 and Vi1361, from the vermivorous cone snail Conus virgo, found off the southern Indian coast, is presented. The peptides, whose masses differ only by 2 Da, possess two disulfide bonds and an amidated C-terminus. Simple chemical modifications and enzymatic cleavage coupled with matrix assisted laser desorption ionization (MALDI) mass spectrometric analysis aided in establishing the sequences of Vi1359, ZCCITIPECCRI-NH2, and Vi1361, ZCCPTMPECCRI-NH2, Which differ only at residues 4 and 6 (Z = pyroglutamic acid). The presence of the pyroglutamyl residue at the N-terminus was unambiguously identified by chemical hydrolysis of the cyclic amide, followed by esterification. The presence of Ile residues in both the peptides was confirmed from high-energy collision induced dissociation (CID) studies, using the observation Of W-n- and d(n)-ions as a diagnostic. Differential cysteine labeling, in conjunction with MALDI-MS/MS, permitted establishment of disulfide connectivity in both peptides as Cys2-Cys9 and Cys3-Cys10. The cysteine pattern clearly reveals that the peptides belong to the class of T-superfamily conotoxins, in particular the T-1 superfamily.

Unfolding of Plasmodium falciparum Triosephosphate Isomerase in Urea and Guanidinium Chloride: Evidence for a Novel Disulfide Exchange Reaction in a Covalently Cross-Linked Mutant†

The conformational stability of Plasmodium falciparum triosephosphate isomerase (TIMWT) enzyme has been investigated in urea and guanidinium chloride (GdmCl) solutions using circular dichroism, fluorescence, and size-exclusion chromatography. The dimeric enzyme is remarkably stable in urea solutions. It retains considerable secondary, tertiary, and quaternary structure even in 8 M urea. In contrast, the unfolding transition is complete by 2.4 M GdmCl. Although the secondary as well as the tertiary interactions melt before the perturbation of the quaternary structure, these studies imply that the dissociation of the dimer into monomers ultimately leads to the collapse of the structure, suggesting that the interfacial interactions play a major role in determining multimeric protein stability. The C-m(urea)/C-m(GdmCl) ratio (where C-m is the concentration of the denaturant required at the transition midpoint) is unusually high for triosephosphate isomerase as compared to other monomeric and dimeric proteins. A disulfide crosslinked mutant protein (Y74C) engineered to form two disulfide cross-links across the interface (13-74') and (13'-74) is dramatically destablized in urea. The unfolding transition is complete by 6 M urea and involves a novel mechanism of dimer dissociation through intramolecular thiol-disulfide exchange.

Photoswitchable multivalent sugar ligands: Synthesis, isomerization, and lectin binding studies of azobenzene-glycopyranoside derivatives

Coating of azobenzene chromophore with multivalent sugar ligands has been accomplished. Such sugar coating allows the study of the isomerization properties of this chromophore in aqueous solutions. The predominantly cis-isomer-containing photostationary state (PS) mixture of these azobenzene derivatives is found to be stable for hours. The rate constants for their isomerization, as well as the Arrhenius activation energies, are determined experimentally. An assessment of the lectin binding properties of the lactoside bearing isomeric azobenzene derivatives, by isothermal calorimetric methods, reveals the existence of an unusual cooperativity in their binding to lectin peanut agglutinin. Thermodynamic parameters evaluated for the trans and the PS mixture are discussed, in detail, for the lactoside bearing bivalent azobenzene derivative.

