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En la presente investigación se simula la actividad antidrepanocitaria en muestras biológicas (sangre total) frente a nanopartículas sólidas lipídicas cargadas con vainillina (NSL- V) a través de las mediciones de los tiempos de relajación magnética de las moléculas de hidrógeno. Para este fin se empleó sangre proveniente del Banco de Sangre del Hospital “Dr. Antonio María Béguez César” perteneciente a pacientes con Anemia Drepanocítica (AD) y se preparó un grupo control y tres grupos experimentales (Grupo 1 (G1): 300 μl de sangre total SS. Grupo 2: 150 μl de sangre total SS y 150 μl de formulación de NSL con vainillina. Grupo 3 (G3): 150 μl de sangre total SS y 150 μl de formulación de vainillina. Grupo 4 (G4):150 μl de sangre total SS y 150 μl de formulación de NSL) y cada uno se realizó por triplicado. Se obtuvieron nanopartículas sólidas lipídicas con valores de eficiencia de encapsulación superiores al 50%, lo que demuestra una adecuada incorporación de vainillina en estos nanosistemas. Mediante el empleo de la técnica RMN se demostró que la vainillina encapsulada en NSL aumenta los valores del tiempo de demora (Td), lo que es indicativo de la inhibición de la formación de los polímeros de HbS en los eritrocitos falciformes avalando el uso de nanopartículas sólidas lipídicas como vehículos para aumentar la eficacia de la vainillina en el tratamiento de la Anemia Drepanocítica.

Electrochemical treatment has been suggested as an effective alternative to local cancer therapy. Nevertheless, its effectiveness decreases when highly aggressive primary tumors are treated. The aim of this research was to understand the growth kinetics of the highly aggressive and metastatic primary F3II tumor growing in male and female BALB/c/Cenp mice under electrochemical treatment. Different amounts of electric charge (6, 9, and 18 C) were used. Two electrodes were inserted into the base, perpendicular to the tumor's long axis, keeping about 1 cm distance between them. Results have shown that the F3II tumor is highly sensitive to direct current. The overall effectiveness (complete response + partial response) of this physical agent was ≥75.0% and observed in 59.3% (16/27) of treated F3II tumors. Complete remission of treated tumors was observed in 22.2% (6/27). An unexpected result was the death of 11 direct current-treated animals (eight females and three males). It is concluded that direct current may be addressed to significantly affect highly aggressive and metastatic primary tumor growth kinetics, including the tumor complete response. Bioelectromagnetics. 39:460-475, 2018. © 2018 Wiley Periodicals, Inc.

En esta investigación se analizan los conocimientos del profesorado en activo respecto a las características, barreras y facilitadores del alumnado con altas capacidades, a través de un estudio cualitativo realizado a 30 docentes de Educación General Básica. Se aplicó una entrevista semiestructurada y se sistematizaron las respuestas mediante un análisis categórico, pues interesaba conocer con cierta libertad y apertura las percepciones que los docentes. Los resultados señalan que existe un desconocimiento sobre las Altas Capacidades, pues persisten mitos y el profesorado no da el valor necesario a la capacitación, situación que implica que se tomen medidas urgentes para atender a este grupo y a sus necesidades educativas especiales

Interactions of proline-rich motifs with SH3 domains are present in signal transduction and other important cell processes. Analysis of structural and thermodynamic data suggest a relevant role of water molecules in these protein–protein interactions. To determine whether or not the SH3 domain of the Fyn tyrosine kinase shows the same behaviour, the crystal structures of its complexes with two high-affinity synthetic peptides, VSL12 and APP12, which are class I and II peptides, respectively, have been solved. In the class I complexes two water molecules were found at the binding interface that were not present in the class II complexes. The structures suggest a role of these water molecules in facilitating conformational changes in the SH3 domain to allow the binding of the class I or II peptides. In the third binding pocket these changes modify the cation–π and salt-bridge interactions that determine the affinity of the binding. Comparison of the water molecules involved in the binding of the peptides with previous reported hydration spots suggests a different pattern for the SH3 domains of the Src tyrosine kinase family.

