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The thermal degradation of rabbit skin glue, a collagen-based proteinaceous material used as a paint binder in paintings, was investigated in this paper. Paint reconstructions of the glue on its own or mixed with azurite (Cu3(CO3)2(OH)2), calcium carbonate (CaCO3), hematite (Fe2O3·nH2O) and red lead (Pb3O4) were analysed using a thermoanalytical approach. This method enabled us to investigate the interactions between the glue and pigments before and after artificial indoor light ageing. The study was carried out using differential scanning calorimetry, thermogravimetry and thermogravimetry/FTIR analysis already successfully employed to characterize the paint binders. The results highlighted that all the inorganic pigments interact with rabbit skin glue, thus decreasing the thermal stability of the binder. Light ageing further decreased the thermal stability of pigmented paint replicas, suggesting a moderate increase in the rate of the degradation.

The naturalized dyes (ND) and the traditional acid dyes (ADs) were compared by studying the different behavior during the leather dyeing process. NDs are glyconjugated compounds synthesized by the covalent union of a dye species with a natural sugar (e.g. lactose) able to confer water-soluble properties to the dye molecule as a whole. The interactions between the dyes and the leather proteins were studied by FT-IR spectroscopy and thermogravimetric (TG) analyses. The protein cross-linking of the dyed leather samples was investigated by studying the 1654/1690 cm(-1) peak height ratio and a deconvolution procedure of the amide I peak. The helix secondary structure was the predominant component of the leather proteins of the samples dyed with low concentrations of NDs (2%), while the beta-sheets prevailed when leather samples were dyed with the traditional ADs and high concentrations of NDs (>5%). The data were discussed with respect to TG results. (C) 2015 Elsevier Ltd. All rights reserved.

In this work we present an advantageous method for the simultaneous separation and detection of Tl(I) and Tl(III) species through ion chromatography coupled with on-line inductively coupled plasma - mass spectrometry. Chromatographic separation between Tl(III) and Tl(I) was achieved in less than two minutes. The method was validated by recovery experiments on real samples, and by comparing the sum of the concentrations of individual Tl species with total thallium values obtained from continuous flow ICP-MS. The experimental procedure offers an accurate, sensitive and interference-free method for Tl speciation at trace levels in environmental samples. This allowed us to investigate the Tl speciation in acid mine drainages (AMD), surface waters and springs in a mining catchment in Valdicastello Carducci (Tuscany, Italy), where severe Tl contamination ad been evidenced previously. This study shows for the first time that Tl(III), in addition to Tl(I), is present in considerable amounts in water samples affected by acid mining outflow, raising the question of the origin of this thermodynamically unstable species.

The present work describes a non invasive lactate sensing in sweat during workout. The sensing system is based on a non-equilibrium potentiometric measure performed using disposable, chemically modified, screen printed carbon electrodes (SPCEs) that can be wetted with sweat during the exercise. The potentiometric signal, which is proportional to lactate concentration in sweat, is produced by a redox reaction activated by UV radiation, as opposed to the enzymatic reaction employed in traditional, blood-based measuring devices. The sensing system exhibits chemical selectivity toward lactate with linearity from 1 mM up to 180 mM. The dynamic linear range is suitable for measurement of lactate in sweat, which is more than 10 times concentrated than hematic lactate and reaches more than 100 mM in sweat during workout. The noninvasive measure can be repeated many times during exercise and during the recovery time in order to get personal information on the physiological and training status as well as on the physical performance. The device was successfully applied to several human subjects for the measurement of sweat lactate during prolonged cycling exercise. During the exercise sweat was simultaneously sampled on filter paper and extracted in water, and the lactate was determined by HPLC for method validation. The lactate concentration changes during the exercise reflected the intensity of physical effort. This method has perspectives in many sport disciplines as well as in health care and biomedical area.

We described the hyphenation of photo-induced chemical vapor generation with high performance liquid chromatography–atomic fluorescence spectrometry (HPLC–AFS) for the quantification of inorganic mercury, methylmercury (MeHg) and ethylmercury (EtHg). In the developed procedure, formic acid in mobile phase was used for the photodecomposition of organomercury compounds and reduction of Hg 2 + to mercury vapor under microwave/ultraviolet (MW/UV) irradiation. We optimized the proposed method studying the influence of several operating parameters, including the type of organic acid and its concentration, MW power, composition of HPLC mobile phase and catalytic action of TiO 2 nanoparticles. Under the optimized conditions, the limits of detection were 0.15, 0.15 and 0.35 μg L − 1 for inorganic mercury, MeHg and EtHg, respectively. The developed method was validated by determination of the main analytical figures of merit and applied to the analysis of three certified reference materials. The online interfacing of liquid chromatography with photochemical-vapor generation–atomic fluorescence for mercury determination is simple, environmentally friendly, and represents an attractive alternative to the conventional tetrahydroborate (THB) system.

Different metal substituted (Y, Sn and Ce) MCM-41 materials were synthesized and detemplated by a low temperature surfactant removal methodology. All metal substituted materials showed an increase in the d(100) lattice parameter compared to the parent MCM-41 matrices. The increase depends on both the metal type and amount that is successfully incorporated by direct conventional hydrothermal synthesis. The metal modified MCM-41 materials were detemplated by an ultrasound assisted (US) ion-exchange process using methanol as the solvent (NH4NO3/US/MeOH). The effect of the ultrasound amplitude, extraction time and salt concentration were explored, and optimal values were determined for Y-MCM-41 detemplation (40 mM of NH4NO3, 60% of US amplitude and 15 min of adiabatic treatment). The removal percentage achieved with these values was in the following order: Y (97.7%) > Ce (94.4%) > Sn ;(92.1%) > Si (90.3%). Several techniques (SAXS, FTIR, TGA, H-1 MAS, Si-29 HPDEC MAS NMR and N-2 physisorption) demonstrated that the mesoporous materials keep their hexagonal structure and high surface area after the NH4NO3/US/MeOH surfactant extraction. Moreover, the thermal shrinkage of the structure was reduced in the following order: Si (0.6%) < Sn (4%) < Ce (5%) < Y (9%) < calcined samples (from 9 to 15%). The surfactant recovered was successfully recycled in a consecutive microwave assisted hydrothermal synthesis cycle (MW-HT). The synergy of different strategies (MW-HT synthesis, NH4NO3/US/MeOH surfactant removal and surfactant recovery) produces considerable time, energy and cost abatement, environmental impact reduction and promising scale up projections in the eco-friendly synthesis of MCM-41 materials.