Energetics of carbohydrate binding by a 14 kDa S-type mammalian lectin

The thermodynamics of the binding of derivatives of galactose and lactose to a 14 kDa beta-galactoside-binding lectin (L-14) from sheep spleen has been studied in 10 nM phosphate/150 mM NaCl/10 mM beta-mercaptoethanol buffer, pH 7.4, and in the temperature range 285-300 K using titration calorimetry. The single-site binding constants of various sugars for the lectin were in the following order: N-acetyl-lactosamine thiodigalactoside > 4-methylumbelliferyl lactoside > lactose > 4-methylumbelliferyl alpha-D-galactoside > methyl-alpha-galactose > methyl-beta-galactose. Reactions were essentially enthalpically driven with the binding enthalpies ranging from -53.8 kJ/mol for thiodigalactoside at 301 K to -2.2 kJ/mol for galactose at 300 K, indicating that hydrogen-bonding and van der Waals interactions provide the major stabilization for these reactions. However, the binding of 4-methylumbelliferyl-alpha-D-galactose displays relatively favourable entropic contributions, indicating the existence of a non-polar site adjacent to the galactose-binding subsite. From the increments in the enthalpies for the binding of lactose, N-acetyl-lactosamine and thiodigalactoside relative to methyl-beta-galactose, the contribution of glucose binding in the subsite adjacent to that for galactose shows that glucose makes a major contribution to the stability of L-14 disaccharide complexes. Observation of enthalpy-entropy compensation for the recognition of saccharides such as lactose by L-14 and the absence of it for monosaccharides such as galactose, together with the lack of appreciable changes in the heat capacity (delta Cp), indicate that reorganization of water plays an important role in these reactions.

Thermodynamic and kinetic studies on the mechanism of binding of methylumbelliferyl galactosides to the basic agglutinin from winged bean (Psophocarpus tetragonolobus)

The binding of winged bean basic agglutinin (WBA I) to 4-methylumbelliferyl (MeUmb) galactosides was examined by extrinsic fluorescence titration and stopped-flow spectrofluorimetry. Upon binding to WBA I, MeUmb alpha-galactosides show quenching in fluorescence intensity, decrease in UV absorbance with a concomitant blue shift, and decrease in fluorescence excited-state lifetimes. However, their beta-analogues show enhancement in fluorescence intensity, increase in UV absorbance with a red shift, and an increase in fluorescence excited-state lifetimes. This implies that the umbelliferyl groups of alpha- and beta-galactosides experience non-polar and polar microenvironments, respectively, upon binding to WBA I. Replacement of the anomeric hydroxyl group of galactose by 4-methylumbelliferyl moiety increases the affinity of resulting saccharides. Substitution of C-2 hydroxyl of galactose by an acetamido group leads to increased affinity due to a favorable entropy change. This suggests that acetamido group of MeUmb-alpha/beta-GalNAc binds to a relatively non-polar subsite of WBA I. Most interestingly, this substitution also reduces the association rate constants dramatically. Inspection of the activation parameters reveals that the enthalpy of activation is the limiting factor for the differences in the forward rate constants for these saccharides and the entropic contribution to the activation energy is small

Thermodynamics of monosaccharide binding to concanavalin A, pea (Pisum sativum) lectin, and lentil (Lens culinaris) lectin

Titration calorimetry measurements of the binding of methyl alpha-D-mannopyranoside (Me alpha Man), D-mannopyranoside (Man), methyl alpha-D-glucopyranoside (Me alpha Glu), and D-glucopyranoside (Glu) to concanavalin A (Con A), pea lectin, and lentil lectin were performed at 281 and 292 K in 0.01 M dimethylglutaric acid-NaOH buffer (pH 6.9) containing 0.15 M NaCl and Mn+2 and Ca+2 ions. The site binding enthalpies, delta H, are the same at both temperatures and range from -28.4 +/- 0.9 (Me alpha Man) to -16.6 +/- 0.5 kJ mol-1 (Glu) for Con A, from -26.2 +/- 1.1 (Me alpha Man) to -12.8 +/- 0.4 kJ mol-1 (Me alpha Glu) for pea lectin, and from -16.6 +/- 0.7 (Me alpha Man) to -8.0 +/- 0.2 kJ mol-1 (Me alpha Glu) for lentil lectin. The site binding constants range from 17 +/- 1 x 10(3) M-1 (Me alpha Man to Con A at 281.2 K) to 230 +/- 20 M-1 (Glu to lentil lectin at 292.6 K) and exhibit high specificity for Con A where they are in the Me alpha Man:Man:Me alpha Glu:Glu ratio of 21:4:5:1, while the corresponding ratio is 5:2:1.5:1 for pea lectin and 4:2:2:1 for lentil lectin. The higher specificity for Con A indicates more interactions between the amino acid residues at the binding site and the carbohydrate ligand than for the pea and lentil lectin-carbohydrate complexes. The carbohydrate-lectin binding results exhibit enthalpy-entropy compensation in that delta Hb (kJ mol-1) = -1.67 +/- 0.06 x 10(4) + (1.30 +/- 0.12)T(K) delta Sb (J mol-1K-1). Differential scanning calorimetry measurements on the thermal denaturation of the lectins and their carbohydrate complexes show that the Con A tetramer dissociates into monomers, while the pea and lentil lectin dimers dissociate into two submonomer fragments. At the denaturation temperature, one carbohydrate binds to each monomer of Con A and the pea and lentil lectins. Complexation with the carbohydrate increases the denaturation temperature of the lectin and the magnitude of the increases yield binding constants in agreement with the determinations from titration calorimetry.