Phycoerythrin is the major light-harvesting pigment-protein of the red algae Porphyridium cruentum and is widely used as fluorescent probe and analytical reagent. Additionally this protein has a potential application as natural dye in food industry. Nevertheless the knowledge of the functional properties of this alga protein is limited, hindering its application as food additive. In this article we report a biophysical characterization of B-phycoerythrin from Porphyridium cruentum (B-PE) in order to study its stability and spectral properties in a broad range of pHs. This information can help in its potential application as colorant in the food industry. Spectroscopic data obtained in this work show that B-PE has a stronger functional stability in the pH range 4.0–10.0, and Size Exclusion Chromatography suggests that the protein maintains a (αβ)6-γ oligomeric structure in that range of pHs. At pH 7.0, an apparent T m value of 77.5 ± 0.5 °C was calculated. At this pH, the protein is highly stable with a loss of only 20 % of its spectral properties (absorbance and fluorescence) after 25 days at room temperature. These results indicate that B-PE is more stable in a broad range of pHs than other phycoerythrin proteins, which would facilitate its use in the food industry.

B-phycoerythrin from the red alga Porphyridium cruentum was crystallized using the technique of capillary counter-diffusion. Crystals belonging to the space group R3 with almost identical unit cell constants and diffracting to 1.85 and 1.70 Å were obtained at pH values of 5 and 8, respectively. The most important difference between structures is the presence of the residue His88α in two different conformations at pH 8. This residue is placed next to the chromophore phycoerythrobilin PEB82α and the new conformation results in the relocation of the hydrogen-bond network and hydration around PEB82α, which probably contributes to the observed pH dependence of the optical spectrum associated with this chromophore. Comparison with the structures of B-phycoerythrin from other red algae shows differences in the conformation of the A-ring of the chromophore PEB139α. This conformational difference in B-phycoerythrin from P. cruentum enables the formation of several hydrogen bonds that connect PEB139α with the chromophore PEB158β at the (αβ)(3) hexamer association interface. The possible influence of these structural differences on the optical spectrum and the ability of the protein to perform energy transfer are discussed, with the two pH-dependent conformations of His88α and PEB82α being proposed as representing critical structural features that are correlated with the pH dependence of the optical spectrum and transient optical states during energy transfer.

alpha-Spectrin SH3-domain (Spc-SH3) crystallization is characterized by very fast growth of the crystals in the presence of ammonium sulfate as a precipitant agent. The origin of this behaviour can be attributed to the presence of a proline residue that participates in a crystal contact mimicking the binding of proline-rich sequences to SH3 domains. This residue, Pro20, is located in the RT loop and is the main contact in one of the interfaces present in the orthorhombic Spc-SH3 crystal structures. In order to understand the molecular interactions that are responsible for the very fast crystal growth of the wild-type (WT) Spc-SH3 crystals, the crystal structure of a triple mutant in which the residues Ser19-Pro20-Arg21 in the RT loop have been replaced by Gly19-Asp20-Ser21 (GDS Spc-SH3 mutant) has been solved. The removal of the critical proline residue results in slower nucleation of the Spc-SH3 crystals and a different arrangement of the protein molecules in the unit cell, leading to a crystal that belongs to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = b = 42.231, c = 93.655 A, and that diffracts to 1.45 A resolution. For both WT Spc-SH3 and the GDS mutant, light-scattering experiments showed that a dimer was formed in solution within a few minutes of the addition of 2 M ammonium sulfate at pH 6.5 and allowed the proposal of a mechanism for the nucleation and crystal growth of Spc-SH3 in which the Pro20 residue plays a key role in the rate of crystal growth.

We report two methods that have allowed the attachment of glu- cose, mannose and lactose to one or both of the cyclopentadienyl rings of ferrocene. The resulting ferrocene-car- bohydrate conjugates were synthesised by the reaction of thioglycosides with ferrocenemethanol and 1,1'-ferrocene- dimethanol in acidic media. A second method based on the regiospecific cop- per(I)-catalysed cycloaddition of prop- argyl glycoside, azidomethyl and bis- Arocene as well as azi- doethyl glycoside and ethynylferrocene was also used and led to the synthesis of 1,2,3-triazole-containing glycoconju- gates. The electrochemical behaviour of the synthesised glycoconjugates was investigated. In addition, their binding interactions with b-cyclodextrin were studied by means of NMR spectrosco- py, isothermal titration calorimetry, and cyclic and differential pulse vol- tammetric experiments. These tech- niques allowed the determination of the thermodynamic parameters of the complexes, the stability constants for the complexes formed with both the neutral and the oxidised states of the ferrocenyl glycoconjugates, the mode of inclusion and the diffusion coeffi- cients for both the glycoconjugates and the complexes.

