Microanalysis of the surface concentration of sulfate groups at polystyrene particle by isothermal titration calorimetry

 Microanalysis of sulfate groups at polystyrene particle surfaces, which were derived as persulfate initiator fragments, was carried out with isothermal titration calorimetry, and compared with a conventional conductometric titration. The quantitative analysis was possible even with an extremely small number of polystyrene particles have 10 μmol sulfate groups. Received: 15 December 1998 Accepted in revised form: 24 February 1999     

Estimation of water absorption state within ionized carboxylated polymer particles with high sensitive differential scanning calorimetry

Glass transition temperature (Tg) of submicron-sized, carboxylated polymer particles dispersed in aqueous media, which were prepared by emulsion copolymerization of styrene, iso-butyl methacrylate, or methyl methacrylate with methacrylic acid, was measured at alkali or acidic pH region with a power compensation-type high sensitive differential scanning calorimeter. The Tg of relatively hydrophilic polymer particles was obviously decreased by the neutralization of the carboxyl groups with KOH, whereas that of hydrophobic polymer particles was not changed though water was absorbed therein. These results indicate that water absorption state, which means not only the amount of water absorption but also the heterogeneity of the ionized carboxylated polymer particles, markedly depends on the hydrophilicity of their base polymers. This strongly supports the formation mechanism of multihollow particles by the stepwise alkali/acid or the alkali/cooling treatments of carboxylated polymer particles proposed by the authors.Part CCLXIX of the series, “Studies on Suspension and Emulsion.”     

Applications of isothermal titration calorimetry to measure enzyme kinetics and activity in complex solutions

The application of isothermal titration calorimetry (ITC) was tested towards measurements of enzyme kinetics in complex solutions containing high concentrations of proteins. Such investigations are important, due to the increasing interest in biochemical reactions in physiological relevant media as well as the application of enzymes in industrial processes. In contrast to spectral methods, measurements performed with ITC, are independent of the optical properties of solutions, making it possible to measure enzyme kinetics in concentrated solutions of macromolecules. In this study the kinetic properties of hexokinase was investigated in concentrated protein solutions (BSA). It was found, that the quality of the measured kinetic data was independent of protein concentration in the investigated range (0-250 mg BSA ml⁻¹). All results could be accounted for by Michaelis-Menten's approach and both k_cat and K_M decreased with increasing protein concentrations. The decrease in K_M with increasing protein concentration was ascribed to an increase in the ratio of activity coefficients between the native enzyme and the enzyme-substrate complex. The decrease in k_cat with increasing protein concentrations indicates that crowding by BSA effect the conformational changes/rehydration that accompanies catalysis and/or diffusion of product from the enzyme-product complex. The methodology is discussed together with an analysis of the experimental results.     

Complexation of triptolide and its succinate derivative with cyclodextrins: affinity capillary electrophoresis, isothermal titration calorimetry and 1H NMR studies

The complexation of the triptolide PG490 and its succinate derivative PG490-88Na with various cyclodextrins was studied using three complementary techniques: affinity capillary electrophoresis (ACE), isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR). The apparent binding constants of the complexes formed between the drugs and 8 CDs (α-CD, β-CD, γ-CD, HP-α-CD, HP-β-CD, HP-γ-CD, CM-β-CD and amino-β-CD) were determined by ACE through linear Scott's plots. The apparent and averaged binding constants of the complexes formed between PG490-88 and β-CD, γ-CD, HP-α-CD, HP-β-CD or HP-γ-CD are contained in the narrow range 135-167 M(-1). For the anionic CM-β-CD and cationic amino-β-CD, these constants are 38 and 278 M(-1), respectively, which is in accordance with electrostatic repulsions or attractions with the succinate moiety. ITC and NMR investigations for the binding constants determinations were performed for 2 CDs allowing high complexation: HP-β-CD and amino-β-CD. The three techniques provided similar results. ITC and NMR, in contrast to ACE, allowed to study the complexes formed between the neutral compound PG490 and neutral cyclodextrins. A more advanced characterization of the PG 490-88Na/amino-β-CD complex, which displays the highest apparent binding constant, was undertaken using NMR spectroscopy. The 1:1 stoichiometry of the complex was established by (1)H NMR 1D and selective 1D TOCSY experiments using the continuous variation method. Moreover, the 1D and 2D ROESY experiments revealed the inclusion of the isopropyl moiety of the triptolide derivative in the hydrophobic CD cavity. Altogether, the data provide strong evidences that the two triptolide compounds can be efficiently complexed with CD.     

