The 13C chemical shifts of amino acids in aqueous solution containing organic solvents: Application to the secondary structure characterization of peptides in aqueous trifluoroethanol solution

Summary The ¹³C chemical shifts for all of the protonated carbons of the 20 common amino acid residues in the protected linear pentapeptide Gly-Gly-X-Gly-Gly have been obtained in water at low pH as well as in aqueous solution containing 10, 20 and 30% acetonitrile or trifluoroethanol. Dioxane was used as an internal reference and its carbon chemical shift value was found to be 66.6 ppm relative to external TMS in water. Comparison of the different referencing methods for ¹³C chemical shifts in organic cosolvent mixtures showed that an external standard (either TMS or TSP capillary) was the most appropriate. In the present study, external TSP was chosen to define the 0 ppm of the ¹³C chemical shift scale. When the difference in referencing the dioxane carbon resonance is taken into account, the carbon chemical shift values of the amino acids in aqueous solution are similar to those previously reported (Richarz and Wüthrich (1978) Biopolymers, 17, 2133–2141; Howarth and Lilley (1979) Prog. NMR Spectrosc., 12, 1–40). The pentapeptides studied were assumed to be in a random coil conformation and the measured ¹³C chemical shifts were used as reference values to correlate carbon chemical shifts with the secondary structure of two well-characterized peptides, bombesin and the 1–29 amino acid fragment of Nle²⁷ human growth hormone-releasing factor. In both cases, the C chemical shifts exhibited a characteristic positive deviation from the random coil values, which indicates the presence of -helices.     

Improving Tenoxicam Solubility and Bioavailability by Cosolvent System

The formulation study of tenoxicam, a poorly water-soluble drug, was developed by use of a ternary cosolvent system and has significantly enhanced the solubility. Additionally, the relative bioavailability of testing formulation was also evaluated by New Zealand rabbit with a single i.m. injection. The three-phase diagram for dimethylsulfoxide (DMSO)/propylene glycol/water, DMSO/ethanol/water, and DMSO/polyethoxylated castor oil/ethanol system was developed. The volume ratio of 5:4:1 in the DMSO/polyethoxylated castor oil/ethanol system resulted in a more suitable vehicle than other systems, with a high solubility (20.73 mg/ml) and low viscosity (10.0 Cp). A pharmacokinetic study of bioequivalence (F _rel = 0.89) was also obtained. The present study not only provides a novel strategy improving tenoxicam solubility but also helps further scientific knowledge for the development of parenteral formulations.     

Hydrolase-catalyzed β-mannosylations

Transmannosylations catalysed by -mannosidase from snail viscera or -galactosidase from Aspergillus oryzae were accomplished with 4-nitrophenyl D -mannopyranoside as donor substrate. With suitable hydrophobic acceptor molecules preferentially 1-4-linked disaccharides were obtained. The activities of both glycosidases in buffer cosolvent mixtures were determined, and conditions for their immobilization were elaborated and optimized. A model of the enzymic transfer mechanism is suggested.     

Solvational tuning of the unfolding, aggregation and amyloidogenesis of insulin

Solvational perturbations, accomplished by the addition of the three model cosolvents glycerol, ethanol and trifluoroethanol, exert pronounced and diversified effects on the unfolding, non-native assembly and fibril formation of the amyloidogenic protein insulin. Fluorescence, CD and UV-spectroscopic methods as well as atomic force microscopy imaging have been employed to reveal distinct structural and kinetic features upon the aggregation of insulin under different solvational perturbations, which ultimately manifest in morphological variations of mature aggregates and fibrils. In particular, fluorescence anisotropy studies proved to be very valuable in characterizing the corresponding aggregation nuclei. Glycerol stabilizes, through enhanced hydration, native oligomerization and retards fibrillar aggregation at all concentrations studied (up to 40% (w/w)). In contrast, both monoalcohols facilitate the formation of aggregation-prone intermediates by destabilization of the native assembly. The reversal from a kosmotropic to a merely chaotropic solvational behaviour can explain the accelerating effect on ordered fibrillation of low concentrations and the inhibitory nature of high concentrations of ethanol and trifluoroethanol, ultimately leading to amorphous aggregate structures. Mechanistically, dimer dissociation under stabilizing and nucleation under destabilizing conditions have been identified to be the rate-limiting steps that account for the non-monotonic concentration effects of the monoalcohols on the aggregation kinetics. A rationale as to how solvational constraints can tune the stability of the species on the native self-assembly and non-native aggregation pathway, and the energetic barriers that need to be overcome for the required structural interconversions has been put forward. We may propose that the concept of perturbed solvation is generally applicable to phenomena that are related to pathogenic amyloidogenesis of proteins and, in general, solvational effects, besides other aspects of the cellular environment, may play a significant role in a reshaping of the folding/aggregation funnel of proteins.     

Effects of Cosolvent and Temperature on the Desorption of Polynuclear Aromatic Hydrocarbons from Contaminated Sediments of Chien-Jen River,Taiwan

This study evaluated the effects of the water-miscible cosolvent and temperature on the sorption-desorption of polynuclear aromatic hydrocarbons (PAHs) from contaminated sediments in Chien-Jen River, Taiwan. Sediment samples from five sampling stations of downstream section were utilized in this study. Phenanthrene and anthracene were selected as target compounds. The cosolvent effect on sorption of phenanthrene and anthracene was examined by the addition of various volume fractions of methanol (i.e., 0.3, 0.5, 0.7, and 0.9, respectively) in the sediment/water systems. The utility of the log-linear cosolvency model for predicting PAH sorption from solvent mixtures was evaluated. An inverse relationship was observed for sorption coefficients of phenanthrene and anthracene as a function of increasing cosolvent. The effect of temperature on sorption of phenanthrene and anthracene was conducted at temperature from 10°C to 40°C. The use of elevated temperatures in desorption experiments increased the PAH release from sediments. It was observed that sorption of phenanthrene and anthracene onto sediments decreased when temperature increased. The decrease of sorption coefficient of phenanthrene was more sensitive than that of anthracene. The magnitude of decreased sorption was attributed by the increased desorption rate constant, solubility, and heterogeneities of sediments.