Potentiometric studies on the complexation of copper(II) by phenolic acids as discrete ligand models of humic substances

Studies on the complexation of copper(II) by phenolic acids, as ligand models of humic substances were done by potentiometry. The acids under study were: 3,4-dihydroxyhydrocinnamic acid or hydrocaffeic acid (1), 3,4-dihydroxyphenylacetic acid (2) and 3,4-dihydroxybenzoic acid or protocatechuic acid (3). Acidity constants of the ligands and the formation constants of metal-ligand complexes were evaluated by computer programs. The carboxylic group of the phenolic acids has different pK_a1 values, being the dissociation constants intrinsically related with the distance between the function and the aromatic nucleus. The results obtained allow concluding that acidity constants of the catechol moiety of the compounds are similar with pK_a2 and pK_a3 values between 9.47-9.41 and 11.55-11.70. The complexation properties of the three ligands towards copper(II) ion are quite similar, being the species found not different either in nature or stability. Although the model ligands have some structural differences no significant differences were found in their complexation properties towards copper(II). So, it can be postulated that complexation process is intrinsically related with the presence of a catechol group.     

Lipase from a Brazilian StrainPenicillium citrinum Cultured in a Simple and Inexpensive Medium st]Heat-Denaturation, Kinetics, and pH Stability

This work is a study of lipase production by a Brazilian strain ofPenicillium citrinum using an inexpensive and simple medium without organic nitrogen sources and of some important industrial properties, including thermostability in relation to ionic strength. The maximal lipase activity (1585 U/L) was obtained whenPenicillium citrinum was cultured on 0.75% ammonium sulfate complemented with minerals salts instead of yeast extract. Although this activity was about 55% lower than that produced in medium with yeast extract (2850 U/L), the specific activity (7.8 U/mg proteins) was higher than that obtained with the yeast extract (4.9 U/mg proteins). The morphology of fungus changed totally, with yeast extract there are smooth, solid, and spherical pellets whereas on ammonium sulfate there are small “hairy” pellets uniformly suspended in the medium. The effect of ferrous (Fe⁺⁺) ions was carried out using medium MA with and without Fe⁺⁺ ions. Lipase production byPenicillium citrinum in medium MA requires Fe⁺⁺ ions, the absence of which caused a decreased of about 50% in the specific activity (3.5 U/mg proteins). The utilization of commercial, locally available oils as carbon sources, such as soybean oil (236 U/L) and corn oil (74 U/L) resulted in lower activity compared to olive oil, showing that lipase production byPenicillium citrinum is specifically induced by olive oil. Potassium concentration in the medium can effects the production of lipase (1 mM (1585 U/L), 10 mM (1290 U/L), and 30 mM (1238 U/L), 50 mM (195 U/L), and 100 mM (2 U/L). The crude culture filtered was susceptable to thermal deactivation. It was stable at pH 6.0, but was not stable at the optimum pH (8.0-8.5) at 50 mM. At the low ionic concentration (1-25 mM) this lipase was stable at low pH (3.5-4.0). The activation energy was 22.4 ±2.2 Kcal. mol 1.     

Automatic sequential determination of the hydrogen peroxide scavenging activity and evaluation of the antioxidant potential by the 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation assay in wines by sequential injection analysis

An automatic system that performs two analytical procedures, allowing the evaluation of the relative antioxidant capacity of wine samples, was developed. Automation was carried out using a sequential injection analysis (SIA) system that allowed, thanks to its versatility, the development of two methodologies. One is based on the decolorization assay of the 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical, using a spectrophotometric detector. A second methodology allowed the evaluation of the hydrogen peroxide scavenging activity by measuring the oxidation of homovanylic acid (HVA) to its fluorescent dimer, using a fluorescent detector.The developed automatic methodologies were evaluated using trolox as standard and subsequently using other antioxidant substances as gallic acid, caffeic acid, ascorbic acid, catechin and taxifolin which are abundant in wine and whose antioxidant activities were compared to that shown by trolox. The spectrophotometric and fluorimetric assays showed linearity intervals between 0.001 and 0.01 mM, and 0.001 and 0.008 mM of trolox, respectively.The evaluation of the antioxidant power of 20 white and red wine samples, from different Portuguese wine producing regions, was carried out sequentially, in the automatic system. The results were expressed in trolox equivalent antioxidant capacity (TEAC) and presented, for the ABTS and hydrogen peroxide scavenging activity methodologies, detection limits of 8.4 × 10⁻⁷ and 1.4 × 10⁻⁴ mM and relative standard deviation (R.S.D. (%)) in the range 0.6-2.4 and 1-1.8, respectively.     

Isocratic mixed mode liquid chromatographic separation of phospholipids with octadecylsilane-silica stationary phases

Molecular species of phosphatidylcholine, phosphatidylethanolamine and phosphatadic acid were resolved by isocratic reversed phase high performance liquid chromatography (HPLC) using mobile phases of methanol-isopropanol containing para-toluenesulfonic acid (p-tsa). Separation by both non-polar fatty acid chain length and by polar head group functionality was achieved concurrently upon a commercially available octadecylsilane (C18) column endcapped with trimethylsilane (C1) groups. Using a mobile phase of 97.5:2.5 methanol:isopropanol with 7OmMpara-toluenesulfonic acid (p-tsa) at a pH of approximately 1, twelve phospholipid species comprised of four tail group classes (dilauroyl-,dimyristoyl-, dipamitoyl- and distearoyl-) and three head group speciations (phosphatidylcholine, phosphatidylethanolamine and phosphatadic acid) were separated. The column was then exposed to the acidic mobile phase for 48 hours continuously during which the bound phase underwent severe acid-induced hydrolysis, after which the separation of the twelve analytes resulted in the separation of the phospholipid species by non-polar tail group alone. The experimental results are discussed in terms of potential separation mechanisms including dependency of the separation on adsorption of the counter ion into the stationary phase, residual acidic silanol group interactions, and potential interactions of the surface active phospholipids with C1 groups.     