Rapid and efficient screening of adsorbent for oligopeptide using molecular docking and isothermal titration calorimetry

A rapid and efficient method based on molecular docking and isothermal titration calorimetry (ITC) was developed to identify effective adsorbents for the target peptide Ser‐Glu‐Ala‐Asp‐His (SEADH). Preliminary screening of five candidate adsorbents using molecular docking revealed that three suitable structures (A1, A2, and A3) either with or without coordination of Zn²⁺ should be effective. The three selected structures were then prepared and further screened by evaluating their affinities for the peptide SEADH using ITC. The screening results revealed that the adsorbent A2 coordinated with Zn²⁺ was the best adsorbent, and subsequent static adsorption experiments confirmed the reliability of the screening method. Further ITC analysis, combined with molecular docking, was performed to provide the possible adsorption mechanism.     

Application of a new method for data analysis of isothermal titration calorimetry in the interaction between human serum albumin and Ni

The interaction of human serum albumin (HAS) with divalent nickel ion was studied by isothermal titration calorimetry (ITC) in 30 mM Tris buffer, pH 7.0. There is a set of eight identical and independent binding sites for nickel ions on the protein at the temperature of 300 K. A new calorimetric data analysis allows the determination of the complete set of thermodynamic parameters. The binding isotherm for nickel-HSA interaction is easily obtained by carrying out two different ITC experiments. In the first experiment, the enthalpy of binding for one mole of nickel ion to one mole of binding site on HSA (ΔH=−36.5 kJ) is obtained, and is used in a second experiment to determine the binding isotherm and to find the number of binding sites (g=8) and the equilibrium constant (K=0.57 μM⁻¹).     

The use of isothermal titration calorimetry to assess the solubility enhancement of simvastatin by a range of surfactants

Surfactants are commonly used to increase the solubility of poorly water soluble drugs but the interactions between drug and surfactant can be complex and quantitative relationships can be hard to derive. One approach is to quantify the thermodynamics of interaction and relate these parameters to known solubility or dissolution rate enhancement data. Isothermal titration calorimetry (ITC) was used to measure the enthalpy and free energy of transfer of a model drug (simvastatin) to a number of surfactant (SDS, HTAB, SDCH and Brij 35) micelles. These data were then compared with the solubility enhancements determined for each surfactant using HPLC assays. As expected, there was correlation between the free energy of transfer for the drug to each surfactant and the solubility enhancement of that surfactant. Although the data set is limited, the results suggest that ITC screening of a range of surfactants against a poorly water soluble drug may allow the selection of the best potential solubilising surfactants.     

Localization of carboxyl groups at surface layer of carboxylated polymer emulsion particles by alkali treatment

For the purpose of localizing carboxyl groups from inside to particle surface, styrene — butyl acrylate — methacrylic acid (74.3/17.0/8.7, mol ratio) terpolymer emulsion was kept under pH 9 at different temperatures. The amount of carboxyl groups at the particle surface, As, was remarkably increased by the alkali-treatment above 35 °C. On the other hand, As value of the alkalitreated emulsion was decreased by keeping under ph 3 above 45 °C, although in the case of the original emulsion without the alkalitreatment, it was not changed by the acid-treatment. These results suggest that a part of polymer segments which have ionized carboxyl groups is dragged out at the surface by an increase in the affinity of the groups against water, and the dragged segments turn back into the inside again when the carboxyl groups are deionized.Part 97 of the series Studies on Suspension and Emulsion.     