We developed a flow injection (FI) method for the determination of thiomersal (sodium ethylmercurithiosalicylate, C9H9HgNaO2S) based on the UV/microwave (MW) photochemical, online oxidation of organic mercury, followed by cold vapor generation atomic fluorescence spectrometry (CVG-AFS) detection. Thiomersal was quantitatively converted in the MW/UV process to Hg(II), with a yield of 97 ± 3%. This reaction was followed by the reduction of Hg(II) to Hg(0) performed in a knotted reaction coil with NaBH4 solution, and AFS detection in an Ar/H2 miniaturized flame. The method was linear in the 0.01–2 μg mL−1 range, with a LOD of 0.003 μg mL−1. This method has been applied to the determination of thiomersal in ophthalmic solutions, with recoveries ranging between 97% and 101%. We found a mercury concentration in commercial ophthalmic solutions ranging between 7.5 and 59.0 μg mL−1.

S-nitrosoglutathione (GSNO) is an endogenous nitrosothiol involved in several pathophysiological processes. A role for GSNO has been envisaged in the expression of inflammatory cytokines such as IL-8; however, conflicting results have been reported. γ-Glutamyltransferase (GGT) enzyme activity can hydrolyze the γ-glutamyl bond present in the GSNO molecule thus greatly accelerating the release of bioactive nitric oxide. Expression of GGT is induced by oxidative stress, and activated neutrophils contribute to GGT increase in cystic fibrosis (CF) lung exudates by releasing GGT-containing microvesicles. This study was aimed at evaluating the effect of GSNO catabolism mediated by GGT on production of IL-8 in CF transmembrane regulation protein-mutated IB3-1 bronchial cells. The rapid, GGT-catalyzed catabolism of GSNO caused a decrease in both basal and lipopolysaccharide-stimulated IL-8 production in IB3-1 cells, by modulating both NF-κB and ERK1/2 pathways, along with a decrease in cell proliferation. In contrast, a slow decomposition of GSNO produced a significant increase in both cell proliferation and expression of IL-8, the latter possibly through p38-mediated stabilization of IL-8 mRNA. Our data suggest that the differential GSNO catabolism mediated by GGT enzyme activity can downregulate the production of IL-8 in CF cells. Hence, the role of GGT activity should be considered when evaluating GSNO for both in vitro and in vivo studies, the more so in the case of GSNO-based therapies for cystic fibrosis.

A dynamic surface tension detector (DSTD) for the multidimensional analysis of polymers and proteins in aqueous solutions is reviewed. The DSTD is briefly compared with other instrumental methods for the measurement and evaluation of time-dependent surface tension assessment. The dynamic nature of the surface tension signal is shown to provide unique selectivity and useful chemical information. The benefits of multidimensional detection using the DSTD for flow injection analysis and high performance liquid chromatography providing selective and sensitive surface activity analysis are discussed. © 2013 Society of Chemical Industry

The degradation of the proteinaceous binders, ovalbumin (OVA) and casein, and their interactions with azurite (Cu(3)(CO(3))(2)(OH)(2)), calcium carbonate (CaCO(3)), hematite (Fe(2)O(3)) and red lead (Pb(3)O(4)) pigments were studied. A multi-analytical approach based on Thermogravimetric Analysis (TG), Differential Scanning Calorimetry (DSC), Fourier Transform Infrared Spectroscopy (FTIR) and Size Exclusion Chromatography (SEC) was used. The research was carried out on a set of paint reconstructions, which were analysed before and after artificial light ageing. We highlighted that in most cases the inorganic pigments interact with both proteins by decreasing their thermal stability and their intermolecular β-sheet content, and that ageing induces aggregation. We hypothesized that pigments intercalate between protein molecules, producing a partial disruption to the protein-protein intermolecular interaction. In the case of casein, these phenomena continued during ageing. In fact, we observed a complete disappearance of intermolecular β-sheets and an increase in intramolecular β-sheets and random coil during ageing. This result is in agreement with the structural properties of casein, whose aggregation is known to be induced by hydrophobic interactions. On the other hand, in aged OVA paint replicas, we observed the formation of new intermolecular β-sheets and an increase in thermostability. In addition FTIR showed oxidation of the side chains of the aged OVA/hematite sample and aged casein pigment samples, and SEC highlighted hydrolysis phenomena in aged carbonate, azurite and red lead/OVA complexes and in aged casein/calcium carbonate and casein/azurite samples.

Halloysite nanotubes (HNTs) are considered as ideal materials for biotechnological and medical applications. An important feature of halloysite is that it has a different surface chemistry on the inner and outer sides of the tubes. This property means that negatively-charged molecules can be selectively loaded inside the halloysite nanoscale its lumen. Loaded HNTs can be used for the controlled or sustained release of proteins, drugs, bioactive molecules and other agents. We studied the interaction between HNTs and bovine serum albumin, ? lactalbumin and ? -lactoglobulin loaded into HTNs using Fourier transform infrared spectroscopy and thermogravimetry. These techniques enabled us to study the protein conformation and thermal stability, respectively, and to estimate the amount of protein loaded into the HNTs. TEM images confirmed the loading of proteins into HTNs.

Superparamagnetic iron oxide nanoparticles (SPIONs) have received increasing interest as contrast media in biomedical imaging and innovative therapeutic tools, in particular for loco-regional ablative treatments and drug delivery. The future of therapeutic applications would strongly benefit from improving the capability of the nanostructured constructs to reach the selected target, in particular beyond the intravascular space. Besides the decoration of SPIONs surface with ad hoc bioactive molecules, external magnetic fields are in principle able to remotely influence SPIONs' physiological biodistribution and concentrate them to a specific anatomical region or portion of a tissue. The reduction of SPIONs administered to the body and the need for defining the effective SPIONs local concentration suggest that PET/CT may be a method to quantitatively detect the nanoparticles accumulation in vivo at low concentration and assess their tridimensional distribution in response to an external magnetic field and in relation to the local anatomy highlighted by CT imaging. Here, we report on the possibility to assess the spatial distribution of magnetically-driven radiolabelled SPIONs in a peripheral tissue (mouse thigh) with microPET/CT imaging. To this aim we labelled SPIONs using

We studied the interactions of rabbit glue, a collagen-based proteinaceous binder, with azurite (Cu3(CO3)2(OH)2), calcium carbonate (CaCO3), hematite (Fe2O3·nH2O), red lead (Pb3O4) and cinnabar (HgS) by Fourier transform infrared spectroscopy (FT-IR). The research was carried out on a set of paint reconstructions, which were analysed before and after artificial light ageing. A deconvolution of the amide I FT-IR absorption peak was performed with a written-in-house LabVIEW program to study the secondary structure of the glue. The changes in the glue conformation highlighted that all the inorganic pigments interact with the proteinaceous binder. The conformational changes were correlated with a loss of stability of the collagen structure, especially after ageing, likely due to the interlayer coordination of metals salts and oxide with protein functional groups. These results were correlated with the lower thermal stability of the glue/pigment mixtures with respect to the pure glue, evidenced by thermogravimetry (TG) and differential scanning calorimetry (DSC) analyses performed in a previous step of this work.

