Titration Calorimetry Research Articles

Plasmodium falciparum dUTPase: Studies on protein stability and binding of deoxyuridine derivatives

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase), a ubiquitous enzyme preventing a deleterious incorporation of uracil into DNA, has been thought of as a novel target for anticancer and antiviral drug design. The interaction of Plasmodium falciparum dUTPase (PfdUTPase) with deoxyuridine derivatives (dU, dUMP, dUDP and dUpNHpp) has been studied thermodynamically by both isothermal titration and differential scanning calorimetry. ITC shows no cooperativity for the binding of these derivatives. Dependencies in the binding thermodynamic parameters (enthalpy, entropy and Gibbs energy changes) with the number of phosphate groups in the nucleotide are obtained, and from the heat capacity changes no significant conformational changes upon binding are inferred. DSC shows PfdUTPase trimer is very stable but denatures irreversibly, with a more complex denaturation profile than other homologous trimeric dUTPases. The presence of magnesium ions does not influence the denaturation profile, while the presence of deoxyuridine derivatives increases the stability. The increase depends upon nucleotide concentration and type, with dUDP having the greater effect.

Sulfur-Containing Hetero-Calix[4]pyrroles as Mercury(II) Cation-Selective Receptors: Thermodynamic Aspects

Two sulfur-containing hybrid calix[4]pyrrole derivatives (III and IV) have been synthesized and fully characterized. Several analytical techniques (1H NMR, conductance measurements, UV-vis spectrophotometry, titration potentiometry, and titration calorimetry) have been used to assess the interaction between these hybrid calixpyrrole receptors and metal cations in acetonitrile and dimethylsulfoxide. The partition constants of calix[4]pyrrole, I, II, and IV in the acetonitrile-hexane solvent system and the solubilities of the ligands in various solvents at 298.15 K were determined. 1H NMR measurements reveal the sites of interaction of calixpyrrole ligands with metal cations in CD3CN. Conductance and UV-vis spectrophotometric measurements were performed to establish the composition of mercury(II) calixpyrrole complexes in acetonitrile at 298.15 K. Titration calorimetry was used to quantitatively assess Hg(II)-calixpyrrole interactions. Thus the thermodynamics of complexation of calixpyrrole ligands with the mercury(II) cation in acetonitrile at 298.15 K are reported. Potentiometric titrations were also used to establish the stepwise stability constants for the complexation of calix[3]thieno[1]pyrrole with the Hg(II) cation in acetonitrile at 298.15 K. The results show that replacement of one or more pyrrole units by thiophene rings in calix[4]pyrrole has tuned significantly the discrimination ability of these ligands between anions and enables the produced hybrid calixpyrroles to bind selectively with Hg(II) in acetonitrile. No interaction was observed between these ligands and other metal cations in acetonitrile.

Host–Guest Complexation of β-, γ-Cyclodextrin with Alkyl Trimethyl Ammonium Bromides in Aqueous Solution

As a continuation of our previous investigation, interactions between cyclodextrin (β-CD), γ-cyclodextrin (γ-CD) and alkyl trimethylammonium bromides in aqueous solutions have been studied with titration calorimetry and 1H NMR at 298.15 K. The results are discussed in terms of the amphiphilic interaction of CD with surfactants and the iceberg structure formed by water molecules existing around the hydrophobic tail of surfactant molecules. The stoichiometry of the β-CD–surfactant system is 1:1 whereas that of the γ-CD–surfactant system is 1:2. The corresponding formation enthalpy (negative) of the complexes of the two systems decreases with an increase in the number of carbon atoms (n) in hydrophobic chain of surfactant molecule, C n H2n+1, whereas the entropy increases with the enlargement of n.

