Homotropic cooperative binding of organic solvent vapors by solid trypsin. (17/350)

Homotropic cooperative binding was observed at vapor sorption of organic solvents (acetonitrile, propionitrile, ethanol, 1-propanol, 2-propanol, nitroethane) by dried solid trypsin from porcine pancreas (0.05 g H2O/g protein). The vapor sorption isotherms were obtained by the static method of gas chromatographic headspace analysis at 298 K for 'vapor solvent+solid trypsin' systems in the absence of the liquid phase. All isotherms have a sigmoidal shape with significant sorbate uptake only above the threshold of sorbate thermodynamic activity. On the sorption isotherms of non-hydroxylic sorbates the saturation of trypsin by organic solvent was observed above the sorbate threshold activity. The formation of inclusion compounds with phase transition between solvent-free and solvent-saturated trypsin is supposed. Approximation of obtained isotherms by the Hill equation gives the inclusion stoichiometry S, inclusion free energy, and the Hill constant N of clathrates. The inclusion stoichiometry S depends significantly on the size and shape of sorbate molecules and changes from S=31 mol of sorbate per mol of trypsin for ethanol to S=6 for nitroethane. The inclusion free energies determined for the standard states of pure liquid sorbate and infinitely dilute solution in toluene are in the range from -0.5 to -1.2 kJ/mol and from -3.1 to -8.1 kJ/mol, respectively, per 1 mol of sorbate. The Hill constants are relatively high: from N=5.6 for 1-propanol to N approximately equal to 10(3) for nitroethane. The implication of the obtained results for the interpretation of solvent effects on the enzyme activity and stability in low-water medium is discussed.  (+info)

Behavior of haloperidol and various phenothiazines on several alkyl bonded phases. (18/350)

Haloperidol and phenothiazines are present in psychiatrical treatments. An analysis in body fluids is tedious because of the presence of demethylated (DM) derivatives of phenothiazines. The behavior of some interfering solutes on alkyl bonded phases has been studied. Phenothiazines and DM derivatives exhibit a very similar behavior with a binary eluent (phosphate buffer-acetonitrile), which precludes an optimization with this system. When a ternary phase is used (phosphate buffer-acetonitrile-methanol), haloperidol and reduced haloperidol behave differently as compared with phenothiazines. In this mode it is possible to unambiguously detect haloperidol that would otherwise interfere. Phenothiazine peaks are characterized by a large tailing. An interesting feature is the comparison between cyclohexyl bonded and octadecyl bonded phases, the former being much more efficient.  (+info)

Quercetin glucuronides but not glucosides are present in human plasma after consumption of quercetin-3-glucoside or quercetin-4'-glucoside. (19/350)

The nature of quercetin conjugates present in blood after consumption of quercetin glucosides is still unclear. In this study, we analyzed plasma of volunteers that had consumed 325 micromol of either quercetin-3-glucoside or quercetin-4'-glucoside as an oral solution. Quercetin metabolites were extracted with acetonitrile/phosphoric acid and these extracts were analyzed using a high performance liquid chromatography with Coularray detection that distinguishes between the glucuronidated and the glucosylated forms of quercetin. No intact quercetin glucosides and only trace amounts of aglycone were found in human plasma, irrespective of the glucoside ingested. This was confirmed by spiking the plasma with glucoside standards. The major components in plasma had the same retention time as quercetin glucuronide standards. These plasma components disappeared after treatment of the plasma with bovine liver beta-glucuronidase, under reformation of quercetin, and showed the same oxidation pattern as the glucuronides. These results suggest that after consumption of quercetin glucosides, quercetin glucuronides are major metabolites in plasma.  (+info)

Crystal structure of alpha-momorcharin in 80% acetonitrile--water mixture. (20/350)

Crystals of alpha-momorcharin (MMC) were cross-linked and soaked in an 80% acetonitrile--water mixture and X-ray data were collected to 2.2 A resolution. MMC is a ribosome-inactivating protein with a sugar chain on Asn-227. In previous studies, the whole conformation of the sugar chain could not be obtained in the aqueous system. Here the structure of MMC in a low water system is shown to be similar to the native one, but the sugar chain on Asn-227 is defined by the electron density map. Several oxygen atoms of the oligosaccharide formed intramolecular hydrogen bonds to the protein moiety. The conformation of the residues in the active center is similar to that in the aqueous system. Our results show conformational alteration of the tetrasaccharide of MMC in organic media. They indicate that the oligosaccharides are more rigid in organic solvents. X-ray determination in organic media may be used to solve some structures of oligosaccharides linked to glycoproteins.  (+info)

Filamentous bacteriophage stability in non-aqueous media. (21/350)