Clay minerals are widely used in pharmaceutical formulations, therefore studying how they interaction with proteins is important because they can alter their biological functions. The interactions of proteins with nanostructured clays and, in general, with nanomaterials should also be studied because nanoparticles are known to interfere with protein amyloid formation, which is implicated in severe neurodegenerative diseases. Kaolinite and halloysite belong to the kaolinite group of minerals with the nominal formula Al2Si2O5(OH)(4) per half unit cell, however they have important structural layer stacking differences. We studied the surface interactions between clays and bovine serum albumin (BSA) by Fourier Transform Infrared Spectroscopy in order to understand the role of clay morphology on protein conformation. We show that the conformational changes of BSA depend on protein concentration and its initial structure, clay morphology and the clay/protein ratio. The surface curvature radius seems to play a key role in the final conformation. Both the curved nanoscale surface of halloysite nanotubes (HNTs) and the flat morphology of kaolinite (Kao) interfere profoundly on the a/b transitions of BSA. BSA conformation also determines the percentage of protein adsorbed on the clay surface.

A geological study evidenced the presence of thallium (Tl) at concentrations of concern in groundwaters near Valdicastello Carducci (Tuscany, Italy). The source of contamination has been identified in the Tl-bearing pyrite ores occurring in the abandoned mining sites of the area. The strongly acidic internal waters flowing in the mining tunnels can reach exceptional Tl concentrations, up to 9000μg/L. In September 2014 Tl contamination was also found in the tap water distributed in the same area (from 2 to 10μg/L). On October 3, 2014 the local authorities imposed a Do Not Drink order to the population. Here we report the results of the exposure study carried out from October 2014 to October 2015, and aimed at quantifying Tl levels in 150 urine and 318 hair samples from the population of Valdicastello Carducci and Pietrasanta. Thallium was quantified by inductively coupled plasma - mass spectrometry (ICP-MS). Urine and hair were chosen as model matrices indicative of different time periods of exposure (short-term and long-term, respectively). Thallium values found in biological samples were correlated with Tl concentrations found in tap water in the living area of each citizen, and with his/her habits. Thallium concentration range found in hair and urine was 1-498ng/g (values in unexposed subjects 0.1-6ng/g) and 0.046-5.44μg/L (reference value for the European population 0.006μg/L), respectively. Results show that Tl levels in biological samples were significantly associated with residency in zones containing elevated water Tl levels. The kinetics of decay of Tl concentration in urine samples was also investigated. At the best of our knowledge, this is the first study on human contamination by Tl through water involving such a high number of samples.

The simultaneous determination of lactate and pyruvate in sweat has been performed using reversed phase high-performance liquid chromatography (RP-HPLC) with UV detection at 220 nm. The calibration curves were linear in the investigated range 0.3 - 350 mm of lactate, 0.003- 1 mm of pyruvate. The sensitivity was good with a limit of detection of 0.03 mm for lactate and 0.001 mm for pyruvate. Recoveries evaluated for the entire procedure were 102 ± 0.1 and 96 ± 0.1 for lactate and pyruvate, respectively. The method was successfully applied to analysis of sweat in 8 athletes at rest (pilocarpine sweating) and during physical exercise.

A method based on reversed-phase high-performance liquid chromatography with fluorescence detector has been developed for the determination of tocopherols in vegetable oils. Oils were diluted in acetonitrile/tetrahydrofuran (THF) and injected directly onto HyPurity C 18 column. Methanol and THF (90:10) mixture was used as a mobile phase. Tocopherols were detected by fluorescence detector. The method had good limit of detection (LOD) (7 ng/g for α-tocopherol and 6 ng/g for β-, γ- and δ-tocopherols) and reproducibility (CV% The method was applied to the determination of α-, (β+γ)- and δ-tocopherols in extra virgin olive oil (EVOO), sunflower, high oleic sunflower oil (HOSO), hazelnut, soy, corn, peanut and almond oil. A new parameter, the ratio of α-/(β+γ)-tocopherol concentration (named α/β tocopherol ratio), in these vegetable oils was systematically assessed and proposed as a first screening marker of the authenticity of EVOO by studying EVOO/sunflower, EVOO/hazelnut and EVOO/peanut mixtures. The use of δ-tocopherol as adulteration marker was also discussed.

An analytical method was developed for the determination of thiols in biological samples. Reverse phase chromatography coupled to ICP quaduropole MS or Orbitrap MS was employed for the separation and detection of thiols. For the determination of total thiols, oxidized thiols were reduced using dithiothreitol (DTT). Reduction efficiencies for species of interest were found to be close to 100%. Reduced thiols were derivatized by p-hydroxymercuribenzoate (PHMB) and then separated on a C8 column. Optimization of the extraction, separation and detection steps of the HPLC-ICP-MS and HPLC-Orbitrap MS methods was carried out. Detection limits for cysteine, homocysteine, selenocysteine, glutathione, selenomethionine and cysteinyl-glycine were found to be 18, 34, 39, 12, 128 and 103fmol, respectively, using HPLC-Orbitrap MS and 730, 1110, 440, 1110 and 580fmol for cysteine, homocysteine, selenocysteine, glutathione, and cysteinyl-glycine using HPLC-ICP-MS. Contrary to expectation, the LODs and RSDs are higher for the HPLC-ICP-MS instrument, therefore HPLC-Orbitrap MS was used for the determination of thiols in yeast samples. Three different brands of baker's yeast and a selenized yeast were analyzed. The GSH and cysteine levels found in these samples ranged from 4.45 to 17.87µmolg-1 and 0.61 to 1.32µmolg-1, respectively. © 2010 Elsevier B.V., National Rehabilitation Center, This work was financially supported by NRC , Canada. Sezgin Bakirdere would like to thank Tubitak for financial support throughout this study. The authors thank Peggy McCooeye for her technical support.

Chromatographic determination of glutathione disulfide (GSSG) without any preliminary reduction has been presented using GSSG derivatization by p-hydroxymercuribenzoate (pHMB) in strong alkaline medium followed by the determination of GS-pHMB complex by reversed phase chromatography coupled to chemical vapour generation and atomic fluorescence detector (RPC-CVGAFS). A detection limit of 35 nM for GSSG (corresponding to 1.8 pmol) detected as GS-pHMB species was achieved based on a signal-to-noise ratio of 3 in buffer and in blood. The proposed method was applied to the determination of GSSG in whole blood and validated by the classical determination of GSSG by derivatization after reduction with dithiothreitol (DTT).

