Solid-vapor interactions: Influence of environmental conditions on the dehydration of carbamazepine dihydrate

The goal of this research was a phenomenological study of the effect of environmental factors on the dehydration behavior of carbamazepine dihydrate. Dehydration experiments were performed in an automated vapor sorption apparatus under a variety of conditions, and weight loss was monitored as a function of time. In addition to lattice water, carbamazepine dihydrate contained a significant amount of physically bound water. Based on the kinetics of water loss, it was possible to differentiate between the removal of physically bound water and the lattice water. The activation energy for the 2 processes was 44 and 88 kJ/mol, respectively. As expected, the dehydration rate of carbamazepine dihydrate decreased with an increase in water vapor pressure. While dehydration at 0% relative humidity (RH) resulted in an amorphous anhydrate, the crystallinity of the anhydrate increased as a function of the RH of dehydration. A method was developed for in situ crystallinity determination of the anhydrate formed. Dehydration in the presence of the ethanol vapor was a 2-step process, and the fraction dehydrated at each step was a function of the ethanol vapor pressure. We hypothesize the formation of an intermediate lower hydrate phase with unknown water stoichiometry. An increase in the ethanol vapor pressure first led to a decrease in the dehydration rate followed by an increase. In summary, the dehydration behavior of carbamazepine dihydrate was evaluated at different vapor pressures of water and ethanol. Using the water sorption apparatus, it was possible to (1) differentiate between the removal of physically bound and lattice water, and (2) develop a method for quantifying, in situ, the crystallinity of the product (anhydrate) phase.     

Study of bovine carbonic anhydrase by 13C-NMR-spectroscopy

The study of internal mobility in enzymes is of considerable importance for the understanding of their catalytic function, which cannot be adequately described as a property of a rigid protein. [13C]NMR spectroscopy permits simultaneous and selective observation of spectral lines from carbon atoms in many different residues in the enzyme with the chemical shift and relaxation parameters sensitive to structure, conformation and local motion. The changes in internal mobility in bovine carbonic anhydrase B (carbonate hydrolase, EC 4.2.1.1) in the native form and at various stages of denaturation are studied. Measurements of the relaxation parameters (T1, T1 rho) and of the NOE of 13C nuclei in the native protein showed that the extensive beta-sheet together with groups in the active center has a considerable internal librational mobility with tau G about 10(-11) s. This librational mobility is fairly uniform for all the alpha-carbons in the native enzyme. The use of a semiempirical modification of the motional theory proposed by Woessner allows to use simultaneously all the relaxation parameters measured in order to determine reliable values of the various correlation times.     

13C-NMR study of CO2-fixation during the heterotrophic growth in Chlorobium thiosulfatophilum

The functioning of the biosynthetic pathways of the amino acids alanine, glycine, aspartic acid, glutamic acid and tyrosine, and of nucleosides in the photosynthetic bacterium Chlorobium thiosulfatophilum during heterotrophic growth on ¹³CO₂ and unlabelled acetate was investigated using ¹³C-NMR as the method for determination of the labelling patterns of the separated substances. On the basis of the analysis of the multiplet structure of the spectra of the tightly-coupled systems, the conclusion was drawn that the Calvin cycle does not function in the experimental conditions used. The labelling pattern of the glutamic acid indicated that about 30% of the amino acid molecules were synthesized through the reactions of the reductive carboxylic acid cycle, the remaining 70% being derived from oxaloacetate and exogenous acetate through the reactions of the Krebs cycle. Labelling patterns of the nucleosides were in agreement with their known biosynthetic pathways.     

13C and 15N NMR and time-resolved fluorescence depolarization study of bovine carbonic anhydrase--4-methylbenzenesulfonamide complex

The influence of the binding of the high-affinity inhibitor, 4-methylbenzenesulfonamide, to the active site of bovine carbonic anhydrase B was studied by 15N- and 13C-NMR spectroscopy. The rotational correlation time dependence on temperature and concentration of the complex was determined by time-resolved fluorescence depolarization measurements. Our experiment provides evidence that the stoichiometry of the interaction of 4-methylbenzenesulfonamide with carbonic anhydrase B is 1:1 and the inhibitor is bound in anionic form. The 15N-NMR relaxation parameters confirm our previous conclusions about the presence of librational motions in the active site of carbonic anhydrase and indicate that the internal motion in the enzyme-inhibitor complex is more restricted than the backbone motion in the uncomplexed native enzyme.     

Nicorandil decreases postischemic actin oxidation

This study examined the hypothesis that preconditioning can decrease postischemic oxidative protein damage. Isolated rat hearts were subjected to 25 min of normothermic global ischemia followed by 45 min of reperfusion. These were compared with hearts pretreated with 20 microM nicorandil or preconditioned with two cycles of ischemia. Changes in the high energy phosphates, ATP and phosphocreatine, were followed using (31)P-NMR spectroscopy. Protein carbonyls were assessed using an immunoblot technique. Postischemic hemodynamic function and high energy phosphates recovered to significantly (p <.05) higher levels in nicorandil-treated and ischemic preconditioned hearts as compared to controls. Postischemic protein carbonyl formation was highest in control reperfused hearts but reduced to intermediate between control and preischemic hearts by ischemic preconditioning and virtually prevented by nicorandil pretreatment, with a prominent band at 43 kDa significantly affected (p <.05). Based on immunoshift and immunoprecipitation studies, this band was identified as a mixture of actin isoforms. These studies support the conclusion that nicorandil diminishes protein oxidative damage in general, and specifically actin oxidation, which in the presence of improved supply of high energy phosphates, leads to enhanced postischemic contractile function.     

13C-NMR study of the glucose synthesis pathways in the bacterium Chlorobium thiosulfatophilum

Photoassimilation of ¹³CO₂ and acetate by the photosynthetic bacterium Chlorobium thiosulfatophilum was investigated using ¹³C-NMR as the method for determination of the labelling pattern of the glucose synthesized by the bacterium. Metabolic pathways functioning in the bacterium were identified by analysis of the multiplet structure of the spectra of tightly coupled systems. The labelling pattern showed that glucose was synthesized in C. thiosulfatophilum mainly by the gluconeogenesis pathway. In agreement with previous investigations, the reserve polysaccharide of C. thiosulfatophilum was shown to be polyglucose, with the glucose units linked predominantly by (1→4)α-glucosidic linkages.