International audience; Triacylglycerol (TAG) lipases have been thoroughly characterized in mammals and microorganisms. By contrast, very little is known on plant TAG lipases. An Arabidopsis cDNA called AtLip1 (At2g15230), which exhibits strong homology to lysosomal acid lipase, was found to drive the synthesis of an active TAG lipase when expressed in the baculovirus system. The lipase had a maximal activity at pH 6 and the specific activity was estimated to be about 45 micromol min(-1) mg(-1) protein using triolein as a substrate. Knock-out mutant analysis showed no phenotype during germination indicating that this enzyme is fully dispensable for TAG storage breakdown during germination. Northern blot analyses indicated that the transcript is present in all tissues tested.

The binding interactions between dimeric glutathione transferase from Schistosoma japonicum (Sj26GST) and bromosulfophthalein (BS) or 8-anilino-1-naphthalene sulfonate (ANS) were characterised by fluorescence spectroscopy and isothermal titration calorimetry (ITC). Both ligands inhibit the enzymatic activity of Sj26GST in a non-competitive form. A stoichiometry of 1 molecule of ligand per mole of dimeric enzyme was obtained for the binding of these ligands. The affinity of BS is higher (K(d)=3.2 microM) than that for ANS (K(d)=195 microM). The thermodynamic parameters obtained by calorimetric titrations are pH-independent in the range of 5.5 to 7.5. The interaction process is enthalpically driven at all the studied temperatures. This enthalpic contribution is larger for the ANS anion than for BS. The strongly favourable enthalpic contribution for the binding of ANS to Sj26GST is compensated by a negative entropy change, due to enthalpy-entropy compensation. DeltaG degrees remains almost invariant over the temperature range studied. The free energy change for the binding of BS to Sj26GST is also favoured by entropic contributions at temperatures below 32 degrees C, thus indicating a strong hydrophobic interaction. Heat capacity change obtained for BS (DeltaC(p) degrees =(-580.3+/-54.2) cal x K(-1) mol(-1)) is twofold larger (in absolute value) than for ANS (DeltaC(p) degrees =(-294.8+/-15.8) cal x K(-1) mol(-1)). Taking together the thermodynamic parameters obtained for these inhibitors, it can be argued that the possible hydrophobic interactions in the binding of these inhibitors to L-site must be accompanied by other interactions whose contribution is enthalpic. Therefore, the non-substrate binding site (designed as ligandin) on Sj26GST may not be fully hydrophobic.

The thermodynamics of binding of both the substrate glutathione (GSH) and the competitive inhibitor S-hexylglutathione to the mutant Y49F of human glutathione S-transferase (hGST P1-1), a key residue at the dimer interface, has been investigated by isothermal titration calorimetry and fluorescence spectroscopy. Calorimetric measurements indicated that the binding of these ligands to both the Y49F mutant and wild-type enzyme is enthalpically favorable and entropically unfavorable over the temperature range studied. The affinity of these ligands for the Y49F mutant is lower than those for the wild-type enzyme due mainly to an entropy change. Therefore, the thermodynamic effect of this mutation is to decrease the entropy loss due to binding. Calorimetric titrations in several buffers with different ionization heat amounts indicate a release of protons when the mutant binds GSH, whereas protons are taken up in binding S-hexylglutathione at pH 6.5. This suggests that the thiol group of GSH releases protons to buffer media during binding and a group with low pKa (such as Asp98) is responsible for the uptake of protons. The temperature dependence of the free energy of binding, DeltaG0, is weak because of the enthalpy-entropy compensation caused by a large heat capacity change. The heat capacity change is -199.5 +/- 26.9 cal K-1 mol-1 for GSH binding and -333.6 +/- 28.8 cal K-1 mol-1 for S-hexylglutathione binding. The thermodynamic parameters are consistent with the mutation Tyr49 --Phe, producing a slight conformational change in the active site.