Methodology for rapid tritium determination in urine

Summary The present work proposes a methodology for a rapid determina-tion of the tritium content in urine. The urine is treated with active carbon and tritium is determined by liquid scintillation counting. Different kinds of active carbon were experimented for the metabolites removal from urine samples. The counting efficiency was calculated by a quench curve using yellow food dye as a quenching agent. It was obtained a procedure which maintained a good reproducibility, low uncertainty and detection limit of 7 Bq/l (10 ml of urine, 120 minutes of counting time and efficiency of 23.9%).     

Direct determination of gallium on polyurethane foam by X-ray fluorescence

Gallium chloride is easily extracted from 6M HCl by comminuted polyether-type polyurethane foam. After the extraction step, the gallium absorbed by the PU foam can be quantitatively determined by X-ray fluorescence. A procedure for the direct determination of gallium absorbed by PU foam by XRFS is thus described. Gallium is determined at levels as low as 60 ng/ml (C(L)), with a calibration sensitivity of 424 cps ml/mug, within a linear range 0.1-2.30 mug/ml. The procedure investigated was successfully applied to determination of gallium in aluminum alloys, bauxite and industrial residue samples.     

X-ray diffraction and solid-state NMR studies of a germanium binuclear complex

A compound formulated as (C4H12N2)[Ge2(pmida)2(OH)2] x 4 H2O (where pmida(4-) = N-(phosphonomethyl)iminodiacetate and C4H12N2(2+) = piperazinedium cation), containing the anionic [Ge2(pmida)2(OH)2]2- complex, has been synthesised by the hydrothermal approach and its structure determined by single-crystal X-ray diffraction analysis. Several high-resolution solid-state magic-angle spinning (MAS) NMR techniques, in particular two-dimensional 1H-X(13C,31P) heteronuclear correlation (HETCOR) and 1H-1H homonuclear correlation (HOMCOR) experiments incorporating a frequency-switched Lee-Goldburg (FS-LG) decoupling scheme, have been employed for the first time in such a material. Using these tools in tandem affords an excellent general approach to study the structure of other inorganic-organic hybrids. We assigned the NMR resonances with the help of C...H and P...H internuclear distances obtained through systematic statistical analyses of the crystallographic data. The compound was further characterised by powder X-ray diffraction techniques, IR and Raman spectroscopy, and by elemental and thermal analyses (thermogravimetric analysis and differential scanning calorimetry).     

Efficient method for visualization and isolation of proteins resolved in polyacrylamide gels

Polyacrylamide gel electrophoresis is a popular method used to purify proteins for reconstitution experiments, amino acid composition and sequence determinations. In this report we describe methods that will be of general use in the isolation and characterization of proteins and the benefits of substituting boric acid for glycine in the electrophoresis tray buffers. We also describe how proteins resolved in a variety of gel systems (including those containing sodium dodecyl sulfate) may be rapidly visualized with 8-anilino-1-naphthalene sulfonic acid and efficiently transferred to a second gel for two-dimensional gel analysis, or isolated by electroelution for subsequent characterization.     

Electrospray ionization quadrupole time-of-flight and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometric analyses to solve micro-heterogeneity in post-translationally modified peptides from Phoneutria nigriventer (Aranea, Ctenidae) venom

Previous studies of the fractionated venom of the Brazilian armed spider Phoneutria nigriventer, obtained by gel filtration, have demonstrated the presence of a fraction PhM, a pool of small peptides (up to 2000 Da) that provoke contractions in smooth muscle of guinea pig ileum. Initial attempts to sequence these peptides were largely unsuccessful because of the low purification yield and the fact that the majority seemed to be blocked at their N-termini. In the present work, analysis of this venom fraction by mass spectrometry has revealed the existence of a highly complex mixture of peptides with molecular weights corresponding to those observed for the muscle-active peptides previously described (800-1800 Da). These peptides appear to be a family of isoforms with some particular features. The amino acid sequences of 15 isoforms have been determined by tandem mass spectrometry (MS/MS) using both electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q/ToFMS) and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-ToF/ToFMS). These molecules contain post-translational modifications such as proteolysis and C-terminal amidation, which combine to generate additional isoforms. All the isoforms sequenced in this study possess an N-terminal pyroglutamic acid residue. A search for sequence similarities with other peptides in databanks revealed that these peptides are structurally related to the tachykinins, a family of neuro-hormone peptides. The data obtained in this study will be essential for the subsequent steps of this research, the synthesis of these peptides and pharmacological characterization of their biological activity.     

Determination of ambroxol in an automated multi-pumping pulsed flow system.

A new flow methodology exploiting the multi-pumping approach was developed for the spectrophotometric determination of ambroxol hydrochloride in pharmaceutical preparations. The flow manifold was implemented by using, exclusively, multiple solenoid-actuated micro-pumps, which acted simultaneously as sample insertion, solutions propelling and reagents commutation units. Linear calibration plots were obtained over an ambroxol concentration ranging from 10 to 200 mg l(-1) (r.s.d. < 0.5%, n = 15) and a sampling rate of about 60 samples per hour (flow rate = 1.92 ml min(-1), sample volume = 80 microl).