BACKGROUND: Filamentous bacteriophage are used as general cloning vectors as well as phage display vectors in order to study ligand-receptor interactions. Exposure to biphasic chloroform-water interface leads to specific contraction of phage, to non-infective I- or S-forms. RESULTS: Upon exposure, phage were inactivated (non-infective) at methanol, ethanol and 1-propanol concentrations inversely dependent upon alcohol hydrophobicity. Infectivity loss of phage at certain concentrations of 1-propanol or ethanol coincided with changes in the spectral properties of the f1 virion in ultraviolet fluorescence and circular dichroism studies. CONCLUSIONS: The alcohols inactivate filamentous phage by a general mechanism--solvation of coat protein--thereby disrupting the capsid in a manner quite different from the previously reported I- and S-forms. The infectivity retention of phagemid pG8H6 in 99% acetonitrile and the relatively high general solvent resistance of the phage strains studied here open up the possibility of employing phage display in non-aqueous media.  (+info)

Tuning of the product spectrum of vanillyl-alcohol oxidase by medium engineering. (22/350)

The flavoenzyme vanillyl-alcohol oxidase (VAO) catalyzes the conversion of 4-alkylphenols through the initial formation of p-quinone methide intermediates. These electrophilic species are stereospecifically attacked by water to yield (R)-1-(4'-hydroxyphenyl)alcohols or rearranged in a competing reaction to 1-(4'-hydroxyphenyl)alkenes. Here, we show that the product spectrum of VAO can be controlled by medium engineering. When the enzymatic conversion of 4-propylphenol was performed in organic solvent, the concentration of the alcohol decreased and the concentration of the cis-alkene, but not the trans-alkene, increased. This change in selectivity occurred in both toluene and acetonitrile and was dependent on the water activity of the reaction medium. A similar shift in alcohol/cis-alkene product ratio was observed when the VAO-mediated conversion of 4-propylphenol was performed in the presence of monovalent anions that bind specifically near the enzyme active site.  (+info)

Massive rhabdomyolysis and acute renal failure after acetonitrile exposure. (23/350)

A case of systemic rhabomyolysis after acetonitrile exposure is reported. A 35-year-old previously healthy man suffered from vomiting, convulsion and consciousness loss 15 hours after exposure to acetonitrile. Since acetonitrile is known to be metabolized into cyanide, antidote therapy against cyanide poisoning was given. On admission, pain and all-over muscle swelling were marked. Although the initial therapy was effective, rhabdomyolysis and then acute renal failure developed. Renal function improved very slowly after six weeks of hemodialysis, but atrophy of the muscles remained. The rhabdomyolysis may have been caused by toxicity of the cyanide itself in combination with hypoxia and convulsion.  (+info)

Insights into the alkaline transformation of ferricytochrome c from (1)H NMR studies in 30% acetonitrile-water. (24/350)

Recently, we found that ferricytochrome c (ferricyt c) undergoes significant structural changes in mixed aqueous-nonaqueous media, resulting in the formation of a mixture of alkaline-like species. The equilibrium composition of this mixture of species is dependent on the dielectric constant of the mixed solvent medium. One-dimensional (1D) and two-dimensional (2D) (1)H nuclear magnetic resonance (NMR) methods have now been used to study these alkaline-like forms in 30% acetonitrile-water solution. A native-like (M80-ligated) III* form, two lysine-ligated forms (IVa* and IVb*), and a hydroxide-ligated form (V*) were observed. Heme proton resonance assignments for these forms were accomplished using 1D (1)H NMR and 2D nuclear Overhauser effect spectroscopy methods at 20 degrees C and 35 degrees C. The chemical exchange between the alkaline forms in 30% acetonitrile solution facilitated heme proton resonance assignments. Based on examination of the heme proton chemical shifts and several highly conserved amino acid residues, the electronic structure, secondary structure, and hydrogen bond network in the vicinity of the heme in the III* form were found to be intact. Similarly, the heme electronic structure of the IVa* form was found to be comparable to that of the IVa form. Differences in the order of the heme methyl resonances in the IVb* form, however, suggest that the heme active site in this form is somewhat different from that observed in aqueous alkaline solution. In addition, resonance assignments for the 8- and 3-methyl heme protons were made for the hydroxide-ligated V* form for the first time. The observation of chemical exchange peaks between all species except IVb* and IVa* or V* was used to propose an exchange pathway between the different forms of ferricyt c in 30% acetonitrile solution. This pathway may be biologically significant because ferricyt c, which resides in the intermembrane space of mitochondria, is exposed to medium of relatively low dielectric constant when it interacts with the mitochondrial membrane.  (+info)