RNA channelling by the archaeal exosome

Exosomes are complexes containing 3' --> 5' exoribonucleases that have important roles in processing, decay and quality control of various RNA molecules. Archaeal exosomes consist of a hexameric core of three active RNase PH subunits (ribosomal RNA processing factor (Rrp)41) and three inactive RNase PH subunits (Rrp42). A trimeric ring of subunits with putative RNA-binding domains (Rrp4/cep1 synthetic lethality (Csl)4) is positioned on top of the hexamer on the opposite side to the RNA degrading sites. Here, we present the 1.6 A resolution crystal structure of the nine-subunit exosome of Sulfolobus solfataricus and the 2.3 A structure of this complex bound to an RNA substrate designed to be partly trimmed rather than completely degraded. The RNA binds both at the active site on one side of the molecule and on the opposite side in the narrowest constriction of the central channel. Multiple substrate-binding sites and the entrapment of the substrate in the central channel provide a rationale for the processive degradation of extended RNAs and the stalling of structured RNAs.

A New Calix[4]pyrrole Derivative and Its Anion (Fluoride)/Cation (Mercury and Silver) Recognition

A new calix[4]pyrrole-based macrocycle, meso-tetramethyl-tetrakis{4-[2-(ethylthio)ethoxy]phenyl}calix[4]pyrrole, 7, has been synthesized and fully characterized. Unlike other calixpyrrole derivatives that show selective interaction with anions, calixpyrrole 7 described in the present work forms stable complexes with both metal cations and anions. The thermodynamics of complexation of this ditopic calixpyrrole derivative with metal cations (Hg2+ and Ag+) and the fluoride anion in nonaqueous solutions have been determined by titration calorimetry, and the host-guest composition has been investigated by using conductance measurements at 298.15 K. 1H NMR studies provide clear evidence about the sites of complexation of 7 with the ionic species, which show that the NH groups are taking part in the complexation of this ligand with the fluoride anion while the sulfur donor atoms are responsible for the interaction with metal cations. Using the present data on 7 and structurally related analogues (1-6), the complexation behavior is discussed comparatively from the thermodynamic point of view. Possessing four sulfur-containing pendent arms, 7 displays an enhanced hosting ability for Hg2+ in acetonitrile. As compared with 1, the calixpyrrole derivative, 7, shows a unique interaction with fluoride among the anions investigated in acetonitrile and dimethyl sulfoxide. As far as the fluoride complex is concerned, the medium effect is assessed in terms of the thermodynamics of the transfer of reactants and product from acetonitrile (reference solvent) to dimethyl sulfoxide.

Phosphoprotein and Phosphopeptide Interactions with the FHA Domain from Arabidopsis Kinase-Associated Protein Phosphatase

FHA domains are phosphoThr recognition modules found in diverse signaling proteins, including kinase-associated protein phosphatase (KAPP) from Arabidopsis thaliana. The kinase-interacting FHA domain (KI-FHA) of KAPP targets it to function as a negative regulator of some receptor-like kinase (RLK) signaling pathways important in plant development and environmental responses. To aid in the identification of potential binding sites for the KI-FHA domain, we predicted (i) the structure of a representative KAPP-binding RLK, CLAVATA1, and (ii) the functional surfaces of RLK kinase domains using evolutionary trace analysis. We selected phosphopeptides from KAPP-binding Arabidopsis RLKs for in vitro studies of association with KI-FHA from KAPP. Three phosphoThr peptide fragments from the kinase domain of CLV1 or BAK1 were found to bind KI-FHA with KD values of 8-20 microM, by NMR or titration calorimetry. Their affinity is driven by favorable enthalpy and solvation entropy gain. Mutagenesis of these three threonine sites suggests Thr546 in the C-lobe of the BAK1 kinase domain to be a principal but not sole site of KI-FHA binding in vitro. The brassinosteroid receptor BRI1 and KAPP are shown to associate in vivo and in vitro. Further genetic studies indicate that KAPP may be a negative regulator of the BRI1 signaling transduction pathway. 15N-Labeled KI-FHA was titrated with the GST-BRI1 kinase domain and monitored by NMR. BRI1 interacts with the same 3/4, 4/5, 6/7, 8/9, and 10/11 recognition loops of KI-FHA, with similar affinity as the phosphoThr peptides.

