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Biomedical subjects

B Giardina

Publications and source records attributed to B Giardina.

At least 199 records · Page 11Linked to original sources
PubMed1987

Xenopus laevis hemoglobin and its hybrids with hemoglobin A+.

Isolated alpha and beta chains from Xenopus laevis hemoglobin have been purified. The isolation procedure yields native alpha chains whose functional behavior has been characterized and compared with that of human alpha chains. Isolated beta chains in the presence of oxygen are characterized by low stability, and hence their functional characterization was limited to the CO binding kinetics. When stoichiometric amounts of the isolated alpha and beta chains are mixed, a tetramer characterized by heme-heme interactions and oxygen affinity comparable to that of the native molecule is readily reconstituted. Moreover, both chains, under appropriate conditions, form stable hybrid tetramers with the partner subunits from human hemoglobin; results on the functional properties of these hybrid hemoglobins are presented and discussed in relation to the stereochemical model of the Root effect.

Animals↗
PubMed1987

Is there a Root effect in Xenopus hemoglobin?

The reaction of Xenopus hemoglobin with oxygen and carbon monoxide has been reinvestigated over the pH range 8.5-6.0, in the absence and presence of organic phosphates (2,3-diphosphoglycerate or inositol hexakisphosphate), to establish if the tetramer can be stabilized in a T-quaternary state by protons and polyphosphate; the equilibrium and kinetic data indicate that Xenopus hemoglobin does exhibit a Root effect. These new results are discussed with reference to those reported by Bridges et al. [(1985) Resp. Physiol. 61, 125-136] on Xenopus blood and, more generally, to the molecular definition and the structural basis of the Root effect as an extreme form of the Bohr effect.

2,3-Diphosphoglycerate↗
PubMed1987

Human hemoglobin cross-linked through the polyphosphate-binding site. Functional properties and evidence for conformers.

The properties of human hemoglobin reacted with 2-nor-2-formylpyridoxal 5'-phosphate, a bifunctional derivative of pyridoxal 5'-phosphate, have been investigated both from an equilibrium and kinetic point of view. The experimental data, interpreted in terms of the two-state allosteric model, indicate that a perturbed R state is characteristic of this modified low ligand affinity hemoglobin. In flash photolysis experiments, a quickly reacting component is always observed, in spite of the lack of dissociation into free dimers; this kinetic behavior is thought to reflect the presence of functionally independent alpha beta dimers, still connected by the flexible cross-link but forming an open hemoglobin tetramer. Two possible models for the interpretation of the kinetics of CO and/or haptoglobin binding are presented and discussed.

Hemoglobins↗
PubMed1987

A microspectroscopic analysis of ligand binding in single erythrocyte.

The kinetics of ligand uptake by erythrocytes has been analyzed by single cell microspectroscopy measurements carried out in parallel with mathematical simulations. The main experimental feature is the linear shape of the ligand binding progress curves, whose slope is dependent on the free ligand concentration in the speciman. From the computer simulation a predominant role has been shown to be played by the ligand diffusion from the extracellular space.

Carbon Monoxide↗
PubMed1986

On the oxygen-linked anion-binding sites in human hemoglobin. Functional properties of human hemoglobin reacted with 4-isothiocyanatobenzenesulphonic acid and its hybrids.

Human hemoglobin, reacted at the four amino termini with 4-isothiocyanatobenzenesulphonic acid (Hb-ICBS), was separated into its constituent chains. Recombination of the ICBS-reacted chains with the unmodified mate chains produced the hybrid tetramers modified at either the beta or the alpha chains: alpha 2 beta 2ICBS and alpha 2ICBS beta 2. All of the modified tetramers show a reduced oxygen affinity and reduced cooperativity; furthermore the oxygen affinity of the Hb-ICBS and alpha 2 beta 2ICBS is unaffected by 2,3-bisphosphoglycerate while the oxygen affinity of alpha 2ICBS beta 2 is decreased in the presence of this organic phosphate. The oxygen affinity of Hb-ICBS and alpha 2ICBS beta 2 is independent of chloride concentration, while the alpha 2 beta 2ICBS hybrid shows a reduced response to this anion. The tetramers alpha 2ICBS beta 2 and alpha 2ICBS beta 2ICBS show a decreased alkaline Bohr effect, which can be rationalized as being due to disruption of the oxygen-linked chloride-binding sites; in the case of alpha 2 beta 2ICBS the Bohr effect is instead (partially) maintained. The functional properties of artificial tetramers have been studied also from a kinetic point of view by CO combination and the results obtained compare satisfactorily with equilibrium data. The possibility of obtaining selectively modified hemoglobins promises to provide further insight into the properties of the oxygen-linked anion-binding sites in hemoglobin.

Anions↗
PubMed1986

Mini-myoglobin: preparation and reaction with oxygen and carbon monoxide.