Analysis of the interactions between human serum albumin/amphiphilic penicillin in different aqueous media: an isothermal titration calorimetry and dynamic light scattering study

The complexation process of the amphiphilic penicillins sodium cloxacillin and sodium dicloxacillin with the protein human serum albumin (HSA) in aqueous buffered solutions of pH 4.5 and 7.4 at 25 °C was investigated through isothermal titration calorimetry (ITC) and dynamic light scattering. ITC experiments were carried out in the very dilute regime and showed that although hydrophobic interactions are the leading forces for complexation, electrostatic interactions also play an important role. The possibility of the formation of hydrogen bonds is also deduced from experimental data. The thermodynamic quantities of the binding mechanism, i.e, the enthalpy, , entropy, , Gibbs energy, , binding constant, and the number of binding sites, n_i, were obtained. The binding was saturable and is characterised by Langmuir adsorption isotherms. From ITC data and following a theoretical model, the number of bound and free penicillin molecules was calculated. From Scatchard plots, and n_i were obtained and compared with those from ITC data. The interaction potential between the HSA–penicillin complexes and their stability were determined at pH 7.4 from the dependence of the diffusion coefficients on protein concentration by application of the DLVO colloidal stability theory. The results indicate decreasing stability of the colloidal dispersion of the drug–protein complexes with increase in the concentration of added drug.     

Kinetics and enthalpy measurements of interaction between beta-amyloid and liposomes by surface plasmon resonance and isothermal titration microcalorimetry

The objective of this research is to understand the interaction mechanism of β-amyloid (Aβ) with cell and were basically divided into two parts. The first part focused on the time-dependent structural changes of Aβ (1-40) by circular dichroism (CD) spectroscopy, thioflavin T (ThT) fluorescence assay, and atomic force microscopy (AFM). The second part emphasized the kinetics and enthalpy of interaction between Aβ (1-40) and liposome by surface plasmon resonance (SPR) and isothermal titration microcalorimetry (ITC). Results obtained from CD, ThT and AFM confirmed the formation of 1 μm fibril after single day incubation. The driving force of kinetic interaction between Aβ and liposomes was revealed by SPR to be electrostatics. Further studies indicated that fresh Aβ has high GM1 affinity. Besides, addition of cholesterol to the liposome could alter membrane fluidity and affect the interactions of fresh Aβ with liposomes especially in the amount of Aβ absorbed and preserving the structure of liposome after adsorbing. Hydrophobicity was found to be the driving force leading to the interaction between Aβ fibrils and liposomes. These reactions are endothermic as supported by ITC measurements. When the composition of liposomes is zwitterionic lipids, the interaction of Aβ with liposomes is predominantly hydrophobic force. In contrast, the driving force of interaction of charged lipids with Aβ is electrostatic.     

In‐depth insight into the methods of plasma protein‐drug interaction studies: Comparison of capillary electrophoresis‐frontal analysis,isothermal titration calorimetry,circular dichroism and equilibrium dialysis

Plasma protein‐drug binding assays are routinely performed during the early stages of drug discovery and development, which creates demand for an automated high‐throughput screening assay to increase laboratory efficiency. A comprehensive comparison of the four methods typically used for determining the binding parameters is presented in this study with respect to the above demand. Capillary electrophoresis‐frontal analysis, isothermal titration calorimetry, circular dichroism and equilibrium dialysis were used to study the affinity of human serum albumin for diclofenac and lidocaine. These model drugs were chosen due to their different physico‐chemical properties and different binding sites on the albumin molecule, also resulting in different binding strength. The binding parameters estimated under the conditions as similar as possible were comparable among all these approaches as well as to the literature values. Besides this, the comparison of the results and especially other considerations demonstrated the benefits and drawbacks of the selected methods, with capillary electrophoresis‐frontal analysis being the best candidate for such studies.     

Estimation of distribution of vinyl groups in micron-sized monodisperse polymer microspheres by bromine titration method

The distribution of vinyl groups in polystyrene/poly(styrene-divinylbenzene) (PS/P(S-DVB)) composite microspheres produced by seeded copolymerization of S and DVB in the presence of 2.1-m monodisperse PS seed particles was estimated from the amount detected with bromine titration method as a function of the reaction time under various conditions.Part CXXIII of the series Studies on Suspension and Emulsion.     