A liquid chromatography method with mass spectrometric detection has been developed for the simultaneous determination of six thiols in the sulfur metabolic pathway, including cysteine (Cys), homocysteine (HCys), glutathione (GSH), cysteinyl-glycine (Cys-Gly), gamma-glutamyl-cysteine (Glu-Cys), and S-adenosyl-homocysteine (AdoHcy). Tris(2-carboxyethyl)phosphine (TCEP) was used as the reducing reagent and p-(hydroxymercuri)benzoate (PHMB) as the derivatization reagent. Thiols were extracted from 3 mg of yeast using water in an ultrasonic bath. The absolute detection limits for the compounds studied were in the subpicomole range. It was found that AdoHcy, Cys, GSH, Cys-Gly, Glu-Cys, and very little HCys were present in the selenium-enriched yeast sample studied, and GSH, Glu-Cys, very little AdoHcy, Cys, and Cys-Gly were present in three bakery yeasts.

Microcalorimetric technique based on heat-output measurement, direct microorganism counting and enzymatic activity determination, have been explored to evaluate the toxic effects of diphenol species (catechol, resorcinol, and hydroquinone) on soil microbial activity. The thermokinetic parameters including growth rate constant (k), inhibitory ratio, half inhibitory concentration and total thermal effect (Q(total)), were calculated and compared using the data obtained from the power-time curves of the microcalorimeter. It was found that addition of high concentrations of diphenol compounds to the soil samples resulted in low microorganism counts. The trend of the number of cultivable microorganisms with increasing concentration of diphenols was similar to specific growth rate k. It appeared that the higher the water soluble carbon (WSC) content, the higher the Q(total) value. The low dehydrogenase and beta-glucosidase found in the soils treated by catechol and hydroquinone was possibly due to their low WSC concentration and high inhibitory effects, respectively. The results reveal the toxicity of the three diphenols in a descending sequence: hydroquinone, resorcinol and catechol. The combination of the three methods is a more comprehensive toxicological investigation of a complex microbiological system. Microcalorimetry is for studying the metabolic growth of microorganisms, the plate counting method is for quantifying the real microbial growth, and the soil enzyme activity is for assessing the intracellular and extracellular activity of microbial biomass. Our proposed methods can provide toxicological information of diphenols to soil microbes from the metabolic, microbial and biochemical point of views which are consistent with and correlated to each other. Crown Copyright (C) 2009 Published by Elsevier B.V. All rights reserved.

The characteristic features of a novel microwave and UV photoreactor, useful for microwave assisted photochemical processes, are presented by examining the results obtained in the photo decoloration of Acid Orange 7 in an aqueous hydrogen peroxide solution. The advantages over other experimental procedures, like reduced consumption of chemicals, energy saving and simpler setup are highlighted. The method enables to activate a chemical reaction with microwaves and UV radiation using an immersed source without the need of a microwave oven. The principles of functioning of the electrodeless source are given and discussed in terms of the experimental output UV energy, for applied microwave powers from 3 W to 60 W at 2450 MHz. This very efficient technical solution increases the control and yield, enabling the operator to work in safety conditions and using ordinary instruments and devices, also with metal parts, directly connected to the reactor. The method is versatile and the scale up for industrial applications is more feasible.

Solvent tolerance is important because it allows microorganisms to grow at high concentrations of organic solvents. Organisms capable of surviving under these extreme conditions have great application in bioremediation of contaminated sites. In our study, four strains of Pseudomonas sp. B1 and J2, Acinetobacter sp. B2 and J6 which were isolated from the activated sludge were used to evaluate the solvent tolerance by microcalorimetry. The strains B2 and J2 showed high tolerance to organic solvent as they could grow well in the medium containing 10 vol% of benzene and 70 vol% of toluene, respectively. The higher the growth rate constant, the higher the solvent tolerance of the strains isolated. The microbial growths obtained by microcalorimetry were in good agreement with the results determined by ODD. The decrease in growth rate constant and the change in total thermal effect in the presence of organic solvents were in agreement with the data reported in the literatures. Strains B1 and B2 degraded about 67% and 94% of 0.1 vol% benzene within 72 h in a medium with benzene as the sole carbon source, respectively. Strains J2 and J6 degraded approximately 92% and 85% of 0.1 vol% toluene within 72 h, respectively. The related degradation genes detected in previous study in these strains highlight an important potential use of those bacteria for the clean-up of benzene and toluene in the environment. Crown Copyright (C) 2008 Published by Elsevier Ltd. All rights reserved.

A multi-channel thermal activity monitor was applied to study soil microbial activity in Wuhan brown sandy soil in the presence of different concentrations of hexavalent chromium ( K 2 Cr 2 O 7 ) . In order to stimulate the soil microbial activity, 5.0 mg of glucose and 5.0 mg of ammonium sulfate were added to a 1.20-g soil sample under a controlled humidity of 35%. The results show that the poisonous species of K 2 Cr 2 O 7 at an half inhibitory concentration ( IC 50 ) value of 4.27 μ g mL - 1 against soil microbe, and an increase of the amount of hexavalent chromium is associated to a decrease in the microbial activity of the soil, probably due to an increase in the toxicity of hexavalent chromium, affecting strongly the life in this soil microbial environment. Our work also suggests that microcalorimetry is a fast, simple and more sensitive method that can be easily performed to study the toxicity of different species of heavy metals on microorganism compared to other biological methods.

Using TAM III multi-channel thermocalorimetry combined with direct microorganism counting (bacteria, actinomycetes and fungi) under laboratory conditions, we determined the microbial population count, resistance and activity toward cadmium (Cd) toxicity in soil. The thermokinetic parameters, which can represent soil microbial activity, were calculated from power-time curves of soil microbial activity obtained by microcalorimetric measurement. Simultaneous application of the two methods showed that growth rate constant (k), peak-heat output power (P max) and the number of living microorganisms decreased with increasing concentration of Cd. Anncrease in Cd concentration resulted in the decrease of the peak-heat output power and increase in the time of the peak of power. However, the relationships between the thermokinetic parameters (k and P max) and the number of microorganism were not linear, but the trend was similar. Our research also suggests that microcalorimetry is a very sensitive, simple and useful technique for in vitro investigation of the effects of toxic heavy metals on soil microbial activity.

A microcalorimetric technique based on bacterial heat-output was explored to evaluate the toxic effect of different diphenol species on the growth of Escherichia coli (E. coli). Power-time curves of the growth metabolism for E. coli in the presence of different diphenol species were studied using a multi-channel microcalorimetric system with an ampoule method at 37 degrees C. The growth rate constant (k), generation time (t(G)), inhibitory ratio (I), half-inhibitory concentration (IC(50)) and the total thermal effect (Q(T)) for E. coli were obtained. The results show that catechol and hydroquinone are more toxic to E. coli than resorcinol. In all cases, the growth rate constants of E. coli (in log phase) decreased as the concentrations of these diphenols increased. Among these diphenols species, catechol was found to be the most poisonous species at an IC(50) of 323.5 micro g/mL against E. coli. Hydroquinone exhibited moderate virulence with an IC(50) of 1196 micro g/mL and resorcinol had the lowest toxicity with an IC(50) of 2113 micro g/mL. The microcalorimetric bioassay can be a quantitative, inexpensive, and versatile method for acute cellular toxicity study.