The role of the hydroxyl group of tyrosine 6 in the binding of Schistosoma japonicum glutathione S-transferase has been investigated by isothermal titration calorimetry (ITC). A site-specific replacement of this residue with phenylalanine produces the Y6F mutant, which shows negative cooperativity for the binding of reduced glutathione (GSH). Calorimetric measurements indicated that the binding of GSH to Y6F dimer is enthalpically driven over the temperature range investigated. A concomitant net uptake of protons upon binding of GSH to Y6F mutant was detected carrying out calorimetric experiments in various buffer systems with different heats of ionization. The entropy change is favorable at temperatures below 26 degrees C for the first site, being entropically favorable at all temperatures studied for the second site. The enthalpy change of binding is strongly temperature-dependent, arising from a large negative DeltaC(o) (p1)=-3.45+/-0.62kJK(-1)mol(-1) for the first site, whereas a small DeltaC(o) (p2)=-0.33+/-0.05kJK(-1)mol(-1) for the second site was obtained. This large heat capacity change is indicative of conformational changes during the binding of substrate.

The synthesis of three first-order dendrimers based on a β-cyclodextrin core containing fourteen Val, Phe and Val-Phe residues is described. The guest binding ability of the tetradecavalent peptidyl β-cyclodextrin derivative has been tested by calorimetric titration and the thermodynamic parameters for the complex formation with adamantanecarboxylic acid were obtained.

In order to evaluate the abil- ity of multivalent glycosides based on a -cyclodextrin core as site-specific mo- lecular carriers, a study on both the inclusion complexation behaviour and lectin binding affinity of branched and hyperbranched -cyclodextrins is pre- sented. A series of cluster galactosides constructed on -cyclodextrin scaffolds containing seven 1-thio--lactose or - lactosylamine bound to the macrocyclic core through different spacer arms were synthesised. In addition, the first syn- thesis of three first-order dendrimers based on a -cyclodextrin core contain- ing fourteen 1-thio---galactose, 1-thio--lactose and 1-thio--melibiose residues was performed. Calorimetric titrations performed at 25C in buffered aqueous solution (pH 7.4) gave the af- finity constants and the thermodynamic parameters for the complex formation of these -cyclodextrin derivatives with guests sodium 8-anilino-1-naphthalene- sulfonate (ANS) and 2-naphthalenesul- fonate, and lectin from peanut (Arachis hypogaea) (PNA). The persubstitution of the primary face of the -cyclodextrin with saccharides led to a slight increase of the binding constant values for the inclusion complexation with ANS rela- tive to the native -cyclodextrin. How- ever, the increase of the steric conges- tion due to the presence of the saccha- ride residues on the narrow rim of the - cyclodextrin may cause a decrease of the binding ability as shown for sodium 2-naphthalenesulfonate. The spacer arms are not passive elements and influence the host binding ability ac- cording to their chemical nature. PNA forms soluble cross-linked complexes with cluster galactosides and lactosides scaffolded on -cyclodextrin but not with cluster galactopyranosylamines or melibiose. Both, perbranched and hy- perbranched -cyclodextrins, form stronger complexes with PNA than the monomeric analogues. However, the use of hyperbranched CDs does not contrib- ute to the improvement of the complex stability relative to heptakis-glycocyclo- dextrin derivatives. Finally, a titration experiment with PNA and a complex formed by a heptakis lactose -cyclo- dextrin derivative with sodium 2-naph- thalenesulfonate showed the formation of a soluble cross-linked complex with stronger affinity constant and higher stoichiometry than those observed for the complex formation of PNA with the same heptakis-lactose -cyclodextrin derivative, suggesting the formation of a three component complex.

The binding properties of a glutathione S-transferase (EC 2.5.1.18) from Schistosoma japonicum to substrate glutathione (GSH) has been investigated by intrinsic fluorescence and isothermal titration calorimetry (ITC) at pH 6.5 over a temperature range of 15-30 degrees C. Calorimetric measurements in various buffer systems with different ionization heats suggest that protons are released during the binding of GSH at pH 6.5. We have also studied the effect of pH on the thermodynamics of GSH-GST interaction. The behaviour shown at different pHs indicates that at least three groups must participate in the exchange of protons. Fluorimetric and calorimetric measurements indicate that GSH binds to two sites in the dimer of 26-kDa glutathione S-transferase from Schistosoma japonicum (SjGST). On the other hand, noncooperativity for substrate binding to SjGST was detected over a temperature range of 15-30 degrees C. Among thermodynamic parameters, whereas DeltaG degrees remains practically invariant as a function of temperature, DeltaH and DeltaS degrees both decrease with an increase in temperature. While the binding is enthalpically favorable at all temperatures studied, at temperatures below 25 degrees C, DeltaG degrees is also favoured by entropic contributions. As the temperature increases, the entropic contributions progressively decrease, attaining a value of zero at 24.3 degrees C, and then becoming unfavorable. During this transition, the enthalpic contributions become progressively favorable, resulting in an enthalpy-entropy compensation. The temperature dependence of the enthalpy change yields the heat capacity change (DeltaCp degrees ) of -0.238 +/- 0.04 kcal per K per mol of GSH bound.