A domain of 108 amino acid residues (32 to 139), obtained by digestion of horse heart apomyoglobin with clostripain, was found to bind protoheme in a 1 to 1 molar ratio. This domain is 33 amino acid residues larger than the protein segment encoded by the central exon in seal myoglobin. Flash photolysis experiments have shown that reconstituted "mini-myoglobin" is very similar to myoglobin in the combination reaction with carbon monoxide and with oxygen, and in the oxygen replacement reaction by carbon monoxide. These experiments provide for the first time direct evidence for the presence of a structural and functional domain, closely corresponding to the segment encoded by the central exon of the myoglobin gene, which contains the information for binding the natural heme and for maintaining the native folding typical of a respiratory protein.

Animals↗
PubMed1986

Thermodynamic characterization of the allosteric transition in trout hemoglobin.

The pH induced spectral changes in the Soret region occurring in the carbomonoxy derivative of trout HbIV have been measured under carefully controlled conditions of temperature and organic phosphate concentration. Parallel experiments on the kinetics of carbon monoxide dissociation by NO replacement have been performed over the same pH range. Both sets of results agree satisfactorily with a thermodynamic scheme independently drawn on the basis of functional data previously analyzed within the framework of a two state allosteric model. Thus, the whole body of data strongly supports the idea that the spectral changes are themselves an indication of the pH induced structural transition, thereby reflecting the stabilization of the liganded T-state of the molecule at low pH. This allows us to definitely conclude that the functional changes induced in trout HbIV-CO by protons are associated with a red shift of the Soret absorption band.

Allosteric Regulation↗
PubMed1986

Discrimination of tertiary and quaternary Bohr effect in the O2 binding of Helix pomatia beta-hemocyanin.

Simultaneous determination of proton uptake and oxygen binding has been carried out on Helix pomatia beta-hemocyanin under equilibrium conditions in the absence of buffer and at different initial pH values. Oxygen-binding isotherms of unbuffered H. pomatia beta-hemocyanin, in the presence of phenol red as pH indicator, have been determined employing a thin-layer apparatus. Application of this very accurate technique allows monitoring of proton uptake (or release) coupled to O2-binding also at extremes of saturation which are often difficult to explore and analyze. The data have been analyzed within the framework of the cooperon model (M. Brunori, M. Coletta and E. Di Cera, Biophys. Chem. 23 (1986) 215) and compared with those obtained in the presence of buffer. Comparison of pH changes with ligand binding of the T state over all the saturation range has allowed us to discriminate and obtain quantitative estimates of the Bohr protons associated with both oxygenation of the T state and quaternary allosteric transition; no protons are taken up or released during oxygenation of the R state. These results differ quantitatively from those obtained in the presence of buffer, which alters significantly the T state contribution to the overall Bohr effect.

Journal Article↗
PubMed1985

Kinetics of the reaction of intraerythrocytic haemoglobin by single cell microspectroscopy: effect of shape and osmolarity.

The kinetics of the reaction of CO with intraerythrocytic haemoglobin has been studied in single red blood cells (RBC) using a scanning microspectrophotometer and a photochemical perturbation method. Measurements have been carried out using red blood cells from man and camel (Camelus dromedarius), the latter at different osmotic pressures. Camel RBC, which are smaller and different in shape compared to human RBC, are known to remain intact even at an osmolarity 6-times lower than physiological (280-290 mosm/l), swelling up to twice their normal volume. The results show that the recombination time course is affected by diffusion of CO through a stagnant layer of solvent around the cell membrane, but that it is also influenced by other parameters such as intracellular diffusion of ligand and haemoglobin.

Animals↗
PubMed1985

Tadpole Xenopus laevis hemoglobin. Correlation between structure and functional properties.

Perutz & Brunori (1982) proposed that the COOH-terminal His and Ser F9 of the beta-chains of fish and amphibian hemoglobins are responsible for their Root effect and part of their alkaline Bohr effect. Analysis of the kinetics of carbon monoxide binding by hemoglobin from the tadpole of Xenopus laevis supports that model and suggests an explanation for the absence of an alkaline Bohr effect in many aquatic Anura and Urodela.

Amino Acid Sequence↗
PubMed1984

Oxygen binding to Octolasium complanatum erythrocruorin. Modulation of homo- and heterotropic interactions by cations.

The functional properties of erythrocruorin from Octolasium complanatum (a common earthworm of Central Italy) have been characterized in great detail. Special attention has been given to the reciprocal effects of the various ligands, namely oxygen, cations and protons. The data obtained under a variety of experimental conditions bring out the dominant role played by cations in the modulation of both homotropic and heterotropic interactions. In this respect, the most interesting observation concerns the unusual interplay between protons and cations that occurs in this erythrocruorin, the first respiratory pigment in which the Bohr effect is due totally to the O2-linked binding of an allosteric effector. The oxygen binding data collected under the various experimental conditions have been analyzed in terms of a modified two-state model, which takes into account the fact that allosteric effectors may also influence the ligand binding properties of the state that they stabilize. The analysis shows that the number of interacting sites necessary for the observed co-operativity in O2 binding is much smaller than the number of heme groups carried by the whole molecule, in accordance with previous findings on hemocyanins, the other class of giant respiratory pigments. Moreover, the analysis indicates that the dimensions of these "functional constellations" are under the control of allosteric effectors.