A novel method for the determination of salivary thiocyanate is presented. Thiocyanate was converted into ethyl thiocyanate by single-step aqueous derivatization based on triethyloxonium tetrafluoroborate and measured by gas chromatography-mass spectrometry (15min runtime). The ethyl thiocyanate derivative is volatile and can be sampled from the headspace. The derivatization chemistry proposed allows for separation of the analyte from saliva matrix whose introduction in the measurement system is avoided. Quantitation of the analyte was obtained by isotope dilution, employing a 13C-enriched thiocyanate as internal standard. Technical details and fundamental aspects of derivatization chemistry and calibration strategy are presented.The method was validated by comparison with a standard method based on ion chromatography. The two independent methodologies produced results in agreement within 3%. Also a three level spike recovery test was carried out for validation purpose and quantitative recoveries were attained. The method is fast, simple, safe, and sensitive. Measurement of a 1. mL volume 50. ng/g of thiocyanate standard produced a signal-to-noise ratio of 250 for the analytical peak. This method is therefore suitable for ultra-trace determination of thiocyanate (low part-per-billion range). For the application described the full detection potential of the method was not required and the sample preparation presented has been designed for quantitation of saliva samples containing 1-400. ?g/g of thiocyanate with a combined standard uncertainty of 2% relative for saliva samples containing 25. ?g/g of thiocyanate. This method was applied for the determination of thiocyanate in human saliva samples.

This report describes all the activities carried out under le project LIFE+ "Low cost sorbent for reducing mercury emissions- SOREME" (LIFE11 ENV/IT/000109) over the period between 01/01/2013 and 31/12/2014. The present project aims at demonstrating the use of a innovative sorbent obtained from a pyrolytic process of wasted tyres, demonstrating its capacity of adsorbing mercury. Briefly, the aims of SOREME project are: i) to optimise and demonstrate the functioning of the pyrolytic plant (action B1) and of the innovative sorbent continuous production (action B2); ii) to carry out many different tests of mercury absorption at laboratory level (action B3), semi-industrial level (action B4), and at industrial level (action B5).

The structure of the silk from cocoons of two common spiders, Araneus diadematus (family Araneidae) and Achaearanea tepidariorum (family Theridiidae) was investigated by means of (13)C solid state NMR and FT-IR spectroscopies. The combined use of these two techniques allowed us to highlight differences in the two samples. The cocoon silk of Achaearanea tepidariorum is essentially constituted by helical and beta-sheet structures, whereas that of Araneus diadematus shows a more complex structure, containing also beta-strands and beta-turns. Moreover, the former silk is essentially crystalline while the latter contains more mobile domains. The structural differences of the two cocoon silks are ascribed to the different habitat of the two species.

A calcium carbonate (calcite) bladder calculus was found in the pelvis of an adult female buried in a Mesolithic cave-tomb on the coast of Sicily; it was dated by 1 4 C to around 6500 BC. Its chemical composition, which was not known before, was determined by means of Fourier transform-infared microspectroscopy (FT-IR-M) using the high-pressure diamond cell, a device that makes it possible to analyse a small amount of the sample (1-2 μg). In this way, the analysis of all the laminations of the calculus was performed without destroying the sample. Although calcite was the main component, carbonate apatite was also detected in the nucleus and in a more external layer.

Background Current research has indicated that small natural compounds could interfere with β-amyloid fibril growth and have the ability to disassemble preformed folded structures. Ferulic acid (FA), which possesses both hydrophilic and hydrophobic moieties and binds to peptides/proteins, is a potential candidate against amyloidogenesis. The molecular mechanisms connected to this action have not been elucidated in detail yet. Methods Here the effects of FA on preformed fibrils are investigated by means of a concerted experimental–computational approach. Spectroscopic techniques, such as FTIR, fluorescence, size exclusion chromatography and confocal microscopy in combination with molecular dynamics simulations are used to identify those features which play a key role in the destabilization of the aggregates. Results Experimental findings highlight that FA has disruptive effects on the fibrils. The computational analysis suggests that dissociation of peptides from the amyloid superstructures could take place along the fibril axis and be primarily determined by the cooperative rupture of the backbone hydrogen bonds and of the Asp-Lys salt bridges. Conclusion FA clusters could induce a sort of stabilization and tightening of the fibril structure in the short term and its disruption in the long term, inhibiting further fibril re-assembly through FA screening effects. General significance The combination of experimental and computational techniques could be successfully used to identify the disrupting action of FA on preformed Aβ fibrils in water solution.

Hydrophobic interaction chromatography coupled online with chemical vapour atomic fluorescence spectrometry (HIC–CVGAFS) has been optimized for the analysis of thiolic proteins in denaturing conditions. Proteins are pre-column simultaneously denatured and derivatized in phosphate buffer solution containing 8.0 mol dm−3 urea and p-hydroxymercurybenzoate (PHMB) and the derivatized denatured proteins are separated on a silica HIC Eichrom Propyl column in the presence of 8.0 M urea in the mobile phase. Post-column online reaction of derivatized denatured proteins with bromine, generated in situ by KBr/KBrO3 in HCl medium, allowed the fast conversion of the uncomplexed PHMB and of the PHMB bound to proteins to inorganic mercury also in presence of urea. Hg2+, present in solution as Hg2+–urea complex, is selectively detected by AFS in a Ar/H2 miniaturized flame after sodium borohydride reduction to Hg. Under optimized conditions, online bromine treatment gives a 100±2% recovery of both free and protein-complexed PHMB. Denatured glyceraldehyde-3-phosphate dehydrogenase, aldolase, lactate dehydrogenase, trioso phosphate isomerase and β-lactoglobulin have been examined. As the sensitivity and limit of detection of proteins in the HIC–CVGAFS apparatus depends on number of SH groups reacting with PHMB, the denaturation process, which increases the number of PHMB-reactive thiolic groups in proteins, improves the analytical performances of the described system in protein analysis. The detection limit for the denatured proteins examined was found in the range of 10−10–10−12 mol dm−3, depending on the considered protein, with linear calibration curves spanning over four decades of concentration.