Synthesis, Solution Thermodynamics, and X‐ray Study of CuII [12]Metallacrown‐4 with GABA Hydroxamic Acid: An Unprecedented Crystal Structure of a [12]MC‐4 with a γ‐Aminohydroxamate

The solution equilibria of gamma-aminobutanehydroxamic acid (GABAha) with H+ and Cu2+ were investigated by potentiometry, titration calorimetry, spectrophotometry, NMR spectroscopy, and ESI-MS. The thermodynamic parameters of the CuII [12]metallacrown-4 obtained for GABAha were compared with those of the corresponding complexes of (S)-alpha-Alaha and beta-Alaha. The stability (-DeltaG0) sequence was beta-Alaha>>alpha-Alaha>GABAha, whereas the order of formation enthalpies (-DeltaH0) was beta-Alaha>>GABAha>alpha-Alaha. These data were interpreted on the basis of the dimensions of the chelate rings and the planarity of the metallamacrocycles. The CuII [12]metallacrown-4 ([12]MC-4) complex of GABAha was isolated and its crystal structure, which is the first reported for a [12]MC-4 of a gamma-aminohydroxamic acid, fully supports the structural features interpreted from the thermodynamic data.

Mechanism and Driving Force of NO Transfer from S-Nitrosothiol to Cobalt(II) Porphyrin: A Detailed Thermodynamic and Kinetic Study

The thermodynamics and kinetics of NO transfer from S-nitrosotriphenylmethanethiol (Ph(3)CSNO) to a series of alpha,beta,gamma,delta-tetraphenylporphinatocobalt(II) derivatives [T(G)PPCoII], generating the nitrosyl cobalt atom center adducts [T(G)PPCoIINO], in benzonitrile were investigated using titration calorimetry and stopped-flow UV-vis spectrophotometry, respectively. The estimation of the energy change for each elementary step in the possible NO transfer pathways suggests that the most likely route is a concerted process of the homolytic S-NO bond dissociation and the formation of the Co-NO bond. The kinetic investigation on the NO transfer shows that the second-order rate constants at room temperature cover the range from 0.76 x 10(4) to 4.58 x 10(4) M(-1) s(-1), and the reaction rate was mainly governed by activation enthalpy. Hammett-type linear free-energy analysis indicates that the NO moiety in Ph(3)CSNO is a Lewis acid and the T(G)PPCoII is a Lewis base; the main driving force for the NO transfer is electrostatic charge attraction rather than the spin-spin coupling interaction. The effective charge distribution on the cobalt atom in the cobalt porphyrin at the various stages, the reactant [T(G)PPCoII], the transition-state, and the product [T(G)PPCoIINO], was estimated to show that the cobalt atom carries relative effective positive charges of 2.000 in the reactant [T(G)PPCoII], 2.350 in the transition state, and 2.503 in the product [T(G)PPCoIINO], which indicates that the concerted NO transfer from Ph(3)CSNO to T(G)PPCoII with the release of the Ph(3)CS* radical was actually performed by the initial negative charge (-0.350) transfer from T(G)PPCoII to Ph(3)CSNO to form the transition state and was followed by homolytic S-NO bond dissociation of Ph(3)CSNO with a further negative charge (-0.153) transfer from T(G)PPCoII to the NO group to form the final product T(G)PPCoIINO. It is evident that these important thermodynamic and kinetic results would be helpful in understanding the nature of the interaction between RSNO and metal porphyrins in both chemical and biochemical systems.

Thermodynamic Study for the Protonation of Branched Poly(ethylenimine) in NaCl(aq) and Its Dependence on Ionic Strength

In this paper, we investigated the interactions between poly(ethylenimine) [BPEI] and the proton by using (H+-glass electrode) potentiometry and titration calorimetry. The measurements were carried out in NaCl aqueous solutions, at T = 298 K in a wide range of ionic strength (up to I ≈ 5.0 mol·L-1). The protonation constants were calculated by following different approaches: (a) first, all the protonation constants were considered as true constants (considering BPEI as a simple low molecular weight tetramine); (b) second, the log KH3 was considered as a linear function of p̄ (p̄ = protonation degree); and (c) the log KH3 was considered as a nonlinear function of p̄. The difference in the standard deviation on the fit between (a) and (b) calculations is quite significant, while no further improvement is observed by using the (c) model. The protonation enthalpies were calculated from the calorimetric titrations. In these calculations, all the protonation steps of BPEI were considered as independent of p̄. The dependence of protonation constants and enthalpies on ionic strength was modeled using the Specific ion Interaction Theory (SIT).

