Characterization of dendrimer properties by capillary electrophoresis and their use as pseudostationary phases

The general properties of dendrimers and in particular their electrolytic characteristics that are relevant in electrokinetic separations, are described. In order to confirm theoretical considerations on commercial dendrimer charge and hydrodynamic radius, several capillary zone electrophoresis (CZE) experiments were performed. Electrophoretic mobilities measured at different pH values indicated a sensible increase of dendrimer hydrodynamic radius at pH values lower than 2.5. This was probably due to the Coulombic repulsion of charged amine groups of the inner dendrimer shells. The principal reasons that should address the use of dendrimers as pseudostationary phases in micellar electrokinetic chromatography (MEKC) are discussed. Moreover, a survey of different separations performed utilizing dendrimers in MEKC as well as of several future plausible uses of various classes of dendrimers is presented.     

Affinity capillary electrophoresis study of the linkage existing between proton and zinc ion binding to bacitracin A1

Measurements by capillary electrophoresis (CE) of bacitracin A(1) effective mobility at different pH values permitted to estimate the five acidic dissociation constants and the Stokes radii at different protonation stages of the macrocyclic dodecapeptide. The pK(a) values were 3.6 and 4.4 for the two carboxylic groups of the lateral chains of D-Asp-11 and D-Glu-4, respectively, 6.4 for the aza-atom of the imidazole ring of His-10, 7.6 for the amino group of N-terminal Ile-1 and 9.7 for the delta-amino group of D-Orn-7, very close to the values obtained by other researchers by titration experiments. In agreement with a rigid macrocyclic structure the Stokes radii of different protonated forms ranged only between 14.3 and 14.8 A. Best fitting procedures performed on experimental mobility measured at two different pH values (5.50 and 6.72) in the presence of increasing Zn(+2) concentration allowed confirming the model that assumes the binding of Zn(+2) to P(0) peptide form with a 1.5 x 10(3) M(-1) intrinsic association constant. Following to Zn(+2) binding, the pK(a) of the amino group of N-terminal Ile-1 is shifted from 7.6 to 5.9 and the Stokes radius is reduced of about 3 A. The mean charge of the bacitracin A(1)-Zn(+2) complex resulted +1.67 and +1.12 at pH 5.50 and 6.72, respectively. These results suggest that the amino group of N-terminal Ile-1 is not essential for Zn(+2) binding.     

Dynamic processes in the Venice region outlined by environmental isotopes

Research carried out in the last 40 years has shown the scientific importance of groundwater circulation both in the Northern Adriatic sea bed and within the uppermost sedimentary layers of the Venice lagoon and of the Venice plain. Hydrodynamic processes are strictly controlled by a well-cemented sedimentary horizon lying under and around Venice ('caranto'), which plays the role of regional aquitard. This layer was attributed to the subaerial cementation of the Flandrian (8-10 ka Before Present) sedimentary surface. The caranto is generalised as a continuum horizon, being an easy explanation for several environmental, hydrogeological and geotechnical problems, e.g., a base layer for landfills, a confining layer for deep aquifers and the best substratum for locating the oak wooden pile-dwelling needed to support the largest buildings. The preservation of the isotope signal within the deep aquifers and aquiclude system records the changes in surface and groundwater characteristics and suggests the present and past recharge regimes. In this region, the heavily perturbed hydrodynamic conditions do not allow for the use of isotopic signals to derive a correct reconstruction of the present recharge. The perturbations induced by the intensive anthropogenic activity force to follow climate evolution by considering deep groundwater and pore waters. In addition, the presence of carbonatic rocks inside terrigeneous sediments affects the reconstruction of the past. Results indicate that carbonatic rocks are created by seepage, through the sediments, of gaseous carbon compounds from decaying organic layers. The gas interactions with the intra-sedimentary saline and fresh waters produce CO2, inducing the cementation of the sediments.     

Identification of RFLP G6PD mutations by using microcapillary electrophoretic chips (Experion)

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most well-known human genetic defects, identified in more than 400 million individuals in the world. To date, no commercial kits are available for the mutation screening of this disease. Seventy G6PD-deficient Italian individuals admitted to the Laboratory of Clinical Molecular Biology of Hospital "Agostino Gemelli" of Rome were screened for the most frequent Italian mutations, by means of allele-specific PCR, followed by restriction fragment length electrophoresis. The present study compares two techniques for the identification of restriction patterns: agarose gel electrophoresis versus Experion system. When the first screening was negative, the entire G6PD gene was sequenced using the ABI 3100 Avant Instrumentation. The G6PD variants identified and their frequencies were the following: G6PD Mediterranean (75.7%), G6PD Seattle (7.1%), G6PD A(-) 202 + 376 (7.1%), and G6PD Cassano (2.8%). In addition, we identified by direct sequencing two new mutations, namely Buenos Aires and Rignano. With the Experion method, the size band determination was more accurate than that obtained by gel electrophoresis. The Experion system resulted as a valid, easy, and reproducible diagnostic method for the screening of G6PD mutation as compared with the agarose electrophoretic analysis.     