Thermodynamics of the binding of galactopyranoside derivatives to the basic lectin from winged bean (Psophocarpus tetrogonolobus)

The thermodynamics of the binding of D-galactopyranoside (Gal), 2-acetamido-2-deoxygalactopyranoside (GalNAc), methyl-alpha-D-galactopyranoside, and methyl-beta-D-galactopyranoside to the basic agglutinin from winged bean (WBAI) in 0.02 M sodium phosphate and 0.15 M sodium chloride buffer have been investigated from 298.15 to 333.15 K by titration calorimetry and at the denaturation temperature by differential scanning calorimetry (DSC). WBAI is a dimer with two binding sites. The titration calorimetry yielded single-site binding constants ranging from 0.56 +/- 0.14 x 10(3) M-1 for Gal at 323.15 K to 7.2 +/- 0.5 x 10(3) M-1 for GalNAc at 298.15 K and binding enthalpies ranging from -28.0 +/- 2.0 kJ mol-1 for GalNAc at 298.15 K to -14.3 +/- 0.1 kJ mol-1 for methyl-beta-D-galactopyranoside at 322.65 K. The denaturation transition consisted of two overlapping peaks over the pH range 5.6-7.4. Fits of the differential scanning calorimetry data to a two-state transition model showed that the low temperature transition (341.6 +/- 0.4 K at pH 7.4) consisted of two domains unfolding as a single entity while the higher temperature transition (347.8 +/- 0.6 K at pH 7.4) is of the remaining WBAI dimer unfolding into two monomers. Both transitions shift to higher temperatures and higher calorimetric enthalpies with increase in added ligand concentration at pH 7.4. Analysis of the temperature increase as a function of added ligand concentration suggests that one ligand binds to the two domains unfolding at 341.6 +/- 0.6 K and one ligand binds to the domain unfolding at 347.8 +/- 0.6 K.

Erythrocyte-binding studies on an acidic lectin from winged bean (Psophocarpus tetragonolobus)

n acidic lectin (WBA II) was isolated to homogeneity from the crude seed extract of the winged bean (Psophocarpus tetragonolobus) by affinity chromatography on lactosylaminoethyl-Bio-Gel. Binding of WBA II to human erythrocytes of type-A, -B and -O blood groups showed the presence of 10(5) receptors/cell, with high association constants (10(6)-10(8) M-1). Competitive binding studies with blood-group-specific lectins reveal that WBA II binds to H- and T-antigenic determinants on human erythrocytes. Affinity-chromatographic studies using A-, B-, H- and T-antigenic determinants coupled to an insoluble matrix confirm the specificity of WBA II towards H- and T-antigenic determinants. Inhibition of the binding of WBA II by various sugars show that N-acetylgalactosamine and T-antigenic disaccharide (Thomsen-Friedenreich antigen, Gal beta 1-3GalNAc) are the most potent mono- and di-saccharide inhibitors respectively. In addition, inhibition of the binding of WBA II to erythrocytes by dog intestine H-fucolipid prove that the lectin binds to H-antigenic determinant.