The binding of three competitive glutathione analogue inhibitors ( S -alkylglutathione derivatives) to glutathione S -transferase from Schistosoma japonicum , SjGST, has been investigated by isothermal titration microcalorimetry at pH 6.5 over a temperature range of 15–30°C. Calorimetric measurements in various buffer systems with different ionization heats suggest that no protons are exchanged during the binding of S -alkylglutathione derivatives. Thus, at pH 6.5, the protons released during the binding of substrate may be from its thiol group. Calorimetric analyses show that S -methyl-, S -butyl-, and S -octylglutathione bind to two equal and independent sites in the dimer of SjGST. The affinity of these inhibitors to SjGST is greater as the number of methylene groups in the hydrocarbon side chain increases. In all cases studied, Δ G 0 remains invariant as a function of temperature, while Δ H b and Δ S 0 both decrease as the temperature increases. The binding of three S -alkylglutathione derivatives to the enzyme is enthalpically favourable at all temperatures studied. The temperature dependence of the enthalpy change yields negative heat capacity changes, which become less negative as the length of the side chain increases.

Bovine lung angiotensin I-converting enzyme is a monomer with two active sites, in its two (N and C) homologous domains. A process is described for the preparative isolation of the ACE N-domain to either the ACE soluble monomer or ACE aggregated one. After exposure to moderate heat for 7 hours and in presence of a protease, ACE N-domain was obtained as a whole, and only fragments of the C-domain. N-domain purified was separated by Sephacryl S-300 HR chromatography, and a recovery of 80% was obtained. Molecular mass was estimated to be 100 kDa by sodium dodecyl sulfate gel electrophoresis. After purifying and partially sequencing this domain, we have investigated some catalytic properties and the inhibition by captopril.

In the Src Homology 3 domain (SH3) the RT and n-Src loops form a pocket that accounts for the specificity and affinity in binding of proline rich motifs (PRMs), while the distal and diverging turns play a key role in the folding of the protein. We have solved the structure of a chimeric mutant c-Src-SH3 domain where specific residues at the RT- and n-Src-loops have been replaced by those present in the corresponding Abl-SH3 domain. Crystals of the chimeric protein show a single molecule in the asymmetric unit, which appears in an unfolded-like structure that upon generation of the symmetry related molecules reveals the presence of a domain swapped dimer where both, RT- and n-Src loops, act as hinge loops. In contrast, the fold of the diverging type II β-turn and the distal loop are well conserved. Our results are the first evidence for the presence of a structured diverging type II β-turn in an unfolded-like intermediate of the c-Src-SH3 domain, which can be stabilized by interactions from the β-strands of the same polypeptide chain or from a neighboring one. Futhermore, this crystallographic structure opens a unique opportunity to study the effect of the amino acid sequence of the hinge loops on the 3D domain swapping process of c-Src-SH3.

The α-spectrin SH3 domain (Spc-SH3) is a small modular domain which has been broadly used as a model protein in folding studies and these studies have sometimes been supported by structural information obtained from the coordinates of Spc-SH3 mutants. The structure of B5/D48G, a multiple mutant designed to improve the hydrophobic core and as a consequence the protein stability, has been solved at 1 Å resolution. The crystals belonged to the orthorhombic space groupP212121, with unit-cell parametersa= 24.79,b= 37.23,c= 62.95 Å. This mutant also bears a D48G substitution in the distal loop and this mutation has also been reported to increase the stability of the protein by itself. The structure of the B5/D48G mutant shows a highly packed hydrophobic core and a more ordered distal loop compared with previous Spc-SH3 structures.