Low-Affinity Signature of the Rat β-Parvalbumin CD Site. Evidence for Remote Determinants

Although rat beta-parvalbumin and chicken parvalbumin 3 (CPV3) are identical at 74 of 108 residues, rat beta exhibits perceptibly lower Ca2+ and Mg2+ affinities. At 25 degrees C, in Hepes-buffered saline, at pH 7.4, the overall deltadeltaG degrees ' values are 2.0 and 3.9 kcal/mol, respectively. These differences primarily reflect the disparate behavior of the CD sites in the two proteins. Their respective binding constants for Ca2+, for example, are 1.5 x 10(6) and 2.4 x 10(7) M-1. The extent to which this differential behavior is dictated by local and remote sequence differences is unknown. To explore this question, we performed mutagenesis on rat beta, substituting the corresponding CPV3 codon for residues 49, 50, 57, 58, 59, and 60. The resulting CD site is identical to CPV3 at 27 of 30 positions. The mutations were introduced in four stages, replacing residues 49 and 50 (yielding beta 49/50), then 57 and 58 (beta 49/50/57/58), then 59 (beta 49/50/57/58/59), and finally 60 (beta 49/50/57/58/59/60). Apoprotein stability was examined by scanning calorimetry and chemical denaturation and divalent ion affinity by titration calorimetry. All four variants exhibit elevated Tm values and are between 0.13 and 0.39 kcal/mol more stable at 25 degrees C. Although all four proteins display heightened divalent ion affinity, the increases are small. The maximal deltadeltaG degrees ' values, observed for 49/50/57/58/59/60, are just -0.56 and -0.96 kcal/mol for Ca2+ and Mg2+, respectively. Evidently, structural features beyond the metal ion-binding motif contribute to the unusual divalent ion-binding behavior associated with the rat beta CD site.

Structural Reorganization and the Cooperative Binding of Single-stranded Telomere DNA in Sterkiella nova

In Sterkiella nova, alpha and beta telomere proteins bind cooperatively with single-stranded DNA to form a ternary alpha.beta.DNA complex. Association of telomere protein subunits is DNA-dependent, and alpha-beta association enhances DNA affinity. To further understand the molecular basis for binding cooperativity, we characterized several possible stepwise assembly pathways using isothermal titration calorimetry. In one path, alpha and DNA first form a stable alpha.DNA complex followed by the addition of beta in a second step. Binding energy accumulates with nearly equal free energy of association for each of these steps. Heat capacity is nonetheless dramatically different, with DeltaCp = -305 +/- 3 cal mol(-1) K(-1) for alpha binding with DNA and DeltaCp = -2010 +/- 20 cal mol(-1) K(-1) for the addition of beta to complete the alpha.beta.DNA complex. By examining alternate routes including titration of single-stranded DNA with a preformed alpha.beta complex, a significant portion of binding energy and heat capacity could be assigned to structural reorganization involving protein-protein interactions and repositioning of the DNA. Structural reorganization probably affords a mechanism to regulate high affinity binding of telomere single-stranded DNA with important implications for telomere biology. Regulation of telomere complex dissociation is thought to involve post-translational modifications in the lysine-rich C-terminal portion of beta. We observed no difference in binding energetics or crystal structure when comparing complexes prepared with full-length beta or a C-terminally truncated form, supporting interesting parallels between the intrinsically disordered regions of histones and this portion of beta.

