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Biomedical subjects

M Castagnola

Publications and source records attributed to M Castagnola.

At least 37 records · Page 2Linked to original sources
PubMed1998

Thermodynamics of inositol hexakisphosphate interaction with human oxyhemoglobin.

The interaction of inositol hexakisphosphate (IHP) with oxygenated human adult hemoglobin (Hb) was investigated at 25 degreesC. The affinity of IHP for oxygenated Hb is strongly pH-dependent, and potentiometric measurements of proton uptake and release upon IHP addition have shown that over the range between pH 8.0 and pH 6.0 in oxygenated Hb there are three groups of residues that change their pKa values after IHP addition, likely because of their interaction with negative charges of the heterotropic effector. On the basis of previous calculations on the electrostatic properties of human Hb (Matthew, J. B., Hanania, G. I. H., and Gurd, F. R. N. (1979) Biochemistry 18, 1919-1928; Lee, A. W.-m., Karplus, M., Poyart, C., and Bursaux, E. (1988) Biochemistry 27, 1285-1301), two of these groups might be Val1beta and His143beta, which are located in the beta1beta2 dyad axis, where they have been also proposed to interact with 2,3-diphosphoglycerate, whereas the third group does not appear easily identifiable. Calorimetric measurements of the heat associated with IHP binding at different pH values over the same range indicate that IHP binding is mostly enthalpy-driven at pH < 7 and mostly entropy-driven at pH > 7.

Adult↗
PubMed1998

Glycated human hemoglobin (HbA1c): functional characteristics and molecular modeling studies.

A minor hemoglobin component of human red blood cell hemolysate, HbA1c, is the result of the non-enzymatic reaction of glucose with the alpha-amino groups of the valine residues at the N-terminus of the beta-chains of human hemoglobin. In this paper, the effect of protons, chloride and 2,3-diphosphoglycerate (DPG) on the functional properties of HbA1c has been investigated in some details. Moreover, the structural modifications induced on the native molecule by the sugar moieties, studied by computer modeling, do agree with the observed functional alterations. In particular, the functional results indicate that: (a) the low-affinity conformation (or T-state) of HbA1c is destabilized by the chemical modification per se; (b) the Bohr effect is reduced with respect to that of native HbA0; (c) the affinity of the T-state of HbA1c for 2,3-diphosphoglycerate is about 2.6 x lower than that of the corresponding conformational state of HbA0, while the R-state is less affected with, the affinity being 1.7 x lower. At the structural level, computer modeling studies show that the two sugar moieties are asymmetrically disposed within the 2,3-diphosphoglycerate binding site. In addition, molecular mechanics and dynamics calculations concerning the interaction with 2,3-diphosphoglycerate indicate that while in HbA0 the effector can assume two different stable orientations, in glycated Hb only one orientation is possible. All together, the results show that glycation of the Val 1 residues of both beta-chains does not impair the binding of DPG but imposes a different mode of binding by changing the internal geometry of the complex and the surface distribution of the positive electrostatic potential within the binding pocket.

Chemical Phenomena↗
PubMed1998

Predictive model for capillary electrophoretic peptide mobility in 2,2,2-trifluoroethanol-water solution.

Using capillary electrophoresis (CE) on a set of 21 peptides with a molecular mass ranging from about 350 to 1850 Da, the Stokes radii at different protonation stages and the acidic dissociation constants in water and in a 2,2,2-trifluoroethanol (TFE) water mixture (30% v/v) were determined. These results permitted us to establish separately the reliability of semiempirical models utilized for the prediction of peptide size and charge at different acidic pHapp (pHapp range: 2.00-4.25). The data obtained on size and charge were utilized in order to provide suitable mobility predictions on the basis of the charge-to-size ratio. The best predictive conditions for size and charge were found at the most acidic range of pHapp studied (2.00-2.25), either in water or a TFE-water mixture, and reliable predictive equations for peptide mobility were established at this pHapp.

Electrophoresis, Capillary↗
PubMed1998

The pH dependence of predictive models relating electrophoretic mobility to peptide chemico-physical properties in capillary zone electrophoresis.