Ferrocene with a beta-cyclodextrin unit bound to one or both cyclopentadienyl rings through the secondary face were conveniently synthesized by regiospecific copper(I)-catalyzed cycloaddition of 2-O-propargyl-beta-cyclodextrin to azidomethyl or bis(azidomethyl)ferrocene. The supramolecular behavior of the synthesized conjugates in both the absence and presence of bile salts (sodium cholate, deoxycholate, and chenodeoxycholate) was studied by using electrochemical methods (cyclic and differential pulse voltammetry), isothermal titration calorimetry, and NMR spectroscopy (PGSE, CPMG, and 2D-ROESY). These techniques allowed the determination of stability constants, mode of inclusion, and diffusion coefficients for complexes formed with the neutral and, in some cases, the oxidized states of the ferrocenyl conjugates. It was found that the ferrocenyl conjugate with one beta-cyclodextrin unit forms a redox-controllable head-to-head homodimer in aqueous solution. The ferrocene-bis(beta-cyclodextrin) conjugate is present in two distinguishable forms in aqueous solution, each one having a different half-wave oxidation potential for the oxidation of the ferrocene. By contrast, only one distinguishable form for the oxidized state of the ferrocene-beta-cyclodextrin conjugate is detectable. The redox-sensing abilities of the synthesized conjugates towards the bile salts were evaluated based on the observed guest-induced changes in both the half-wave potential and the current peak intensity of the electroactive moiety.

dUTPase (deoxyuridine 5'-triphosphate nucleotide hydrolase) is an enzyme responsible for maintaining low levels of intracellular dUTP and thus prevents uracil incorporation into DNA by DNA polymerases during replication and repair processes. The thermodynamics of binding for both dUTP and dUMP (deoxyuridine 5'-monophosphate) to the D80A mutant form of Trypanosoma cruzi dUTPase have been investigated by fluorescence spectroscopy and high-sensitivity isothermal titration calorimetry. In the presence of magnesium, approximately a 30-fold decrease in the value of the k(cat) and a 15-fold increase in the K(m) for dUTP hydrolysis was calculated while a 5-fold decrease was observed in the affinity for dUMP. In the absence of magnesium, the affinity for dUTP binding was similar for both enzymes while that for dUMP was lowered 3-fold as a consequence of the mutation. Calorimetric titrations in several buffers with different ionization heats rendered similar proton exchanges during the binding of dUMP. Thus, apparently the side chain of Asp 80 does not seem to vary its protonation state during the binding process. The enthalpy change values for the D80A mutant hardly change with temperature and, in addition, were Mg(2+) independent. We conclude that the D80A mutation induces only a slight conformational change in the active site yet results in a significant alteration of nucleotide binding and modifies the ability of the enzyme to discriminate between dUTP and dUMP when magnesium is present.

The SH3 domain of the c-Src tyrosine kinase (c-Src-SH3) aggregates to form intertwined dimers and amyloid fibrils at mild acid pHs. In this work, we show that a single mutation of residue Gln128 of this SH3 domain has a significant effect on: (i) its thermal stability; and (ii) its propensity to form amyloid fibrils. The Gln128Glu mutant forms amyloid fibrils at neutral pH but not at mild acid pH, while Gln128Lys and Gln128Arg mutants do not form these aggregates under any of the conditions assayed. We have also solved the crystallographic structures of the wild-type (WT) and Gln128Glu, Gln128Lys and Gln128Arg mutants from crystals obtained at different pHs. At pH 5.0, crystals belong to the hexagonal space group P6522 and the asymmetric unit is formed by one chain of the protomer of the c-Src-SH3 domain in an open conformation. At pH 7.0, crystals belong to the orthorhombic space group P212121, with two molecules at the asymmetric unit showing the characteristic fold of the SH3 domain. Analysis of these crystallographic structures shows that the residue at position 128 is connected to Glu106 at the diverging β-turn through a cluster of water molecules. Changes in this hydrogen-bond network lead to the displacement of the c-Src-SH3 distal loop, resulting also in conformational changes of Leu100 that might be related to the binding of proline rich motifs. Our findings show that electrostatic interactions and solvation of residues close to the folding nucleation site of the c-Src-SH3 domain might play an important role during the folding reaction and the amyloid fibril formation., This research was funded by the Spanish Ministry of Science and Innovation and Ministry of Economy and Competitiveness and FEDER (EU): BIO2009-13261-C02-01/02 (ACA); BIO2012-39922-C02-01/02 (ACA); CTQ2013-4493 (JLN) and CSD2008-00005 (JLN); Andalusian Regional Government (Spain) and FEDER (EU): P09-CVI-5063 (ACA); and Valentian Regional Government (Spain) and FEDER (EU): Prometeo 2013/018 (JLN). Data collection was supported by European Synchrotron Radiation Facility (ESRF), Grenoble, France: BAG proposals MX-1406 (ACA) and MX-1541 (ACA); and ALBA (Barcelona, Spain) proposals 2012010072 (ACA) and 2012100378 (ACA).

