Loredana Cassiano

Human erythrocyte metabolism is modulated by the O2‐linked transition of hemoglobin

The metabolic behaviour of human erythrocytes has been investigated with particular regard to the effect of their oxygenation state. Experiments performed at high phosphate concentration (80 mM) within the pH range 7.0–7.8 on erythrocytes at high (HOS) and low (LOS) oxygen saturation showed that at any pH value: (1) glucose consumption was independent of the oxygenation state; (2) pentose phosphate pathway (PPP) flux was about 2 times higher in the HOS than in the LOS state. At low phosphate concentration (1.0 mM) the PPP flux doubled in HOS as well as in LOS erythrocytes, whereas the decrease in glucose consumption was more marked in the HOS state. Metabolism of LOS erythrocytes approached that of HOS erythrocytes under the following conditions: (1) erythrocytes having band 3 modified by 4,4′‐diisothiocyanatostilbene‐2,2′‐disulphonic acid; (2) CO‐saturated erythrocytes. These data support the hypothesis of a modulation of the relative rates of PPP and glycolysis achieved through competition between deoxy‐hemoglobin (deoxy‐Hb) and glycolytic enzymes for the cytoplasmic domain of band 3.

Peptide analysis by capillary (zone) electrophoresis

In this review various aspects concerning the application of capillary (zone) electrophoresis for peptide analysis will be critically examined. First, the basic instrumental requirements of CE apparatus and the strategies employed to enhance sensitivity in the analysis of underivatized sample are described. Multidimensional separative techniques of complex peptide mixtures that use CE as final step and the coupling of CE with mass spectrometry are subsequently discussed. A theoretical section describes the relationships existing between peptide mobility and the pH of the separation buffer. These relationships evidence that proton dissociation constants and Stokes radius at different protonation stages can be calculated by measuring the electrophoretic mobility at different pH values. Investigation of peptide mobility dependence on pH allows us to establish the optimum conditions, in terms of resolution, for peptide separation. Subsequently, a critical discussion about semiempirical models predicting peptide mobility as a function of chemico-physical peptide properties is presented. A section is devoted to the description of principles of peptide affinity capillary electrophoresis, underlining the similarity with peptide-proton interaction. CE separations performed in aquo-organic solvents are also critically discussed, showing the good performance obtained by using water-2,2,2-trifluoroethanol solutions. Finally, selected CE applications for the determination of peptide chemico-physical properties and conventional analysis, like peptide mapping, are reported.

Ion-exchange electrokinetic capillary chromatography with starbust (pamam) dendrimers: a route towards high-performance electrokinetic capillary chromatography

Abstract The use of different generations of Starburst (pamam) dendrimers provides a new opportunity for ion-exchange electrokinetic capillary chromatography. The uniform surface charge density and the structural homogeneity of these macroelectrolytes provide a decrease of plate height in comparison with micellar electrokinetic capillary chromatography with cationic surfactants. In addition, the use of different dendrimer generations allows the separation selectivity to be modulated.

Optimization of phenylthiohydantoinamino acid separation by micellar electrokinetic capillary chromatography

Optimization of phenylthiohydantion (PTH)-amino acid separation by micellar electrokinetic capillary chromatography was achieved by the use of a weighted variable-size simplex algorithm. The optimization procedure concerned the pH of the aqueous buffer, the sodium dodecyl sulphate concentration and the percentage of organic solvent; the organic solvent used was either methanol or acetonitrile. In both instances the optimization procedure led to very similar final experimental conditions and migration order and times of the PTH-amino acids, showing that the organic solvent probably provides a better polydispersity of the micellar phase. The elution pattern observed in the two instances suggests that ionic interactions and polar repartition play a role in the separation mechanism, but other types of interaction cannot be excluded.

Effect of 2,2,2-trifluoroethanol on capillary zone electrophoretic peptide separations

The use of 2,2,2-trifluoroethanol-water mixtures for peptide separations by capillary zone electrophoresis (CZE) displays some advantages over aqueous solutions. First, the increase in viscosity reduces and stabilizes the running current and facilitates heat dispersion, with a consequent improvement in the number of theoretical plates. Second, the decrease in the dielectric constant leads to a modification of the dissociation constants of the ionizable groups. The consequence is a change in selectivity that, for several favourable peptide pairs, provides an increase in resolution. Third, the interaction trifluoroethanol with the peptide modifies the Stokes radius in a manner strongly dependent on the peptide sequence. This can also be utilized for an increase in CZE performance. Fourth, the structural properties of 2,2,2-trifluoroethanol are particularly useful for an improvement in the separation of large apolar peptides. Finally, the use of trifluoroethanol strongly stabilizes the capillary coating.

Capillary zone electrophoresis of peptides: prediction of the electrophoretic mobility and resolution.