Multidimensional analysis of denatured milk proteins is reported using high-performance liquid chromatography (HPLC) combined with dynamic surface tension detection (DSTD). A hydrophobic interaction chromatography (HIC) column (a TSK-Gel Phenyl-5PW column, TosoBiosep), in the presence of 3.0 M guanidine hydrochloride (GdmHCl) as denaturing agent is employed as the mobile phase. Dynamic surface tension is measured through the differential pressure across the liquid-air interface of repeatedly growing and detaching drops. Continuous surface tension measurement throughout the entire drop growth (50 ms to 4 s) is achieved, for each eluting drop of 4 s length, providing insight into both the kinetic and thermodynamic behavior of molecular orientation processes at the liquid-air interface. An automated calibration procedure and data analysis method is applied with the DSTD system, which allows two unique solvents to be used, the HIC mobile phase for the sample and a second solvent (water for example) for the standard, permitting real-time dynamic surface tension data to be obtained. Three-dimensional data is obtained, with surface tension as a function of drop time first converted to surface pressure, which is plotted as a function of the chromatographic elution time axis. Experiments were initially performed using flow injection analysis (FIA) with the DSTD system for investigating commercial single standard milk proteins (alpha-lactalbumin, beta-lactoglobulin, alpha-, beta-, kappa-casein and a casein mixture) denatured by GdmHCl. These FIA-DSTD experiments allowed the separation and detection conditions to be optimized for the HIC-DSTD experiments. Thus, the HIC-DSTD system has been optimized and successfully applied to the selective analysis of surface-active casein fractions (alpha s1- and beta-casein) in a commercial casein mixture, raw milk samples (cow's, ewe's and goat's milk) and other diary products (yogurt, stracchino, mozzarella, parmesan cheese and chocolate cream). The different samples were readily distinguished based upon the selectivity provided by the HIC-DSTD method. The selectivity advantage of using DSTD relative to absorbance detection is also demonstrated.

A drop-based dynamic surface-tension detector (DSTD) has been used to study the dynamic surface tension behavior of proteins denatured in guanidine thiocyanate (GndSCN). The dynamic surface tension at the air-liquid interface is obtained by measuring the internal pressure of drops that grow and detach at a specified rate. In the method the sample of interest is injected and subsequently flows to the DSTD-sensing capillary tip. For this work, a novel DSTD calibration procedure utilizing two distinct mobile phases is applied. Here, the mobile phases are aqueous with different constituents, for example GndSCN and phosphate buffer, either added or omitted. The dual-mobile phase calibration procedure gives the analyst the capability of making protein measurements in a GndSCN-phosphate buffer mobile phase, while measuring a calibration standard in another mobile phase, such as water, in which the surface tension of the calibration standard is readily available. Results are presented with drop volumes of either 2 microL (i.e. 2-s drops) or 7 microL (i.e. 7-s drops) for proteins varying in molar mass from 12,000 to 330,000 g mol(-1). We demonstrate that the DSTD can be used to determine the molar mass of proteins denatured in GndSCN. The method applies a regime where the denatured protein is detected by surface-active properties, and selectivity with regard to molar mass is contained in the dynamic component of the DSTD signal. The dynamic surface pressure signals of the denatured proteins suggest that diffusion plays a large role in the kinetics of the surface activity. The limit of detection for the denatured proteins studied ranged from 3 mg L(-1) to 14 mg L(-1). The DSTD, coupled with the novel dual-mobile phase calibration procedure, can be used to investigate the fundamental properties of proteins. Insight into the behavior at the air-liquid interface for native and denatured proteins is achieved; this is a novel tool for studying protein denaturation, complementary to other common approaches such as spectroscopy and calorimetry. Furthermore, the reported method could be widely applied to the study of effects on the interfacial properties of proteins after a variety of chemical and physical modifications that are possible with the dual-mobile phase calibration procedure.

Separation and determination of denatured alpha-, beta- and kappa-caseins by hydrophobic interaction chromatography (HIC) was improved by using a TSK-Gel Ether-5PW column (Tosoh Biosep). The method, already proposed and performed by a TSK-Gel Phenyl-5PW column (Tosoh Biosep), is based on fast and easy solubilization of commercial and real samples by 4.0 M guanidine thiocyanate and HIC analysis in the presence of 8.0 M urea in the mobile phase. Employment of the less hydrophobic ether phase had the main advantage of separating casein fractions in less than 22 min and, additionally, of separating a-casein in kappaS1- and alphaS2 -casein fractions. The method has been validated by the analysis of reference skim milk powder (BCR-063R) certified for total nitrogen content. A linear relationship between the concentration of casein and peak area (UV absorbance detector at 280 nm) has been obtained over the concentration range of 0.5-40 microM. The detection limit for alpha-, beta- and kappa-caseins ranged between 0.33 and 0.65 microM. The precision of the method was evaluated, the RSDs for alphaS1-, alphaS2-, beta- and kappa-casein determination ranging between 2.3 and 5.5% for standard solutions and between 4.4 and 6.2% for real sample solutions. The mean value of casein content found in eight aliquots of BCR-063R calculated with respect to the total protein content (estimated on the basis of certified total nitrogen content) was 78.3 +/- 6.1%. Results of linear fitting of standard additions data of alphaS1-, alphaS2-, beta- and kappa-caseins to BCR-063R were compared with linear fitting of alphaS1-, alphaS2-, beta- and kappa-casein calibration data. The method was applied to commercial caseins and to 30 real, raw samples. A statistical comparison was performed between results on quantitation of alpha-, beta- and kappa-caseins obtained by TSK-Gel Ether-5PW and TSK-Gel Phenyl-5PW HIC columns, showing more accurate results for chromatographic analysis performed by the ether column.

The absolute and relative quantitation of proteins plays a fundamental role in modern proteomics, as it is the key to understand still unresolved biological questions in medical and pharmaceutical applications. Highly sensitive analytical methods are required to quantify proteins in biological samples and to correlate their concentration levels with several diseases. Enzyme-linked immunosorbent assay (ELISA) and Western blot represent specific strategies for protein quantitation. However, these approaches are impractical for quantitative studies: the availability of high quality ELISAs for biomarker candidates is limited, and the performance characteristics of many commercially marketed ELISAs are poorly documented or unknown. The development of ELISA or Western blot is also expensive and time-consuming. The limitations of these strategies, combined with the large numbers of biomarker candidates emerging from genomic and proteomic discovery studies, have created the need for alternative strategies for quantitation of targeted proteins. A widely explored approach to identify and quantify intact proteins is based on (i) the detection of endogenous metals covalently bound to the protein structure or (ii) the labelling of proteins with metallic probes. The development of several hyphenated analytical techniques for metal quantitation has led to new possibilities for the quantitative analysis of proteins. In the present review, we attempt to provide a full coverage of current methodologies for protein quantitation based on the detection of endogenous metal(loid)s or chemical labeling with metal(loid)s, highlighting, to the best of our knowledge, their merits and limits.

This is the final report on the activities carried out under le project LIFE+ "Environmentally friendly natural products instead of cloroparaffines in the fatting phase of the tanning cycle - ECOFATTING" (LIFE10 ENV/IT/000364) over the period between 01/01/2012 and 31/12/2013. The present project aims at demonstrating the use of an innovative technology for the fatting phase of the leather tanning process, with the goal of producing new or existing products with a significantly higher eco-sustainability profile. The specific technical goal of the project consists in the demonstration of use of a new category of products of natural origin, capable of substituting chlorosulfonated currently used in the fatting phases during the leather tanning cycle.