In order to evaluate the ability of multivalent glycosides based on a beta-cyclodextrin core as site-specific molecular carriers, a study on both the inclusion complexation behaviour and lectin binding affinity of branched and hyperbranched beta-cyclodextrins is presented. A series of cluster galactosides constructed on beta-cyclodextrin scaffolds containing seven 1-thio-beta-lactose or beta-lactosylamine bound to the macrocyclic core through different spacer arms were synthesised. In addition, the first synthesis of three first-order dendrimers based on a beta-cyclodextrin core containing fourteen 1-thio-beta-D-galactose, 1-thio-beta-lactose and 1-thio-beta-melibiose residues was performed. Calorimetric titrations performed at 25 degrees C in buffered aqueous solution (pH 7.4) gave the affinity constants and the thermodynamic parameters for the complex formation of these beta-cyclodextrin derivatives with guests sodium 8-anilino-1-naphthalenesulfonate (ANS) and 2-naphthalenesulfonate, and lectin from peanut (Arachis hypogaea) (PNA). The persubstitution of the primary face of the beta-cyclodextrin with saccharides led to a slight increase of the binding constant values for the inclusion complexation with ANS relative to the native beta-cyclodextrin. However, the increase of the steric congestion due to the presence of the saccharide residues on the narrow rim of the beta-cyclodextrin may cause a decrease of the binding ability as shown for sodium 2-naphthalenesulfonate. The spacer arms are not passive elements and influence the host binding ability according to their chemical nature. PNA forms soluble cross-linked complexes with cluster galactosides and lactosides scaffolded on beta-cyclodextrin but not with cluster galactopyranosylamines or melibiose. Both, perbranched and hyperbranched beta-cyclodextrins, form stronger complexes with PNA than the monomeric analogues. However, the use of hyperbranched CDs does not contribute to the improvement of the complex stability relative to heptakis-glycocyclodextrin derivatives. Finally, a titration experiment with PNA and a complex formed by a heptakis lactose beta-cyclodextrin derivative with sodium 2-naphthalenesulfonate showed the formation of a soluble cross-linked complex with stronger affinity constant and higher stoichiometry than those observed for the complex formation of PNA with the same heptakis-lactose beta-cyclodextrin derivative, suggesting the formation of a three component complex.

The binding affinity of a series of electroactive glycoconjugates, based on a ferrocene core bearing alpha-mannose units on one or both of its cyclopentadienyl rings, to lectin Con A was studied by isothermal titration calorimetry (ITC) and voltammetry. Voltammetric measurements were performed by differential pulse adsorptive stripping voltammetry (DPAdSV). Upon complexation of ferrocene-mannose conjugates with Con A, voltammograms showed a decrease of the peak current. Both the monomannosylated ferrocene and the bis(mannosylated) ferrocene derivatives form more stable complexes with Con A than methyl alpha-D-mannopyranoside. Bis(mannosylated) ferrocene conjugates were found to bind to Con A with enhanced affinity due to the multivalent effect. A comparison of the thermodynamic data obtained by ITC and voltammetry is presented.

High-sensitivity titration calorimetry is used to measure changes in enthalpy, heat capacity and protonation for the binding of captopril to the angiotensin I-converting enzyme (ACE; EC 3.4.15.1). The affinity of ACE to captopril is high and changes slightly with the pH, because the number of protons linked to binding is low. The determination of the enthalpy change at different pH values suggests that the protonated group in the captopril-ACE complex exhibits a heat protonation of approximately −30 kJ/mol. This value agrees with the protonation of an imidazole group. The residues which may become protonated in the complex could be two histidines existing in two active sites, which are joined to the amino acids coordinated to Zn 2+ . Calorimetric measurements indicate that captopril binds to two sites in the monomer of ACE, this binding being enthalpically unfavorable and being dominated by a large positive entropy change. Thus, binding is favored by both electrostatic and hydrophobic interactions. The temperature dependence of the free energy of binding Δ G ° is weak because of the enthalpy-entropy compensation caused by a large heat capacity change, Δ C p =−4.3±0.1 kJ/K/mol of monomeric ACE. The strong favorable binding entropy and the negative Δ C p indicate both a large contribution to binding due to hydrophobic effects, which seem to originate from dehydration of the ligand-protein interface, and slight conformational changes in the vicinity of the active sites.