Quantitative Dissection of the Notch:CSL Interaction: Insights into the Notch-mediated Transcriptional Switch

Complex formation between the intracellular domain of the Notch receptor (NICD) and the transcription factor CSL is indispensable for transcriptional activation. To understand how NICD displaces CSL-associated co-repressors, we have quantified the binding of different Notch1 ICD regions to a key interaction domain (the beta trefoil domain, or BTD) of human CSL. Electrophoresis, scattering, and titration calorimetry indicate that NICD and BTD combine to form a 1:1 heterodimer. Neither the Notch1 ankyrin domain (ANK) nor C-terminal region contributes binding energy towards BTD. In contrast, binding energy is attributed largely to a short segment including the conserved WFP sequence motif within the RAM region (the ∼140 residue polypeptide segment N-terminal to the ANK domain); substitution of this motif substantially reduces affinity. Short (≤25 residues) WFP-containing peptides encoded by the four mammalian Notch genes have similar affinities to BTD; thus, activity differences between paralogues either result from other regions of NICD and CSL or from differences in interaction with downstream components. The importance of RAM was demonstrated by the ability of a short RAM peptides to dissociate NICD:CSL interaction in cellular lysates. These results support an emerging molecular mechanism for the displacement of co-repressors from DNA-bound CSL by NICD.

Determination of the Substrate Specificities of N-Acetyl-d-glucosaminyltransferase

Heparan sulfate plays a wide range of physiological and pathological roles. Heparan sulfate consists of glucosamine and glucuronic/iduronic acid repeating disaccharides with various sulfations. Synthesis of structurally defined heparan sulfate oligosaccharides remains a challenge. Access to nonsulfated and unepimerized heparan sulfate backbone structures represents an essential step toward de novo enzymatic synthesis of heparan sulfate. The nonsulfated, unepimerized backbone heparan sulfate is similar to the capsular polysaccharide from Escherichia coli strain K5. The biosynthesis of this capsular polysaccharide involves in N-acetylglucosaminyltransferase (KfiA) and d-glucuronyltransferase (KfiC). In this study, we report the characterization of purified KfiA. KfiA was expressed in a C-terminal six-His fusion protein in BL21 star cells coexpressing chaperone proteins GroEL and GroES. The recombinant KfiA was purified to homogeneity with a Ni-agarose column. The binding affinities of various UDP-sugars for KfiA were determined using isothermal calorimetry titration, indicating that both the N-acetyl group and sugar type may be essential for donor substrates to bind KfiA. Kinetic analysis of KfiA toward different sizes of oligosaccharide revealed that KfiA is less sensitive to the size of the acceptor substrates. The results from this study open a new approach for the synthesis of the heparan sulfate backbone.

Cobalt(II) chloro complexation in N-methylformamide–N, N-dimethylformamide mixtures

Cobalt(II) chloro complexation has been studied by titration calorimetry and spectrophotometry in solvent mixtures of N-methylformamide (NMF) and N,N-dimethylformamide (DMF). It revealed that a series of mononuclear CoClnn (2–n)+ (n=1–4) complexes are formed in the mixtures of NMF mole fraction x NMF=0.05 and 0.25, and the CoCl+, CoCl3 – and CoCl4 2– complexes in the mixture of x NMF=0.5, and their formation constants, enthalpies and entropies were obtained. As compared with DMF, the complexation is markedly suppressed in the mixtures, as well as in NMF. The decreasing formation constant of CoCl+ with the NMF content is mainly ascribed to the decreasing formation entropy. DMF is aprotic and thus less-structured, whereas NMF is protic to form hydrogen- bonded clusters. In DMF-NMF mixtures, solvent clusters in neat NMF are ruptured to yield new clusters involving DMF, the structure of which depends on the solvent composition. The entropy of formation of CoCl+ will be discussed in relation to the liquid structure of DMF, NMF and their mixtures.

Activity coefficients at infinite dilution and enthalpies of solution of methanol, 1-butanol, and 1-hexanol in 1-hexyl-3-methyl-imidazolium bis(trifluoromethyl-sulfonyl) imide

Activity coefficients at infinite dilution γ i ∞ of methanol, 1-butanol, and 1-hexanol in the ionic liquid 1-hexyl-3-methyl-imidazolium bis(trifluoromethyl-sulfonyl) imide ([HMIM][NTf 2]) have been determined by gas chromatography using the ionic liquids as stationary phase. The measurements were carried out at different temperatures between 298 K and 396 K. From the temperature dependence of the limiting activity coefficients partial molar excess enthalpies at infinite dilution H i E, ∞ of the alcohol in the ionic liquid have been derived. Additionally, enthalpies of solution of the same alcohols in the [HMIM][NTf 2] have been measured at T = 298.15 K in the range of low concentrations using titration calorimetry. Results at infinite dilution, H i E, ∞ , are compared with those indirectly obtained from activity coefficients in infinite dilution γ i ∞ . Within the experimental error of both methods thermodynamic consistency has been confirmed.