We optimized a hyphenated system based on size exclusion chromatography coupled to a microwave/UV mercury oxidation system and an atomic fluorescence detector (SEC-CVG-AFS) for the online oxidation of free and protein-complexed p-hydroxymercuribenzoic acid (pHMB) without the employment of chemical oxidizing agents. This system has been applied to the study of labeling of thiolic groups of native ovalbumin (OVA) as a function of protein concentration. We found that the protein concentration strongly affects the species distribution of OVA, the number of thiolic groups titrated in each species, and thus, the accuracy in the determination of the total number of thiolic groups. The amount of titrated sulfhydryl groups in the protein concentration range investigated (5-100 mu mol L-1) varied from 2.40 +/- 0.01 to 1.85 +/- 0.05 for the monomeric form of OVA and from 4.63 +/- 0.01 to 5.63 +/- 0.05 for the total OVA, which represents more than four theoretical number of reduced Cys. This information is important from the analytical point of view because it suggests that, unless to operate with diluted concentration of protein, the number of titrated thiolic groups results from both the aspecific interaction of the probe with aggregates species and to the specific bond of the probe with the accessible -SH groups.

We developed a novel and straightforward derivatization method for the determination of lactate by reversed phase high-performance liquid chromatography (RP-HPLC) with fluorescence and UV detection in biological matrices as urine and saliva. The derivatization of lactate was achieved employing 9-chloromethyl anthracene (9-CMA) as fluorescence reagent, which has never been previously used to obtain a lactate derivative. Lactate reacts with 9-CMA with high selectivity in a very short time, without requiring extraction procedures from the aqueous solution, and the reaction reaches 70% completion in 30 min. The ester derivative obtained can be easily determined by RP-HPLC with fluorescence detection at 410 nm (lambda(ex)=365 nm) and UV detection at 365 nm. The method was also optimized in order to allow for the simultaneous determination of lactate and creatinine for the application to urine samples. The lactate calibration curve was linear in the investigated range 2 x 10(-4)-3 x 10(-2) mM and the limit of detection, calculated as three times the standard deviation of the blank divided by the calibration curve slope, was 50 nM for both fluorescence and UV detection. Intra-day and inter-day repeatability were lower than 5% and 6%, respectively. The method proposed was successfully applied to the analysis of urine and saliva samples.

This report represents the activities carried out under le project LIFE+ "Low cost sorbent for reducing mercury emissions- SOREME" (LIFE11 ENV/IT/000109) over the period between 01/01/2013 and 31/05/2014. The present project aims at demonstrating the use of a innovative sorbent obtained from a pyrolytic process of wasted tyres, demonstrating its capacity of adsorbing mercury. Briefly, the aims of SOREME project are: i) to optimise and demonstrate the functioning of the pyrolytic plant (action B1) and of the innovative sorbent continuous production (action B2); ii) to carry out many different tests of mercury absorption at laboratory level (action B3), semi-industrial level (action B4), and at industrial level (action B5).

Zeolite Beta, modified with some salts of alkali and alkaline earth metals (K, Zn, Sn), was tested in the hydrothermal heterogeneous catalytic decomposition of microcrystalline cellulose. The reactions were microwave-assisted, where the microwaves were issued by an in situ coaxial applicator. Zeolites were subjected to an ion-exchange process which determines the loss of crystallinity in the following order: Sn-Beta-IE > K-Beta > Zn-Beta > acid form H-Beta. The interaction between zeolites and microwaves was studied by irradiating zeolite powder under constant power and the heating response was in the following order: K-Beta > NH4-Beta > Sn-Beta-IE ≈ Zn-Beta > H-Beta > alumina. These results show that the nature of the counterion strongly affects the absorption of microwaves. The catalytic activity of the different systems on the cellulose decomposition was studied, and resulted in the following order: H-Beta > K-Beta > Zn-Beta > Sn-Beta-IE > alumina, when the reaction medium contained 5 mM HCl. The most active catalyst was the acid zeolite Beta and the identified product distribution under the investigated conditions was (mol yield %): levulinic acid (22.3), glucose (12.1), lactic acid (4.1), formic acid (6.6), 5-(hydroxymethyl) furfural (14.6), acetic acid (15.2) and furfuraldehyde (3.1). The effect of temperature, time and the heterogeneous catalyst reuse (H-Beta) on the yields of different products was investigated. The use of MW radiation with a coaxial applicator instead of conventional heating gave clear advantages in the decrease of the reaction time (45 min) and in terms of yield enhancement (78.6% under the best conditions).

We propose ATR-FTIR spectroscopy for the characterization of the spectral changes in alkyd resin from the Griffin Alkyd Fast Drying Oil Colour range (Winsor & Newton), occurring over 550 days (similar to 18 months) of natural ageing and over six months of artificial ageing under an acetic acid atmosphere. Acetic acid is one of the atmospheric pollutants found inside museums in concentrations that can have a significant effect on the works exhibited. During natural ageing we observed an increase and broadening of the OH group band around 3300 cm(-1) and an increase in bands in the region 1730-1680 cm(-1) due to carbonyl stretching. We found a broad band around 1635 cm(-1) likely due to C=O stretching vibrations of beta dichetons. These spectral changes are the result of autooxidation reactions during natural ageing and cross-linking, which then form f alcohols and carbonyl species. The increase in absorbance at 1635 cm(-1) was selected as a parameter to monitor the ageing process of paintings prepared with FDOC, without the need for any extractive procedure. FTIR spectra of paint replicas kept under an acetic acid atmosphere indicated the chemical groups involved in the reaction with acid, thus suggesting which spectral FTIR regions could be investigated in order to follow any degradation in real paintings. A red paint sample from a hyper-realistic artwork ("Racconta stone", 2003) by the Italian painter Patrizia Zara was investigated by FTIR in order to evaluate the effects of 10 years natural ageing on alkyd colours. The results obtained suggested that after the end of chemical drying (autooxidation), alkyd colours are very stable

The effects of tetrahydroborate and acid concentration and the presence Of L-cysteine and thiourea were investigated in the determination of As, Bi and Sn using continuous flow hydride generation atomic fluorescence spectrometry (HG AFS). The aim was to find conditions allowing the control of those effects exerting negative influence on the analytical performance of the HG AFS apparatus. The effects taken into account were: (i) the radiation scattering generated by carryover of solution from the gas-liquid separator to the atomizer; GO the introduction of molecular species generated by tetrahydroborate decomposition into the atomizer; and (iii) interference effects arising from other elements in the sample matrix and from different acids. The effects (i) and (ii) could be controlled using mild reaction conditions in the HG stage. The effect of FIG conditions on carryover was studied by radiation scattering experiments without hydride atomization. Compromised HG conditions were found by studying the effects of tetrahydroborate (0.1-20 g l(-1)) and acid (0.01-7 mol l(-1)) concentration, and the addition of L-cysteine (10 g l(-1)) and thiourea (0.1, mol l(-1)) on the HG AFS signals. The effect of optical filters was investigated with the aim of improving the signal-to-noise ratio. Optical filters with peak wavelengths of 190 and 220 nm provided an improvement of detection limits by factors of approximately 4 and 2 for As and Te, respectively. Under optimized conditions the detection limits were 6, 5, 3, 2, 2 and 9 ng l(-1) for As, Sb, Bi, Sn, Se and Te, respectively. Good tolerance to various acid compositions and sample matrices was obtained by using L-cysteine or thiourea as masking agents. Determination of arsenic in sediment and copper certified reference materials, and of bismuth in steel, sediment, soil and ore certified reference material is reported. (9) 2001 Elsevier Science B.V. All rights reserved.