We applied best fitting procedures to capillary electrophoresis (CE) mobility values, measured at varying acidic pH, of a set of 21 peptides with a molecular mass ranging from about 350 to 1850 Da. This method allowed the contemporary measurements of C-terminus and carboxylic group of the side-chain of aspartic and glutamic acid dissociation constants and of peptide Stokes radius at different protonation stages. Stokes radius was related to peptide molecular mass M at the power of a fractional coefficient, and best correlation was found at pH 2.25, the fractional coefficient being equal to 0.68. This value is close to that proposed by R. E. Offord (Nature 1966, 211, 591-593), who suggested a proportionality between the polymer Stokes radius and M(2/3). The coefficient value decreases at higher pH, reaching a value of 0.58 at pH 4.25, corresponding to a mean peptide conformational transition towards more compact structures as a consequence of C-terminus dissociation. The measurement of the dissociation constants of each peptide allowed us to determine the percentage error on peptide charge predictions performed utilizing mean dissociation constants. Even for the charge, the best predictive performance is obtained at the most acidic edge of the range of the pH studied, mainly at pH 2.25. Conclusively, this study shows that the best performance of predictive models for peptide CE mobility is obtainable in the very acidic pH range (2.25-2.50) and in the absence of electroosmotic flow, and that a satisfactory predictive equation of peptide electrophoretic mobility (m2V(-1)s(-1) is given by mu = 85.4(Z/M(0.68))10(-8).

Amino Acid Sequence↗
PubMed1997

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.

Buffers↗
PubMed1996

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.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1996

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.

Adrenocorticotropic Hormone↗
PubMed1996

Oxygen transport by fetal bovine hemoglobin.

The functional properties of fetal bovine hemoglobin have been studied as a function of temperature, chloride and 2,3-diphosphoglycerate (DPG) concentration. The fetal bovine erythrocyte has six times the concentration of the allosteric modulator DPG compared with the adult cell, and yet the oxygen affinity of the fetal hemoglobin still exceeds that of the adult molecule at the respective physiological concentration of DPG and at physiological temperature. We find that the allosteric modulator strongly affects the enthalpy of oxygen for the fetal hemoglobin but not for the adult protein. We propose that this may be an important mechanism for the exchange of heat from mother to fetus. In particular, under stripped conditions the oxygen affinity of fetal bovine Hb is considerably higher than that of the adult hemoglobin. Due to the higher DPG concentration that characterizes fetal bovine erythrocytes this difference is almost abolished in the presence of the respective physiological concentration of DPG and at 20 degrees C. However, on going from 20 degrees C to 37 degrees C, the difference in O2 affinity between the two hemoglobins is restored, as it should if oxygen has to be transferred from maternal to fetal blood, by virtue of the lower overall heat of oxygenation (delta H) displayed by fetal Hb when in the presence of DPG at physiological concentration. This behavior is reminiscent of that of human fetal Hb and outlines the role of temperature and of its interplay with heterotropic ligands in the modulation of hemoglobin function to fully meet the physiological needs of the organism.

2,3-Diphosphoglycerate↗
PubMed1996

Determination of peptide dissociation constants and Stokes radius at different protonation stages by capillary electrophoresis.

Peptide electrophoretic mobility measured by capillary zone electrophoresis can be regarded as deriving from the mean of mobilities of different protonated forms, each one participating according to its charge. Stokes radius and relative percentage. The percentage is a function of the peptide dissociation constant and solution pH. Therefore, mobility modifications due to pH variations can be related to peptide dissociation constant, charge, and Stokes radius throughout general binding equations. Thus, not only can peptide dissociation constants be measured, but information about Stokes radius modifications linked to proton loss can also be obtained with picomoles of peptide.

Adrenocorticotropic Hormone↗
PubMed1995

Functional and spectroscopic evidence for a conformational transition in ferrous liganded human hemoglobin.

The effect of pH and/or organic phosphate on the kinetics of O2 dissociation from oxygenated human hemoglobin and on the spectroscopic features shown by the ferrous nitrosylated derivative of human hemoglobin has been investigated. The results indicate the occurrence of conformational change(s) taking place in liganded ferrous hemoglobin, bringing about a faster kinetic ligand dissociation process, accompanied by the appearance of peculiar spectroscopic features of the ferrous nitrosylated hemoglobin. Altogether these functional and spectroscopic observations indicate the existence of a nesting of tertiary and/or quaternary conformations in the liganded forms of human hemoglobin which may account for the action of heterotropic effectors, such as protons and organic phosphates.

Ferrous Compounds↗
PubMed1995

The use of capillary electrophoresis for the determination of hemoglobin variants.

The application of capillary electrophoresis and related techniques for the detection of hemoglobin variants is described. Capillary zone electrophoresis (CZE) was applied for the analysis of intact tetrameric hemoglobin. CZE under denaturing conditions was used for the separation of globin chains. Both CZE and micellar electrokinetic capillary chromatography were applied for a fast and sensitive separation of tryptic digests and for the analysis of amino acid derivatives.

Amino Acids↗
PubMed1995

Effects of gemfibrozil on the oxygen transport properties of erythrocytes.

