Babur Z. Chowdhry

Sensing the heat: the application of isothermal titration calorimetry to thermodynamic studies of biomolecular interactions

Biomolecular interactions can be defined by combining thermodynamic data on the energetic properties of the interaction with high-resolution structural data. The development of high sensitivity isothermal titration calorimetric equipment provides a dramatic advance in the gathering of thermodynamic data, and the interactions between biological macromolecules can now be described with unprecedented accuracy.

Raman spectra of carotenoids in natural products

Resonance Raman spectra of naturally occurring carotenoids have been obtained from nautilus, periwinkle (Littorina littorea) and clam shells under 514.5 nm excitation and these spectra are compared with the resonance Raman spectra obtained in situ from tomatoes, carrots, red peppers and saffron. The tomatoes, carrots and red peppers gave rise to resonance Raman spectra exhibiting a nu1 band at ca. 1520 cm(-1), in keeping with its assignment to carotenoids with ca. nine conjugated carbon-carbon double bonds in their main chains, whereas the resonance Raman spectrum of saffron showed a nu1 band at 1537 cm(-1) which can be assigned to crocetin, having seven conjugated carbon-carbon double bonds. A correlation between nu1 wavenumber location and effective conjugated chain length has been used to interpret the data obtained from the shells, and the wavenumber position (1522 cm(-1)) of the nu1 band of the carotenoid in the orange clam shell suggests that it contains nine conjugated double bonds in the main chain. However, the black periwinkle and nautilus shells exhibit nu1 bands at 1504 and 1496 cm(-1), respectively. On the basis of the correlation between nu1 wavenumber location and effective conjugated chain length, this indicates that they contain carotenoids with longer conjugated chains, the former having ca. 11 double bonds and the latter ca. 13 or even more. Raman spectra of the nautilus, periwinkle and clam shells also exhibited a strong band at 1085 cm(-1) and a doublet with components at 701 and 705 cm(-1), which can be assigned to biogenic calcium carbonate in the aragonite crystallographic form.

Colloidal copolymer microgels of N-isopropylacrylamide and acrylic acid: pH, ionic strength and temperature effects

Aqueous colloidal microgels have been prepared, based on poly(N-isopropylacrylamide)[poly(NIPAM)], cross-linked with bisacrylamide, containing 5% w/w acrylic acid (AAc) as a comonomer. Transmission electron micrographs of the microgels show that the copolymer microgels are monodisperse spheres. The size of the microgel particles containing 5% AAc has been studied, using dynamic light scattering, as a function of pH (3–10), ionic strength (10–4–10–1M NaCl) and temperature (20–75 °C). The hydrodynamic diameter of the copolymer microgels decrease both with increasing ionic strength (at pH 6 and 25 °C) and reversibly with increasing temperature at pH values of 2.6, 3.4 and 6.5. However, under isothermal conditions in 10–3M NaCl at 25 °C, the hydrodynamic diameter increases in going from pH 3.3–9.4. In addition, the temperature-induced conformational changes in the polymer chains have been followed using high-sensitivity differential scanning calorimetry (HSDSC). A comparison is made with the behaviour of poly(NIPAM) microgel particles, not containing AAc. Explanations are offered to account for the pronounced difference in the physico-chemical properties observed.

Parsing free energies of drug-DNA interactions

Publisher Summary This chapter discusses that a number of clinically important small molecules appear to act by binding directly to DNA, and subsequently inhibiting gene expression or replication by interfering with the enzymes that catalyze these functions. Thermodynamics provides quantitative information that can helps elucidate the principal driving forces for the interaction, which can provide insight to guide possible chemical modifications that might enhance both DNA-drug binding affinity and base sequence specificity. It reviews the power of thermodynamics is that it provides quantitative information that is independent of the details for the underlying molecular processes. Advances in instrumentation have enabled the acquisition of reliable thermodynamic data for reactions of biological interest. In parallel, significant advances have been made in the interpretation of thermodynamic data, making it possible to begin to parse free energy values into the contributions from a variety of forces and interactions. Considerable insight into the forces that drive association reactions can be gleaned from such interpretations. The chapter describes a framework and specific details for the application of these methods to DNA-drug binding reactions. While only relatively few compounds have thus far been examined by these approaches, it is clear from these initial successes that valuable new molecular insights will emerge as more DNA-binding drugs are also discussed.

Polycyclic aromatic hydrocarbon extraction from a coal tar-contaminated soil using aqueous solutions of nonionic surfactants

The efficiency and kinetics of surfactant facilitated extraction of phenanthrene and anthracence - two exemplar PAH compounds - from a coal tar-contaminated soil, using five surfactants have been investigated. Three of the surfactants used were ethylene oxide/propylene oxide block copolymers in which the propylene oxide (PO) block was of constant length but the ethylene oxide (EO) block lengths were varied. Steady state extraction values - for PAH removal - were obtained after 50 hours for four of the five surfactants. The extraction efficiency - calculated as the fraction of PAH removed in one washing - is shown to be related to the EO/PO ratio for the block copolymers. Apparent soil/aqueous surfactant solution distribution coefficients (K-d) were also obtained and suggest that the surfactants can reduce K-d by several orders of magnitude. Finally the kinetic data is readily fitted to the Elovich equation an essentially empirical equation which has been used, previously, to fit phosphate adsorption data.