A new analytical method is proposed for the determination and characterization of thiolic proteins, based on hydrophobic interaction chromatography (HIC) coupled on-line with cold vapour atomic fluorescence spectrometry (CVAFS). Thiolic groups are derivatized pre-column by p-hydroxymercurybenzoate (PHMB) and the derivatized proteins are separated on a TSKgel Ether-5PW column. Post-column on-line reaction of derivatized proteins with bromine, generated in situ by KBr/KBrO3 in HCl medium, allowed the fast conversion of protein-bound PHMB to inorganic mercury, Hg(II), which is selectively detected by AFS after sodium borohydride reduction to Hg0. Under optimized conditions, on-line bromine treatment gives a 85 ± 2% recovery of both free and protein-complexed PHMB in less than 2.5 s and at room temperature. Glyceraldehyde-3-phosphate dehydrogenase, aldolase, pyruvate kinase, trioso phosphate isomerase and phospho-glucose isomerase have been examined. Sensitivity and limit of detection of proteins depends on the number of –SH groups reacting with PHMB and are in the range of 10−8–10−9 mol dm−3 with calibration curves spanning over four decades of concentration.

A method recently described for the separation of denatured alpha-, beta- and kappa -caseins by hydrophobic interaction chromatography was validated by the analysis of reference skim milk powder (BCR-063R) certificated for total nitrogen content. The method is based on fast and easy solubilization of commercial and real samples by 4.0 M guanidine thiocyanate and elution on a TSK-Gel (R) Phenyl-5PW column (TosoHaas) in the presence of 8.0 M urea in the mobile phase. No preliminary precipitation or separation of the casein fraction is required. A linear relationship between the concentration of casein and peak area (UV absorbance detector at 280 nm) was obtained over the concentration range 0.5-60 muM. The detection limits for alpha-, beta- and kappa -caseins ranged between 0.30 and 0.65 muM. The precision of the method was evaluated; the relative standard deviation for alpha-, beta- and kappa -casein determination ranged between 2.2 and 2.7% for standard solutions and between 3.5 and 6.2% for real sample solutions. The mean casein content found in 10 aliquots of BCR-063R calculated with respect to the total protein content (estimated on the basis of certified total nitrogen content) was 79.1 +/- 2.7%. Results of linear fitting of standard additions data for alpha-, beta- and kappa -caseins to BCR-063R were compared with linear fitting of alpha-, beta- and kappa -casein calibration data. The method was applied to commercial caseins and to 31 real, raw samples [processed cow's milk (pasteurised, UHT-treated), follow-up milk powders, cream, cheeses, casein-free infant formulae, cookies for babies containing milk proteins] with the aim of showing the wide applicability of the method in order to determine alpha-, beta- and kappa -caseins.

Using circular dichroism, fluorescence, and infrared spectroscopies, we studied the secondary structure of purified hamster PrP C in the presence of the mild, nonionic detergent octylglucoside. Under these native conditions, PrP C displayed an unexpectedly high β-sheet component, intermediate between the values previously reported for PrP Sc and an isoform of PrP C isolated in a zwitterionic detergent. The structure of PrP C appeared to depend strongly on the detergent and/or phase. Switching from octylglucoside to zwitterion 3–14 drastically modified PrP secondary structure by increasing the α-helix while abolishing the β-sheet component. In contrast, the conformation of PrP C in zwitterion was highly stable, since reverting to octylglucoside did not restore the original native structure. These and other results show that native PrP C in octylglucoside has some of the conformational characteristics that make the protein susceptible to conversion into PrP Sc . Most importantly, this is the first study to demonstrate the intrinsic plasticity of the full-length native PrP C isolated from animal brains.

The equilibrium adsorption of three nucleic acids: chromosomal DNA, supercoiled plasmid DNA, and 25S rRNA, on the clay minerals, montmorillonite (M) and kaolinite (K), were studied. Adsorption of the nucleic acid on the clays was rapid and maximal after 90 min of contact time. Chromosomal DNA was adsorbed to a greater extent than plasmid DNA and RNA, and the adsorption was also greater on M than on K. Adsorption isotherms were of the L type, and a plateau was reached with all the complexes, with the exception of chromosomal DNA adsorbed on M. To determine where nucleic acids are adsorbed on clay minerals and the nature of the interaction, complexes were studied by X-ray diffraction (X-RD), electron microscopy, and Fourier transform infrared (FT-IR) spectroscopy. X-RD showed that nucleic acids did not penetrate the clay, indicating that the adsorption occurred primarily on the external surfaces of clay particles, as also suggested by electron microscopy observations. FT-IR spectra of clay-tightly bound nucleic acid complexes showed absorption bands that indicate a variation of the nucleic acids status as a consequence of their adsorption on clay. Data obtained suggested that the formation of clay-nucleic acid complex could have an important role in the preservation of genetic material in primeval habitats.

Aldolase, glyceraldehyde 3-phosphate dehydrogenase and ovalbumin were determined by hydrophobic interaction chromatography, which allows the evaluation of the number of –SH groups per molecule of protein in both the native and denatured form. These proteins were chosen as models to show the generality of use of the procedure for analytical and biochemical applications. The experiments were performed by using 203Hg-labelled p-hydroxymercuribenzoate as reagent and known concentrations of proteins. The results were compared with FI-CV-ETAAS measurements and literature data.

Previous Fourier transform infrared (FT-IR) spectroscopic studies on neoplastic and normal cells have shown different band profiles and intensity associated with absorptions of proteins and nucleic acids. In the present study, an interpretation of such differences has been attempted by comparing the spectra of DNA/RNA/protein mixtures with the spectra, particularly, obtained for lymphocytes from B-chronic lymphatic leukemia (B-CLL) patients and normal donors. FT-IR microspectroscopy analysis showed a good agreement between the intensity and the band profile of the spectra of leukemic lymphocytes and those of the binary mixture made up of 75% human serum albumin and 25% DNA. The addition of small amounts of RNA (1–5%) modified the band shape, making it more similar to the spectrum of normal lymphocytes. An attempt was also made to estimate the relative amounts of DNA and RNA. The results demonstrated an increase in the DNA/RNA ratio value in neoplastic lymphocytes with respect to that reported in literature for normal ones.