Thermal behavior of a pharmaceutical solid acetaminophen doped withp-aminophenol

Thermal behavior of a series of acetaminophen (APAP) doped withp-aminophenol (PANP) was studied by differential scanning calorimetry (DSC) to determine whether it exhibited a eutectic system. Within the temperature range of 120 to 200° C, accurately weighed (1–2 mg) samples sealed in hermetic pans were calorimetrically scanned with a low scanning rate of 1° C/min. The mixture formed a single eutectic with the composition ratio APAP/PANP of 0.6/0.4 at a temperature of 138° C, where it liquefied. Melting began as early as at the eutectic point, which was below the melting temperature of APAP (169° C). The melting point as well as heat of APAP fusion was depressed with the increase in doped PANP. It was postulated that there might be a deficit heat of APAP fusion in APAP doped with PANP, which was coincident with the heat consumed by early liquefaction. The deficit heat was used to correct fraction molten in the van’t Hoff law of purity determination. It was found that the purity determination of APAP doped with PANP was comparable to the UV-spectroscopic method up to the maximum doped PANP level of 8 mol percent. It was concluded that DSC was able to approach early heat of liquefaction of APAP doped with PANP. The van’t Hoff law may be applicable to the determination of APAP with the presence of PANP as a eutectic impurity.     

The unfolding enthalpy of the pH 4 molten globule of apomyoglobin measured by isothermal titration calorimetry

The unfolding enthalpy of the pH 4 molten globule from sperm whale apomyoglobin has been measured by isothermal titration calorimetry, using titration to acid pH. The unfolding enthalpy is close to zero at 20 degrees C, in contrast both to the positive values expected for peptide helices and the negative values reported for holomyoglobin and native apomyoglobin. At 20 degrees C, the hydrophobic interaction should make only a small contribution to the unfolding enthalpy according to the liquid hydrocarbon model. Our result indicates that some factor present in the unfolding enthalpies of native proteins makes the unfolding enthalpy of the pH 4 molten globule less positive than expected from data for peptide helices.     

Thermodynamic properties of BPTI variants with highly simplified amino acid sequences

We report the first detailed thermodynamic analysis of simplified proteins by differential scanning calorimetry (DSC). The experiments were carried out with five simplified BPTI variants, whose structures and activities have been reported, in which several residues not essential for specifying the tertiary structure were replaced by alanine. In most aspects, the thermodynamics of simplified proteins were very similar to, if not essentially identical with, those of natural proteins. In particular, they undergo a highly cooperative two-state thermal unfolding process with a large enthalpy change, which is a thermodynamic hallmark of the native state of natural globular proteins. Furthermore, the specific enthalpy and entropy changes upon unfolding at 110 degrees C were close to values invariably observed for small natural globular proteins (55 J g(-1) and ~16 J K(-1) g(-1), respectively). On the other hand, two simplified BPTI variants, BPTI-21 and BPTI-22 (containing 21 and 22 alanine residues), were enthalpically stabilized while entropically destabilized with respect to the reference BPTI-[5,55] molecule. This peculiar type of entropy-enthalpy compensation is in sharp contrast to the usual enthalpy destabilization/entropy stabilization observed in mutational studies of natural proteins. Overall, we conclude that a thermodynamic native state can be achieved by proteins encoded with extensively simplified sequences.     

On the molecular basis of the high affinity binding of basic amino acids to LAOBP,a periplasmic binding protein from Salmonella typhimurium