Different binding thermodynamics of Ni2+, Cu2+, and Zn2+ to bacitracin A1 determined by capillary electrophoresis

Thermodynamics of the binding of Ni(2+), Cu(2+) and Zn(2+) to bacitracin A(1) was studied by capillary electrophoresis measuring the peptide effective mobility at different pH in the presence of increasing concentration of the three ligands. The affinity follows the order Ni(2+) > Cu(2+) > Zn(2+), with association constant values of (2.3 +/- 0.1)x10(4), (4.9 +/- 0.2)x10(3), and (1.5 +/- 0.1)x10(3) M(-1), respectively. The only model able to rationalize mobility data implies that metal ion binds to the P(0) peptide form. Moreover, mobility values indicated a change of bacitracin A(1) acidic properties on Ni(2+) and Cu(2+) binding, with a shift of the pK(a) of N-terminal Ile-1 from 7.6 to about 5 and of the pK(a) of the delta-amino group of D-Orn-7 from 9.7 to about 7. Even though on Zn(2+) binding a shift of the N-terminal Ile-1 pK(a) was observed, restrictions in the pH range suitable for investigation, due to precipitation phenomena, did not allow establish if the shift of D-Orn-7 lateral chain pK(a) also occurred. Nonetheless, if present, the shift should be limited to the 7.8-9.7 range. Mobility data indicated that the Stokes radius of the complexes is ca. 3 A lower than that of the free peptide. The present results indicate that metal-ion binding to bacitracin A(1) is more complex than previously assumed.     

A simplified method for the determination of total homocysteine in plasma by electrospray tandem mass spectrometry

Hyperhomocysteinemia is a risk factor for different diseases. Several methods have been developed to analyze homocysteine and the immunometric ones, although expensive, they are in widespread use. A rapid LC‐MS/MS method for homocysteine assay has been developed for the application of large clinical chemistry routines. Selected reaction monitoring was performed through the transitions m/z 136.0→90.1 for homocysteine and m/z 140.0→94.0 for the internal standard. ESI was used to generate [H+] adduct ions. Chromatographic isocratic separation was achieved using a strong cation exchange column. The mobile phase was methanol/water (20:80 v/v, containing 0.1% formic acid and 1.5 mmol/L ammonium formate in the water phase) at a flow rate of 0.250 mL/min (35°C). Samples treatment consisted in the reduction with DTT and deproteinization with methanol. Recovery, linearity, LOD, LOQ and total imprecision were evaluated to validate the method. Homocysteine values on 100 serum samples were compared with those obtained by HPLC and immunometric methods. The method is robust, selective and precise in the whole range of values studied. Moreover, low reagent cost and easiness of sample treatment make this method useful, not only for research, but also for routine work.     

Association of Molybdenum Ionic Species with Alumina Surface

The adsorption isotherms at 25, 45, and 65 degrees C of molybdenum solutions of concentration ranges between 10(-3) and 3x10(-2) M(Mo) (pH 4-5) on different alumina samples are investigated. The analysis is conducted using a modified Frumkin isotherm which takes a more realistic account of the lateral interaction between adsorbed species and considers that the adsorption takes place on the most basic OH groups on the surface of alumina. The results are discussed in view of the difference in solutions speciation, and the changes in the pH of the remaining supernatant solutions. The solution temperature, PZC of the used aluminas, the configuration of the basic OH groups on their surface, and the pore structure have been shown to intervene effectively. Copyright 2000 Academic Press.     

Effect of a contaminated site (the San Giuliano landfill, Venice, Italy) on the interaction between water bodies in a coastal aquifer system

A complex multiple aquifer system affected by a contaminated site and exposed to tidal effects was investigated to develop the hydrogeological conceptual model of the study case. Two water bodies were identified, from top to down: a surface aquifer unaffected by tidal events, and a semiconfined aquifer partly communicating with the brackish lagoon waters. By using the Cl(-)/SO4(2-) ratio and the DOC (Dissolved Organic Carbon) concentration, as well as by applying the kriking to the piezometric levels results, the groundwater flow directions and the hydraulic gradients within the investigated aquifers were also identified. Based on the overall results, a schematic of the conceptual model for the whole aquifer system was provided which allows to visualize the communication between the different water bodies.     

Capillary electrophoretic study of the binding of zinc(II) ion to bacitracin A1 in water-2,2,2-trifluoroethanol

Binding of Zn(2+) to bacitracin A(1) was studied by capillary electrophoresis in water/2,2,2-trifluoroethanol (70/30 v/v) at different apparent pH values in order to estimate the association constant of metal, the acidic dissociation constants and the Stokes radii of both free and bounded peptide in apolar environment. The Stokes radii of the free peptide species were compared with those in aqueous solution, as obtained in a recent study performed by our group, indicating that apolar environment stabilizes bacitracin A(1) in a conformational structure with the lateral chain of apolar amino acids exposed on the external surface. This conformation of the macrocyclic dodecapeptide is ready to interact with Zn(2+) ion, as pointed out by the strong increase of the association constant measured in water/2,2,2-trifluoroethanol with respect to the value obtained in aqueous solution. In addition, whereas Zn(2+) ion binding in aqueous solution provides a sensible reduction of peptide Stokes radius, no sensible variations following to ion binding were observed in hydro-organic solution. The present results suggest that the apolar environment, rather than the metal ion binding, could be responsible for the conformational transition that brings bacitracin A(1) towards its biologically active structure.*