The determination of the pKa values of some selected peptides of similar size was performed by microtitration, which makes possible an accurate determination of the peptide charge as a function of the solution pH. Capillary zone electrophoresis separation of these peptides on modified capillaries at acidic pH showed that the electrophoretic mobility correlates with the peptide charge. This observation suggests that when an appropriate charge value is used, the basic electrophoretic equation is respected and, at least at a peptide charge value less than 1, the utilization of alternative semi-empirical predictions is not necessary. As a general rule, a peptide separation at acidic pH values is to be preferred to that at basic pH values. In fact, at basic pH a separation in the absence of both electroosmotic flow and of spurious interactions between the peptides and the inner wall of the capillary is difficult, owing to the instability of capillary modification. Further, from the differences in the peptide charge, a prediction of the best resolution as a function of the pH could be obtained; in fact, the resolution, for peptides of similar size and in the absence of electroosmotic flow, is connected to a simple equation, where the principal term depends on the effective charge of the peptides, which is a function of the pH of the solution and the pKa values of the peptides. The predictions of resolution at acidic pH agreed well with the experimental results; the spatial resolution measured in the separation of met- and leu-enkephalin was virtually coincident with the predicted resolution; in the case of a mixture of four model tetrapeptides of sequence GGNA, GGQA, GGDA and GGEA some anomalous results with respect to the predicted resolutions were observed. Nevertheless, an acceptable prediction can also be made in this case.

Modification of capillary electrophoresis selectivity in hydro-organic solutions - Dissociation constants and Stokes radius measurements of peptides in water-2,2,2-trifluoroethanol mixtures

Abstract Peptide electrophoretic mobilities were measured at different acidic pH in water–2,2,2-trifluoroethanol mixtures. Fit of experimental mobilities according to binding equations allowed the calculation of peptide C-terminus dissociation constants and the Stokes radius of the differently protonated forms. Analysis of the data showed that the use of hydro-organic solvents in capillary electrophoresis offers the following principal advantages: (a) a modification of dissociation constants and Stokes radii that are strongly dependent upon peptide sequence and that can be conveniently utilised for selectivity manipulation; (b) an increase of separation performance arising from the stabilisation of particular peptide conformations; (c) a greater solubility of large apolar peptides with respect to aqueous solutions.

Peptide mapping through the coupling of capillary electrophoresis and high-performance liquid chromatography: map prediction of the tryptic digest of myoglobin

The tryptic map of horse myoglobin was analysed through capillary electrophoresis using capillaries modified by a monolayer of acrylamide. The results were reproducible and the map was obtained in less than 30 min from ca. 8 pmol of tryptic digest. The peptide identification was performed using peptides previously identified by high-performance liquid chromatography. The peak areas measured using the two techniques are closely related, and the comparison of elution and migration times shows that the two techniques provide different maps. Furthermore, using the semiempirical relationship suggested by Grossman et al. [Anal. Biochem., 179 (1989) 28], which links the electrophoretic mobility to the charge of the peptide and its number of amino acids, a good agreement between predicted and experimental mobilities was observed.

Analysis of the globins from fast human haemoglobins by isoelectrofocusing on polyacrylamide gel rods

The globins from all fast haemoglobin (Hb) components obtainable by Bio-Rex 70 cation-exchange chromatography were examined by isoelectrofocusing on polyacrylamide gel rods with 8.0 mol/l urea. From this analysis HbA1a1 and HbA1a2 seem to be very heterogeneous components. HbA1b is separable into two components, one of which is varied in both the beta chains. Between HbA1b2 and the well-known HbA1c components two chromatographic peaks are separated, one with a noticeable percentage of glucosylated beta chain and one that probably contains HbF. HbA1c has both beta chains glucosylated, while HbA1x seems to be a beta monoglucosylated Hb form. Finally, the early part of the HbAo peak has a large amount of glucosylation on both alpha and beta chains.

Determination of the non-enzymatic glycation of hemoglobin by isoelectrofocusing of its globin chains

Micro cation exchange chromatography determination of HbA1c does not provide a complete picture of Hb glycation, for it does not determine all the glycated forms of hemoglobin. For the determination of total glycation, we describe here a rod IEF method, which allows the simultaneous quantitation of glycation on alpha and beta globin chains. The method exhibits good sensitivity; it is not affected by artifacts deriving from temperature, hypertriglyceridemia, Hb variants or labile HbA1 (aldiminic Hb). The results obtained indicate that in a normal population approximately 18% of the beta chain and 8% of the alpha chain are glycated. These mean percentages increase in the diabetic to 28% and 12%, respectively. The beta chain is glycated on both valine and lysine residues, while the alpha chain is glycated only on the latter. HbA1 values from micro cation exchange chromatography are significantly related to both alpha and beta glycation. Thus, valinic or lysinic glycation have roughly the same clinical significance.