Error estimates in the determination of rigid body displacements in protein crystals

We report the estimation of random errors in the refinement of the rigid body displacements of the -helices of the enzyme 6-phosphogluconate dehydrogenase. Least-squares refinement of the TLS parameters of the helices has been carried out using X-ray reflection data of 2.1 Å resolution, resulting in anR-factor of 19.5%. Standard deviations were estimated from the normal matrix. The results show that the translational mean-square displacements of nearly all the helices are significant at this resolution. However the libration parameters are only significant when the helices have at least four turns. Screw-rotation tensor values cannot be determined at this resolution.     

Tyrosinase biosensor response as a function of physical properties of organic solvents

The main aim was to investigate the possibility of developing a fast, easily produced biosensor capable of being used in non-aqueous solvents such as n-hexane, chloroform, mixtures thereof and water-saturated chloroform. The research also provided an experimental confirmation of several concepts, described in the literature, concerning enzymatic activity in different types of non-aqueous solvents. The results are decidedly encouraging as regards future possible uses of this sensor to determine soluble substances in non-aqueous solvents.     

Studies about the adsorption on lichen Evernia prunastri by enthalpimetric measurements

Lichens are bioaccumulators of divalent metal ions and the interaction between the lichens and five heavy metals was studied. In order to have a better understanding of the metal-uptake process by the lichens, data from atomic absorption spectroscopy were used to calculate the apparent coordination constants of the lichen–metal ion interactions by means of the Langmuir elaboration and microcalorimetric measurements to obtain enthalpimetric information. The results showed a correlation between the Langmuir constants and enthalpimetric measurements.     

Further development of catalase, tyrosinase and glucose oxidase based organic phase enzyme electrode response as a function of organic solvent properties

Using three enzyme sensors (tyrosinase, catalase and glucose oxidase), capable of functioning also in non-aqueous solvents, we found new correlations between classical indicators, e.g. the log P value of several organic solvents and new empirical indicators such as ;maximum current variation' (MCV) and above all the ;current variation rate' (CVR), the values of which may be monitored with the biosensor considered dipping directly into the organic solvent. The trend of the immobilised specific activity of the tyrosinase enzyme dipping into different organic solvents was evaluated and compared with that determined by the spectrophotometric method. Lastly, an investigation was performed to experimentally verify the relation between hydrophobicity of the solvent and its ability to draw back the water from the enzyme microenvironment using the Karl Fischer method and thermogravimetric analysis to estimate the residual water in the enzyme microenvironment after having treated the enzyme with the organic solvent, then allowing it to dry.     

Further applications of thermoanalytical methods to the detection of potassium and sodium salts of penicillins and cephalosporins

The potassium and sodium salts of some penicillins and cephalosporins were analyzed by thermoanalytical techniques (TG, DTG and DSC); in particular, those antibiotics whose thermal decomposition in oxygen occurs too quickly. The analyses were carried out in atmospheres of static air and oxygen, with and without the addition of ammonium sulphate or ammonium persulphate. It is possible to evaluate the purity of these antibiotics from the resultant sodium or potassium sulphate residue. Better results in terms of accuracy and precision were obtained by the addition of ammonium sulphate or persulphate to the analyzed compounds.     

Electron impact mass spectrometry of some macrocyclic polyether lactones

Electron impact mass spectrometry has been used together with linked scans, exact mass measurements and mass-analysed ion kinetic energy spectrometry to study nine inacrocyclic polyether lactones in detail. The presence of abundant hydrogenated oxirane ions has been observed for all the compounds examined, and confirmed by kinetic energy release measurements.     

Glutamate receptor incorporated in a mixed hybrid bilayer lipid membrane array, as a sensing element of a biosensor working under flowing conditions

The realization of a reliable receptor biosensor requires stable, long-lasting, reconstituted biomembranes able to supply a suitable biomimetic environment where the receptor can properly work after incorporation. To this end, we developed a new method for preparing stable biological membranes that couple the biomimetic properties of BLMs (bilayer lipid membranes) with the high stability of HBMs (hybrid bilayer membranes); this gives rise to an innovative assembly, named MHBLM (mixed hybrid bilayer lipid membrane). The present work deals with the characterization of biosensors achieved by embedding an ionotropic glutamate receptor (GluR) on MHBLM. Thanks to signal (transmembrane current) amplification, which is typical of natural receptors, the biosensor here produced detects glutamate at a level of nmol L(-1). The transmembrane current changes linearly vs glutamate up to 100 nmol L(-1), while the limit of detection is 1 nmol L(-1). In addition, the biosensor response can be modulated both by receptor agonists (glycine) and antagonists (Mg(2+)) as well, and by exploiting the biosensor response, the distribution of different kinds of ionotropic GluR present in the purified sample, and embedded in MHBLM, was also evaluated. Finally, one of the most important aspects of this investigation is represented by the high stability of the biomimetic system, which allows the use of biosensor under flowing conditions, where the solutions flow on both biomembrane faces.     

Behaviour of different eluents and stabilizing agents in the determination of sulphite in water by ion-chromatography

Ion-chromatography has been used for the determination of sulphite in water. The eluents were solutions of Na(2)CO(3) (1.1mM)-NaHCO(3) (1.4mM) or NaHCO(3) (1.0mM)-formaldehyde (0.2% w/w), and formaldehyde, glycerol or fructose was used as stabilizing agent. With the first eluent, fructose or glycerol can be used to stabilize samples against sulphite oxidation, but formaldehyde affects the peak height. On the other hand, formaldehyde can stabilize sulphite in the presence of Fe(III), whereas glycerol and fructose can not. If Fe(III) is present, the second eluent is used and sulphite is eluted directly as hydroxymethanesulphonate; formaldehyde will not then affect the peak height. This eluent allows a good peak separation and is suitable for the sulphite concentration range 0.1-12.0 mg/l.     

Eptastigmine,nicotinamide and nicotinic acid determination using an inhibition enzyme sensor; application to pharmaceutical analysis

An enzyme inhibition biosensor, developed in our laboratory and previously used for the analysis of compounds with anticholinesterase activity (e.g. physostigmine, neostigmine, pyridostigmine nicotine and organophosphorus compounds) has now been tested for the analysis of another recently synthesized cholinesterase inhibitor, i.e. eptastigmine. In addition nicotinic acid and nicotinamide, although displaying weaker inhibition properties, were also tested in pharmaceutical products using the same inhibition enzyme sensor. The biosensor consisted of a hydrogen peroxide amperometric electrode coupled to a functionalised nylon membrane chemically bonding both the enzymes butyrylcholinesterase and choline oxidase; a butyrylcholine standard solution in glycine buffer acted as substrate. The response of the system to all the inhibitors considered was characterised completely and the analysis of several pharmaceutical formulations containing nicotinamide or nicotinic acid was also performed.     

Simultaneous determination of cysteine, cystine and 18 other amino acids in various matrices by high-performance liquid chromatography

An extraction protocol and a reversed-phase high-performance liquid chromatographic method for the simultaneous determination of cysteine, cystine and 18 other amino acids in biological and vegetable samples are described. Among the different methods proposed for amino acid determination, phenylisothiocyanate was used as the reagent for derivatization. Chromatograms obtained in the analysis of standard solutions and actual samples are reported, together with regression equation, correlation coefficient (> 0.999 for all), limit of detection and recoveries (between 86 and 102% for all the examined matrices) for each amino acid. Practical protocol and method applications in normal patients and patients affected by different pathologies, and in algal products are discussed.