Dioxouranium(VI)–carboxylate complexes: A calorimetric and potentiometric investigation of interaction with oxalate at infinite dilution and in NaCl aqueous solution at I = 1.0 mol L −1 and T = 25 °C

In this paper we investigated the interactions between dioxouranium(VI) and oxalate using (H +-glass electrode) potentiometry and titration calorimetry. Potentiometric measurements were carried out in NaCl aqueous solutions and at T = 25 °C in a wide range of experimental conditions (concentrations, ligand/metal molar ratio, pH, titrants) at low ionic strength values ( I ≤ 0.090 mol L −1, without supporting electrolyte) and at I = 1.0 mol L −1; different procedures were employed for the acquisition of experimental data and careful analysis of these data performed. In all cases the speciation model that best fits experimental data takes into account the formation of the binary mononuclear species UO 2(ox) 0, UO 2(ox) 2 2−, UO 2(ox) 3 4− widely reported in literature, the ternary hydroxyl mononuclear species UO 2(ox)OH −, UO 2(ox)(OH) 2 2−, UO 2(ox) 2OH 3−, UO 2(ox) 3OH 5−, the protonated ternary mononuclear species UO 2(ox) 3H 3− and the binuclear species (UO 2) 2(ox) 5 6−. Calorimetric measurements were carried out following similar procedures and in the same experimental conditions as employed for the potentiometric measurements at I = 1.0 mol L −1 in NaCl. The stability of UO 2 2+–oxalate 2− complexes is fairly high and their main contribution to stability is entropic in nature. Some linear empirical relationships were found which make it possible to calculate (i) the contribution of a single bond: Δ G sb ° = − 16.6 ± 0.2 kJ mo l − 1 and T Δ S sb ° = 16.2 ± 0.5 kJ mo l − 1 ; (ii) chelate stabilisation per ring: Δ G r ° = − 1.30 ± 0.04 kJ mo l − 1 and T Δ S r ° = 1.27 ± 0.23 kJ mo l − 1 and (iii) the mean stability of negatively charged Na +-ion pair complexes: log T K = (0.46 ± 0.02)·| z| ( z = charge of complex species), Δ G° = −(2.60 ± 0.1)·| z| kJ mol −1 and TΔ S° = 2.5 ± 0.5 kJ mol −1. Both potentiometric and calorimetric results provide evidence of the penta-coordination of the species UO 2(ox) 3 4−. SIT parameters were calculated from the data at I = 0 and I = 1.02 mol kg −1. Comparisons are made with literature data. An insoluble dioxouranium(VI) ternary complex was synthesised (at I = 1.0 mol L −1 in NaCl) and characterised by thermoanalysis and elemental analysis.

Structural and Thermodynamic Basis for the Enhanced Transcriptional Control by the Human Papillomavirus Strain-16 E2 Protein

Strain 16 of the human papillomavirus is responsible for the largest number of cases of cervical cancers linked to this virus, and the E2 protein is the transcriptional regulator of all viral genes. We present the first structure for the DNA binding domain of HPV16 E2 bound to DNA, and in particular, to a natural cognate sequence. The NMR structure of the protein backbone reveals that the overall conformation remains virtually unchanged, and chemical shift analysis of the protein bound to a shorter DNA duplex uncovered a contact out of the minimal E2 DNA binding site, made by lysine 349. This contact was confirmed by titration calorimetry and mutagenesis, with a contribution of 1.0 kcal mol(-)(1) to binding energy. HPV16 E2 has the highest DNA binding affinity and exerts a strict transcriptional control, translated into the repression of the E6 and E7 oncogenes. These novel features provide the structural and thermodynamic basis for this tight transcriptional control, the loss of which correlates with carcinogenesis.