Studies on the interaction of concanavalin A with glycoproteins

Lectins (phytohaemagglutinin) are known to have the unique property of binding with certain specific sugars, polysaccharides and glycoproteins. Although the kinetics of interaction between lectins and sugar have been extensively studied, the binding characteristics of the lectins with various glycoproteins are not well understood. In this laboratory a systematic study has been initiated in relation to the interaction of lectins with glycoproteins. Concanavalin A is known to bind alpha-glucosides, mannosides and biopolymers having these sugar configurations. A galactose binding protein from caster bean has been purified to homogeneity and was found to contain mannose. This lectin was used as the source of glycoprotein for studying its interaction with concanavalin A. This study showed that the interaction is temperature dependent and the dissociation is time and alpha-methyl glucoside concentration dependent. This has led to speculate a model for cell-lectin interaction. Using concanavalin A it has been shown that all the lysosomal enzymes from brain studied were glycoprotein in nature. Moreover, using Sepharose-bound concanavalin A it has been possible to devise a method by which these lysosomal enzymes could be purified considerably. With the knowledge that the interaction between lectin and glycoprotein is not only dependent on the specific sugar present in the glycoprotein, but also on the nature of the glycoprotein it was possible to develop a novel method for immobilizing various glycoprotein enzymes, such as arylsulphatase A, hyaluronidase and glucose oxidase.

Formation ranges of icosahedral, amorphous and crystalline phases in rapidly solidified Ti-Zr-Hf-Ni alloys

From the quaternary Ti-Zr-Hf-Ni phase diagram. the cross-section at 20 at % Ni was selected for investigation. The icosahedral quasicrystalline, crystalline and amorphous phases were observed to form in nine kinds of rapidly solidified (TixZryHfz)(80)Ni-20 (x + y + z = 1) alloys at different compositions. The quasilattice constants of 0.519 and 0.531 nm were obtained for the icosahedral phase formed in the melt-spun Ti40Zr20Hf20Ni20 and Ti20Zr40Hf20Ni20 alloys. respectively. The icosahedral phase formed in the melt-spun Ti40Zr20Hf20Ni20 alloy especially is thermodynamically stable. The supercooled liquid region of the Ti20Zr20Hf40Ni20 glassy alloy reached 64 K. From these results a comparison of quasicrystal-forming and glass-forming abilities, was carried out. The quasicrystal-forming ability was reduced and glass-forming ability was improved with an increase in Hf and Zr contents in the (TixZryHfz)(80)Ni-20 alloys. On the other hand. an increase in Ti content caused an improvement in quasicrystal-forming ability.

Lattice dynamics of hexagonal ice

The lattice dynamics of hexagonal ice is worked out with the force constants deduced from the experimental elastic constants.

Interface stresses in a composite cylindrical shell under ring loading

The problem of a two-layer circular cylindrical shell subjected to radial ring loading has been solved theoretically. The solution is developed by uniting the elasticity solution through Love function approach for the inner thick shell with the Flügge shell theory for the thin outer shell. Numerical work has been done with a digital computer for different values of shell geometry parameters and material constants. The general behaviour of the composite shell has been studied in the light of these numerical results.

Determination of the orthotropic plate parameters of stiffened plates and grillages in free vibration

It is well known that the analysis of vibration of orthogonally stiffened rectangular plates and grillages may be simplified by replacing the actual structure by an orthotropic plate. This needs a suitable determination of the four elastic rigidity constants Dx, Dy, Dxy, D1 and the mass {Mathematical expression} of the orthotropic plate. A method is developed here for determining these parameters in terms of the sectional properties of the original plate-stiffener combination or the system of interconnected beams. Results of experimental work conducted on aluminium plates agree well with the results of the theory developed here.

A symmetric solution for a cylindrical shell enclosed in an elastic casing

The problem of a long, thin circular cylindrical shell enclosed in an elastic casing and subjected to a ring of radial load on the inner rim is solved using the Love function for the casing in conjunction with Flügge shell theory. Numerical work has been done with a digital computer and the results for stress and displacement fields are given for various values of the shell geometry parameters and material constants.

Studies in the hydrolysis of metal ions -Part I.Copper

The hydrolysis of cupric ion has been studied at various ionic strengths (0·01, 0·05, 0·1 and 0·5 M). The results are analyzed employing 'core + links' theory, log-log plot, normalization plot, and extrapolation method for obtaining the pure mononuclear curve. The stability constants of Cu2(OH)2++, Cu3(OH)4++, Cu(OH)+ and Cu(OH)2 have been reported.