The rational designing of binding abilities in proteins requires an understanding of the relationship between structure and thermodynamics. However, our knowledge of the molecular origin of high‐affinity binding of ligands to proteins is still limited; such is the case for l ‐lysine–l ‐arginine–l ‐ornithine periplasmic binding protein (LAOBP), a periplasmic binding protein from Salmonella typhimurium that binds to l ‐arginine, l ‐lysine, and l ‐ornithine with nanomolar affinity and to l ‐histidine with micromolar affinity. Structural studies indicate that ligand binding induces a large conformational change in LAOBP. In this work, we studied the thermodynamics of l ‐histidine and l ‐arginine binding to LAOBP by isothermal titration calorimetry. For both ligands, the affinity is enthalpically driven, with a binding ΔCp of ~?300 cal mol^?1 K^?1, most of which arises from the burial of protein nonpolar surfaces that accompanies the conformational change. Osmotic stress measurements revealed that several water molecules become sequestered upon complex formation. In addition, LAOBP prefers positively charged ligands in their side chain. An energetic analysis shows that the protein acquires a thermodynamically equivalent state with both ligands. The 1000‐fold higher affinity of LAOBP for l ‐arginine as compared with l ‐histidine is mainly of enthalpic origin and can be ascribed to the formation of an extra pair of hydrogen bonds. Periplasmic binding proteins have evolved diverse energetic strategies for ligand recognition. STM4351, another arginine binding protein from Salmonella, shows an entropy‐driven micromolar affinity toward l ‐arginine. In contrast, our data show that LAOBP achieves nanomolar affinity for the same ligand through enthalpy optimization. Copyright © 2015 John Wiley & Sons, Ltd.     

Thermodynamics of conformational transition and chain association of ι-carrageenan in aqueous solution: Calorimetric and chirooptical data

Isothermal microcalorimetry, differential scanning calorimetry (DSC), and chirooptical data obtained for ι-carrageenan in NaCl, LiCl, and NaI aqueous solutions are presented. The experiments have been performed as a function of concentration both for the polymer and for the simple salt as a cosolute. The experimental findings consistently show the occurrence of a salt-induced disorder-to-order transition. From microcalorimetric experiments the exothermic enthalpy of transition ΔH_tr is obtained as the difference between the theoretical, purely electrostatic ΔH_el enthalpy change and the actual mixing enthalpy ΔH_mix, measured when a ι-carrageenan salt-free solution at constant polymer concentration is mixed with a 1:1 electrolyte solution of variable concentration. In the case of added NaCl, the absolute values of enthalpy changes |ΔH_tr| are in good agreement with those obtained for the opposite process, at comparable polymer and salt concentrations, from DSC melting curves. The microcalorimetric results show that the negative maximum value of ΔH_tr corresponding to the interaction of Li⁺ counterion with ι-carrageenan polyion results to be significantly lower than the corresponding values obtained for Na⁺ counterion. At variance with the microcalorimetric data, chirooptical results show that the salt-induced disorder-to-order transition, occurring in the 0.02–0.2M salt concentration range, appears to be complete at a concentration of about 0.08–0.1M of the simple ion, irrespective of the polymer concentration and of the nature of added electrolyte. © 1998 John Wiley & Sons, Inc. Biopoly 45: 105–117, 1998     

Chemiluminescence during rice seed imbibition at different temperatures.

The chemiluminescence (CL) of rice (Oryza sativa L.) seeds at different temperatures and the CL spectra of rice seed, caryopses and seed coat were studied during early imbibition. Compared with the CL of barley (Hordeum vulgare L.) and soybean (Glycine max L. Merr.) seeds, the CL of rice seeds had a non-linear, logarithmic-like increase of intensity in the temperature range 30-50 degrees C. The Van't Hoff coefficient Q(10) = I(T+10)/I(T) was equal to 2. The emission spectrum of whole rice seed, rice and coat had a greater proportion of red light during early imbibition, which led to the conclusion that the CL of rice seed during early imbibition arises partially from enzyme-catalysed reactions.     

Kinetic and thermodynamic characterization of the cold activity acquired upon single amino-acid substitution near the active site of a thermostable α-glucosidase