Binding of α-Synuclein Affects the Lipid Packing in Bilayers of Small Vesicles

The intracellular deposition of fibrillar aggregates of alpha-synuclein is a characteristic feature of Parkinson disease. Alternatively, as a result of its unusual conformational plasticity, alpha-synuclein may fold into an amphipathic helix upon contact with a lipid-water interface. Using spin label ESR and fluorescence spectroscopy, we show here that alpha-synuclein affects the lipid packing in small unilamellar vesicles. The ESR hyperfine splittings of spin-labeled phospholipid probes revealed that alpha-synuclein induces chain ordering at carbon 14 of the acyl chains below the chain melting phase transition temperature but not in the liquid crystalline state of electroneutral vesicle membranes. Binding of alpha-synuclein leads to an increase in the temperature and cooperativity of the phase transition according to the fluorescence anisotropy of the hydrophobic polyene 1,6-diphenylhexatriene and of the fluorescence emission maxima of the amphiphilic probe 6-dodecanoyl-2-dimethylaminonaphthalene. Binding parameters were obtained from the fluorescence anisotropy measurements in combination with our previous determinations by titration calorimetry (Nuscher, B., Kamp, F., Mehnert, T., Odoy, S., Haass, C., Kahle, P. J., and Beyer, K. (2004) J. Biol. Chem. 279, 21966-21975). We also show that alpha-synuclein interacts with vesicle membranes containing sphingomyelin and cholesterol. We propose that the protein is capable of annealing defects in curved vesicle membranes, which may prevent synaptic vesicles from premature fusion.

Construction of solid–liquid phase diagrams in ternary systems by titration calorimetry

Titration calorimetry was used to construct the solid–liquid equilibrium line in ternary systems containing the solute and an aqueous mixed solvent by measuring the heat of dissolution of the solid solute during successive additions of the liquid solvent. The plot of cumulated heats versus the mole ratio, n solvent/ n solute, yields two (almost) linear increases of different slopes. These two lines represent successively the enthalpy of dissolution then the enthalpy of dilution of the medium; their intersection gives the solubility and the enthalpy of dissolution of the solute. Phase diagrams have been established over the whole concentration range for o-anisaldehyde, 1,3,5-trimethoxybenzene and vanillin, in water + methanol, +ethanol, or + n-propanol at 303, 313 and 318 K.

Activated Peroxide for Enzymatic Control of Wool Shrinkage Part I: Elucidation

The ARS process combines activated peroxide for pretreatment and enzyme for subsequent treatment to provide bleached, biopolished, and shrinkage-resistant wool. The pretreatment step is of particular interest because it combines dicyandiamide (DD) with alkaline peroxide to form peroxycarboximidic acid, a stable and powerful bleaching agent that is stabilized by gluconic acid (GA) when applied at pH 11.5 for 30 minutes at 30 ° C. Analytical methods of analysis provided information showing that the active bleaching component, peroxycarboximidic acid, is formed immediately upon the combination of DD with alkaline H2O2 and that cyanamide forms concomitantly as a co-product. The peroxide pretreatment bath was stable at room temperature for up to 4.5 hours with only 10% peroxide consumed. It should therefore be possible to reconstitute the bleaching bath for further utilization. The eventual alkaline hydrolysis of cyanamide to form urea and the recombination of urea with peroxycarboximidic acid formed the end-product, guanylurea, shown by high performance liquid chromatography associated with electron impact mass spectrometry (LC/EI-MS), Fourier transforminfrared (FT-IR), and 13C NMR. Differential scanning calorimetry and peroxide titration of pretreatment baths provided evidence that GA stabilizes the activated peroxide. Activated DD peroxide pretreatment is essential to the ARS process for it provides a high level of whiteness without loss in fabric properties and it prepares the wool fiber for enzymatic treatment designed to selectively modify the scales of wool to biopolish the fabric surface and provide itch-free, machine-washable wool.