Preparation of polymer particles having ethyleneurea groups at their surfaces by emulsifier-free seeded emulsion polymerization and wet adhesion of its emulsion film

Polymer emulsion having ethyleneurea groups at particle surfaces was produced by emulsifier-free seeded emulsion copolymerization of n-butyl methacrylate (BMA) and methacrylamide ethylethyleneurea (EU) with poly(BMA) seed particles utilizing the starved-fed monomer addition method. This emulsion film, prepared by casting the polymer emulsion on an alkyd resin plate, had a superior adhesion in water, as well as on stainless steel. Such superior wet adhesions seem to be based on a large amount of EU predominantly localized at the particle surfaces.Part CCXLIX of the series "Studies on suspension and emulsion"     

Study on the interaction of amino phosphine ester derivatives with DNA by spectroscopy, modeling and calorimetry

The binding properties of amino phosphate ester derivatives, compound 1 and 2 with calf thymus DNA (CT-DNA) were investigated by UV spectra, fluorescence spectra, molecular modeling and isothermal titration calorimetry (ITC). The intrinsic binding constants K_b of compound 1 and 2 with CT-DNA were determined by fluorescence spectroscopy and ITC, respectively. The results indicated that the two compounds bind to CT-DNA with different binding affinity, which is in the order of compound 1 > compound 2. At the same time, fluorescence spectra suggested that the mechanism of the binding of the two compounds to CT-DNA is a static enhancing type. According to the ethidium bromide displacement experiments, UV spectra, molecular modeling and ITC studies, it can be concluded that compound 1 and 2 are intercalators that can slide into the G–C rich region of CT-DNA. Furthermore, ITC data showed that compound/DNA binding is enthalpy controlled.     

Potentiometric titration behavior of poly(acrylic acid) within a cross-linked polymer network having amide groups

This study aimed to investigate the effect of COOH group distribution within a polymer network having amide groups, with which the COOH could form hydrogen bonds. We employed here two polyelectrolyte gels composed of N-isopropylacrylamide (NIPA) networks, either copolymerized with acrylic acid (AA) or within which poly(acrylic acid) (PAA) was entrapped. Both gels (AA–NIPA ∼ 1:4 mol/mol) were prepared by aqueous red-ox polymerization with N,N’-methylenebisacrylamide as the cross-linker. Finely divided gels in NaCl solutions (0.025 and 0.1 M) were titrated with NaOH and back-titrated with HCl at 25 °C. The results of the copolymer gel (CG) agreed well with those of a linear copolymer and a nanoscale gel which had a similar AA content to CG. However, marked differences were observed in the titration behaviors of the AA-copolymerized and PAA-entrapped gels, mainly due to the hydrogen bonding between the entrapped PAA chain and its surrounding NIPA network.     

Interaction of Pseudomonas putida with kaolinite and montmorillonite: a combination study by equilibrium adsorption, ITC, SEM and FTIR

Equilibrium adsorption along with isothermal titration calorimetry (ITC), Fourier transform infrared spectra (FTIR) and scanning electron microscopy (SEM) techniques were employed to investigate the adsorption of Pseudomonas putida on kaolinite and montmorillonite. A higher affinity as well as larger amounts of adsorption of P. putida was found on kaolinite. The majority of sorbed bacterial cells (88.7%) could be released by water from montmorillonite, while only a small proportion (9.3%) of bacteria desorbed from kaolinite surface. More bacterial cells were observed to form aggregates with kaolinite, while fewer cells were within the larger bacteria–montmorillonite particles. The sorption of bacteria on kaolinite was enthalpically more favorable than that on montmorillonite. Based on our findings, it is proposed that the non-electrostatic forces other than electrostatic force play a more important role in bacterial adsorption by kaolinite and montmorillonite. Adsorption of bacteria on clay minerals resulted in obvious shifts of infrared absorption bands of water molecules, showing the importance of hydrogen bonding in bacteria–clay mineral adsorption. The enthalpies of −4.1 ± 2.1 × 10⁻⁸ and −2.5 ± 1.4 × 10⁻⁸ mJ cell⁻¹ for the adsorption of bacteria on kaolinite and montmorillonite, respectively, at 25 °C and pH 7.0 were firstly reported in this paper. The enthalpy of bacteria–mineral adsorption was higher than that reported previously for bacteria–biomolecule interaction but lower than that of bacterial coaggregation. The bacteria–mineral adsorption enthalpies increased at higher temperature, suggesting that the enthalpy–entropy compensation mechanism could be involved in the adsorption of P. putida on clay minerals. Data obtained in this study would provide valuable information for a better understanding of the mechanisms of mineral–microorganism interactions in soil and associated environments.     