Singular value decomposition of 3-D DNA melting curves reveals complexity in the melting process

Abstract The thermal denaturation of synthetic deoxypolynucleotides of defined sequence was studied by a three dimensional melting technique in which complete UV absorbance spectra were recorded as a function of temperature. The results of such an experiment defined a surface bounded by absorbance, wavelength, and temperature. A matrix of the experimental data was built, and analyzed by the method of singular value decomposition (SVD). SVD provides a rigorous, model-free analytical tool for evaluating the number of significant spectral species required to account for the changes in UV absorbance accompany-ing the duplex – to – single strand transition. For all of the polynucleotides studied (Poly dA – Poly dT; [Poly (dAdT)]2; Poly dG – Poly dC; [Poly(dGdC)]2), SVD indicated the existence of at least 4 – 5 significant spectral species. The DNA melting transition for even these simple repeating sequences cannot, therefore, be a simple two-state process. The basis spectra obtained by SVD analysis were found to be unique for each polynucleotide studied. Differential scanning calorimetry was used to obtain model free estimates for the enthalpy of melting for the polynucleotides studied, with results in good agreement with previously published values.

Characterisation of the aggregation behaviour in a salmeterol and fluticasone propionate inhalation aerosol system.

The nature of the drug-drug aggregation phenomena between salmeterol xinafoate and fluticasone propionate used in a metered-dose inhaler system has been examined. Interactions between the drugs in the solvents 1,1,2-trichlorotrifloroethane (CFC-113) and 1,1,1,2-tetrafluoroethane (HFA-134a) have been characterised using a focused beam reflectance measurement probe by measuring the average floc size of the drug particles individually and in combination as a function of stirrer rate. The floc composition in the CFC-113 system, where the drug particles cream, was determined by high-performance liquid chromatography analysis. The aggregation behaviour of the individual drugs was shown to depend on the physical and chemical properties of both the drug substance and the media. Larger flocs were observed for salmeterol xinafoate compared with fluticasone propionate, while both drugs formed larger aggregates in HFA-134a compared with in CFC-113. The floc composition studies demonstrated that, in the combined formulation in CFC-113, salmeterol xinafoate and fluticasone propionate aggregate together to form hetero-flocs. The interaction between the two drugs was such that they did not separate on creaming, despite having different densities. The average floc size of the combined drug suspension was also found to depend on the dispersion medium.

Multicomponent phase transitions of diacylphosphatidylethanolamine dispersions

The phase transition properties of aqueous suspensions of a series of nonhydrated (not heated above room temperature) and hydrated 1,2 diacylphosphatidylethanolamines (PEs) have been examined by high sensitivity differential scanning calorimetry at scan rates of 0.02-1.0 K min-1. At all scan rates nonhydrated PEs show a single asymmetric transition curve of excess heat capacity as a function of temperature. Multilamellar dispersions of hydrated PEs, however, exhibit transitions with fine structure, which can be fitted as the sum of three two-state component transitions, at scan rates of 0.02-0.1 K min-1, but give only a single asymmetric transition at 1.0 K min-1. At all scan rates the transition(s) of hydrated samples occur at lower temperatures than those of nonhydrated samples. One of the component transitions of hydrated PEs may be analogous to the pretransition that occurs in 1,2 diacylphosphatidylcholines.

Vibrational Spectra of α-Amino Acids in the Zwitterionic State in Aqueous Solution and the Solid State: DFT Calculations and the Influence of Hydrogen Bonding

The zwitterionic forms of the two simplest alpha-amino acids, glycine and l-alanine, in aqueous solution and the solid state have been modeled by DFT calculations. Calculations of the structures in the solid state, using PW91 or PBE functionals, are in good agreement with the reported crystal structures, and the vibrational spectra computed at the optimized geometries provide a good fit to the observed IR and Raman spectra in the solid state. DFT calculations of the structures and vibrational spectra of the zwitterions in aqueous solution at the B3-LYP/cc-pVDZ level were found to require both explicit and implicit solvation models. Explicit solvation was modeled by inclusion of five hydrogen-bonded water molecules attached to each of the five possible hydrogen-bonding sites in the zwitterion and the integration equation formalism polarizable continuum model (IEF-PCM) was employed, providing a satisfactory fit to observed IR and Raman spectra. Band assignments are reported in terms of potential-energy distributions, which differ in some respects to those previously reported for glycine and l-alanine.

Calorimetric techniques in the study of high-order DNA-drug interactions.

Publisher Summary Biophysical studies of DNA–drug (or DNA–ligand) interactions have an established and pivotal role in the rational development of novel ligands directed toward high-order nucleic acid system. To illustrate the importance and power of calorimetry as a biophysical tool this chapter describes how isothermal titration calorimetry (ITC) has been used to determine the energetics of ligand binding to triplex and tetraplex DNA, and how such studies can help to clarify the nature of the interactions and thereby provide feasible binding models. Complementary studies involving DSC can be used to examine nucleic acid stability under given conditions; thus, for example, protocols for using DSC to examine the differential stabilization of tetraplex DNA with Na + and K + ions are also discussed. The genuine strength of calorimetry becomes apparent when it is used in conjunction with parallel techniques such as UV-visible/CD/fluorescence spectrophotometry, NMR spectroscopy, X-ray crystallography, and stopped-flow kinetic methods. Such a multitechnique approach to studies of DNA–drug interactions (or indeed any binding interaction) can provide a comprehensive and cohesive picture for the underlying biomolecular events.