The α-glucosidase of Bacillus sp. SAM1606, a thermophilic bacterium, is a thermostable enzyme that has maximal activity at an apparent optimal temperature between 65 and 70 °C and only very low activity at low temperatures (0–25 °C). In this study, we identified Thr272, which is located adjacent to Glu271 (a catalytic residue) and Gly273 (a determinant of specificity), as a determinant of the optimal temperature, as substitution of Thr272 with other residues significantly altered the temperature–activity profile of the enzyme. Substitution of Thr272 with other amino acids, in particular bulky hydrophobic residues such as valine, methionine and phenylalanine, resulted in a significant downward shift (by 30 °C) of the apparent optimal temperature with an increase in catalytic activity at low temperatures. The observed downward shift of the apparent optimal temperature was not due to instability of the mutants at 40–65 °C, as the mutants were stable at temperatures up to 65 °C. Among the mutants examined, T272V displayed the highest k_cat values at 10–25 °C, which was at least 11-fold greater than the k_cat value observed for the wild-type enzyme. The thermodynamic characteristics of reactions catalyzed by T272V, T272M, T272F, and wild type at 25 °C were examined in greater detail. The T272V, T272M and T272F mutants displayed large K_s (or K_m) values and reduced and values at 25 °C, consistent with the general features of cold adaptation. The observed cold activities of T272V, T272M and T272F most likely arose from local flexibility of the active site at low temperatures due to loss of a Thr272-mediated hydrogen bond. However, this hydrogen-bond loss likely permits reversible conformational changes of the active site to less active forms at elevated temperatures (e.g., 60 °C). This may explain why catalytic activities for T272V, T272M and T272F at high temperatures (e.g., 60 °C) were lower than those at low temperature (e.g., 25 °C), even though the mutant enzymes appeared stable at 60 °C.     

Pharmacologic implications of alpha-adrenoreceptor interactive parameters for epinephrine enantiomers in the rat vas deferens

After alkylation of a fraction of the total alpha-adrenoreceptors by phenoxybenzamine in rat vas deferens, the dissociation constants of (-)- and (+)-epinephrine in functional studies were 7 X 10(-7) M and 2 X 10(-5) M, respectively. In the adrenoreceptor-containing tissue fraction, when 3H-labeled WB4101 was used as the interacting ligand, for each enantiomer two affinity sites were found. Only the low-affinity dissociation constant for each isomer correlates with the constant obtained from the functional studies. If the change in Gibb's free energy, delta G degrees, is calculated from the low-affinity binding constants, the values -8.1 and -6.2 kcal/mol for (-)- and (+)-isomer, respectively, are obtained. The small difference in the value between isomers is consistent with the view that the benzylic hydroxyl group of the (-)-isomer forms a hydrogen bond with the receptor. The interaction of epinephrine with this receptor appears to be driven largely by the entropy of the drug-receptor interaction with only a small nonstereoselective contribution from the enthalpy of interaction.     

Significant discrepancies between van't Hoff and calorimetric enthalpies. II.

Isothermal calorimetric titration of 18-crown-6 ether with BaCl2 in pure aqueous solution over the temperature range 7-40 degrees C gives precise binding constants and enthalpy changes. Nonlinear least-squares fitting of the binding constants to the integrated van't Hoff equation, including a temperature-independent change in heat capacity, leads to van't Hoff enthalpies that differ significantly from the observed calorimetric enthalpies. This perplexing discrepancy appears at present to be very widely occurring.     

Theoretical investigation of the effect of sugar substitution on the antioxidant properties of flavonoids

Natural flavonoids are secondary phenolic plant metabolites known for their bioactivity as antioxidants. The evaluation of this property is generally done by the estimation of their direct free radical-scavenging activity as hydrogen or electron donating compounds. This paper reviews experimental results available in the literature for a selection of flavonoids and compares them with calculated quantities characteristic of the hydrogen or electron donation. For that purpose, bond dissociation energies, ionization potentials and electron transfer enthalpies are computed by using DFT methods and the ONIOM procedure implemented in the ab initio program Gaussian. This process has been chosen because it can be extended to the study of large molecules. When acid dissociation and interaction with the solvent are taken into account, the results present very good concordance with experimental results, enlightening the complexity of the processes involved in the classical assays which measure the ability of compounds to scavenge the (2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt) radical cation (ABTS ⁺) or the 2,2-diphenyl-1-picryl-hydrazyl radical (DPPH^·). This study demonstrates the good accuracy of theoretical calculations in obtaining the relative energies involved in free radical scavenging abilities and its capacity for predictive behaviour. It also highlights the necessity to take into account the pK_a of the compounds and the solvent interaction. The ability of the method to calculate the antioxidant properties of larger molecules are tested on glycosylated flavonoids and the effects of sugar substitution on the antioxidant properties of flavonoids are investigated, pointing out the importance of the charges on the oxygen atoms.