Investigation of the inclusion of the herbicide cycluron in native cyclodextrins by X-ray diffraction,nuclear magnetic resonance spectroscopy and isothermal titration calorimetry

The formation of inclusion complexes between the native cyclodextrins (CDs) and the urea herbicide cycluron has been investigated both in solution and in the solid state. Single-crystal X-ray structures of both the uncomplexed guest and the β-CD·cycluron complex were determined while powder X-ray diffraction was used to confirm complexation between γ-CD and cycluron in the solid state. Solution-state complexation between the herbicide and α-, β- and γ-CD was established using ¹H NMR spectroscopy and isothermal titration calorimetry (ITC). From the ¹H NMR spectroscopic studies 1:1 complex stoichiometry was indicated in all cases and association constant values (K) were determined as 228, 3254 and 155 for the complexes α-CD·cycluron, β-CD·cycluron and γ-CD·cycluron, respectively. Assigning a 1:1 host–guest ratio, the ITC technique produced K values of the same order as those determined using the spectroscopic method. The thermodynamic parameters ΔH, ΔS and ΔG obtained using ITC provide insights into the driving forces involved during complex formation.     

Isothermal titration microcalorimetric method for studying the combined ligand binding with application to the binding of ethylurea and (N,N)dimethylurea on urease

A simple, novel method was introduced for determining equilibrium constants and enthalpies of binding of two different competitive ligands on a macromolecule by isothermal titration microcalorimetry technique. This method was applied to the simultaneous binding of ethylurea (I) and (N,N)dimethylurea (X), on jack-bean urease at pH 7.0 (tris-base; 30 mM) at 27°C. The dissociation equilibrium constants measured by this method were markedly consistent with inhibition constants obtained from assay of enzyme activities in the presence of I and X.     

Synthesis and photophysical properties of metal free, titanium, magnesium and zinc phthalocyanines substituted with a single carboxyl and hexylthio groups

2,3,9,10,16,17,23,24-Octakis(hexylthio)phthalocyanines (4a-6a) and 2-carboxy-9,10,16,17,23,24-hexakis(hexylthio)phthalocyanines (4b-6b) were synthesized using a one-pot method by cyclotetramerization of the phthalonitriles: 4,5-bis(hexylthio)phthalonitrile and carboxylic acid phthalonitrile. 2-Carboxycatecholato-2,3,9,10,16,17,23,24-octakis(hexylthio)phthalocyaninatotitanium(IV) (8) was prepared from 2,3,9,10,16,17,23,24-octakis(hexylthio)phthalocyaninatooxotitanium(IV) (7). The structures of these compounds were characterized by using elemental analyses, UV-Vis, FT-IR, ¹H NMR and mass spectroscopies. Their photophysical properties were also studied. The Φ_F values are 0.12, 0.02, 0.10, 0.06, 0.10, 0.06, 0.65, 0.80 and the Φ_T values are 0.58, 0.56, 0.57, 0.64, 0.22, 0.48, 0.17, 0.12 for 4-8, respectively. The Φ_F value for complex 8 is higher than ever reported for phthalocyanine complexes. The triplet lifetimes (τ_T) values for all the complexes were generally good, ranging from 50 to 310 μs, and generally increased in the presence of the single carboxyl group. These complexes showed reasonable triplet quantum yields and lifetimes, and hence have potential for use as photosensitizers in photodynamic therapy (PDT) of cancer.