1. In the present study we have investigated the effects of the relatively low plasma concentrations of gemfibrozil (GFZ) found in clinical practice on the oxygen dissociation curve (ODC) of erythrocytes. 2. ODCs were measured at 30 degrees C and 37 degrees C and at pH 7.4: a) both on HbA solution and erythrocytes incubated in vitro with gemfibrozil and clofibric acid; b) on erythrocytes from healthy volunteers treated with a single oral dose of gemfibrozil. 3. These experiments showed a significant drug-induced shift of the ODC towards lower O2 affinity values without any significant modification of metabolic parameters of erythrocytes such as intracellular pH and intraerythrocytic levels of ATP and DPG. 4. In our experimental conditions gemfibrozil appears to lower both in vitro and in vivo, the partial pressure of oxygen required to give 50% of the haemes saturated with oxygen (P50) of erythrocytes from the control value of 24 +/- 0.5 mm Hg to 29 +/- 0.5 mm Hg (mean +/- s.d.; P < 0.02 by ANOVA). 5. These data clearly indicate that therapeutic doses of gemfibrozil may influence the oxygen transport properties of red cells. This effect could have relevant pharmacological and toxicological implications.

2,3-Diphosphoglycerate↗
PubMed1995

The multiple functions of hemoglobin.

The aim of this review is to focus and discuss several parallel biological functions of hemoglobin besides its basic function of oxygen transport. In light of the information present in the literature the following possible physiological roles of hemoglobin are discussed: (1) hemoglobin as molecular heat transducer through its oxygenation-deoxygenation cycle, (2) hemoglobin as modulator of erythrocyte metabolism, (3) hemoglobin oxidation as an onset of erythrocyte senescence, (4) hemoglobin and its implication in genetic resistance to malaria, (5) enzymatic activities of hemoglobin and interactions with drugs, and (6) hemoglobin as source of physiological active catabolites.

Amino Acid Sequence↗
PubMed1995

Multiparametric evaluation of functional outcome after pylorus-preserving duodenopancreatectomy.

The aim of the present study was to evaluate the clinical, morphological and functional results obtained in a group of patients previously submitted to Traverso-Longmire pylorus-preserving duodeno-pancreatectomy (PPDP). The study was performed as a clinical, endoscopic, radioisotopic and electro-manometric evaluation. An analysis of the results allowed us to conclude that: 1) most patients show good clinical features, and this becomes more evident as post-operative time progresses; 2) bile reflux gastritis is an infrequent event; 3) gastric emptying times in patients overlap those seen in control subjects, and progressive normalization occurs postoperatively. The best clinical results coexist with normal gastric emptying times; 4) gastrojejunal interdigestive motor activity shows a reduction in phase 3 and a relative increase in phase 2. We argue that the motor activity of the upper gastrointestinal tract can restore, from a functional point of view, the new anatomical situation. On the basis of the good clinical and functional results, pyloric preservation seems to be the most physiological procedure for the restoration of alimentary continuity following duodenopancreatic resection.

Bile Reflux↗
PubMed1994

Hemoglobin function under extreme life conditions.

Considering the variety of species that depend on hemoglobin for oxygen transport, these molecules must execute their primary function under extreme environmental conditions. Hence, a thermodynamic analysis of oxygen binding with hemoglobins from different species reveals a series of adaptive mechanisms which are based on the thermodynamic connection between the binding of heterotropic effectors and the reaction with oxygen. The examples reported, from fishes to human fetus, illustrate how evolution can alter the structural basis of the heterotropic interactions to optimize the oxygenation-deoxygenation cycle in dependence of the physiological needs of the particular organisms. Moreover they show that a thermodynamic analysis of the reaction with oxygen overcomes the meaning of a detailed structural and functional characterization going deeper into the physiology of the specific organism.

Adaptation, Physiological↗
PubMed1994

Functional alterations in adult and fetal hemoglobin Sassari Asp-alpha 126(H9)-->His. The role of alpha 1 alpha 2 contact.

The effects of pH, organic phosphates (2,3-diphosphoglycerate), and temperature on the functional properties of both adult and fetal hemoglobin Sassari alpha (Asp-126-->His) have been studied. The functional properties of the adult variant are characterized by the following: (i) an oxygen affinity higher than that of normal HbA in all the experimental conditions used; (ii) a dramatic reduction of homotropic interactions (n50 very close to unity); and (iii) a significant decrease of the effect of 2,3-diphosphoglycerate, which is 35% lower than that observed on HbA. The fetal variant shows an increased oxygen affinity compared with normal HbF and an almost abolished heme-heme interaction. The molecular basis of these functional differences is discussed in terms of the possible role played by the substitution of alpha (Asp-26-->His) on the stability of the R state of the molecule due to a decreased interaction at the level of alpha 1 alpha 2 contact.

Adult↗
PubMed1994

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.

Amino Acid Sequence↗