Allosteric Regulation↗
PubMed1984

Stereochemistry of ATP and GTP bound to fish haemoglobins. A transferred nuclear overhauser enhancement, 31P-nuclear magnetic resonance, oxygen equilibrium and molecular modelling study.

This study was undertaken in order to test the models of ATP and GTP binding to carp deoxyhaemoglobin proposed by Perutz & Brunori (1982) and to find out why GTP is a more potent allosteric effector than ATP. We have determined the conformations of both nucleoside triphosphates by nuclear magnetic resonance studies and found them to be the same. The purines are in anti conformation about the glycosidic bond that links them to the ribose; the pentose ring is 3'-endo; the P-O5'-C5'-C4' torsion angle lies in the trans domain (180 degrees +/- 20 degrees); the P alpha-O-P beta and P beta-O-P gamma angles are as in the free nucleotides, i.e. the trinucleotide chain is fully extended. Models having this conformation were fitted, first manually and then by energy refinement, to the effector site of an atomic model of human deoxyhaemoglobin in which the side-chains in the NA, EF and H segments had been replaced by those of carp. The results showed the location of the polar groups in carp haemoglobin to be such that (PO4) gamma can accept hydrogen bonds from Val NA1 beta 2 and from Arg H21 beta 1, while (PO4) beta and (PO4) alpha can accept hydrogen bonds from Lys EF6 beta 1 and beta 2. In ATP, the 6-amino group of the purine can donate a hydrogen bond to Glu NA2 beta 1. In GTP, the 2-amino group can donate a hydrogen bond to Glu NA2 beta 1; in addition, Val Na1 beta 1 can donate a hydrogen bond to O2' of the ribose. This additional hydrogen bond may explain why in carp haemoglobin GTP is a stronger allosteric effector than ATP. We have found the influence of the two allosteric effectors on the oxygen affinity of trout IV haemoglobin to be the same, even though the only difference in the lining of the allosteric effector sites lies in the replacement of Glu Na2 beta in carp by Asp in trout IV haemoglobin. Model building then showed that formation of a hydrogen bond between Asp Na2 beta and the 2-amino group of guanine precludes formation of a hydrogen bond between Val NA1 beta and O2' of the ribose or vice versa, which makes the number of hydrogen bonds formed between trout IV haemoglobin and GTP the same as those formed with ATP.

Adenosine Triphosphate↗
PubMed1983

The reaction of trout hemoglobins with isocyanides.

Binding of alkylisocyanides of different bulkiness to the two major components of trout hemolysate is presented. In the case of trout hemoglobin I isocyanide binding is pH-independent, similar to O2 and CO, and the bulkiness of the ligand is related to the endothermicity of ligand binding to the T quaternary state. On the other hand, in trout hemoglobin IV the size of the ligand seems to affect the pH dependence of affinity and cooperativity. A comparison with other ligands, like O2, allows us to hint at possible stereochemical determinants of ligand binding in these two hemoglobins.

Animals↗
PubMed1983

[Microspectrophotometry in the study of physiological and pathological events in erythrocytes].

Microspectrophotometry turns out to be the ideal method for an accurate study of single cells, namely of red blood cells, from the biochemical and physiological standpoint. Hereafter several possible applications are reported: a. the kinetics of gas-exchange (O2 and CO) in human erythrocytes has been made possible by the photosensitivity of the Hb-CO complex. In this way, it has been possible to establish that both ligands recombine with intraerythrocyte hemoglobin following a zero-order kinetic process. This suggested that diffusion of a ligand across an unstirred layer of buffer (approximately 5.10 mu thick) all around the cell could be the rate-limiting step of this recombination. Moreover, an intracellular facilitated oxygen diffusion has been observed and possible physiological implications have been shortly mentioned; b. the kinetics of intracellular polymerization of sickle cell hemoglobin in single red blood cells has been measured by quickly (approximately 1 msec) flashing-off the CO employing a cw Argon ion laser. The polymerization of deoxyhemoglobin has been followed by detecting the increased light scattering of the laser beam itself. The distribution of delay times (td) in several patients has been found significantly correlated to the severity of the disease, supporting the hypothesis that the td is a determinant factor in the pathophysiology of the disease. Possible therapeutic applications of this method are briefly discussed; c. the intraerythrocyte distribution of different hemoglobins of trout (Salmo irideus) among several erythrocytes has been observed by means of different physiochemical properties of single components. These turned out to be homogeneously present in all red blood cells studied.

Animals↗
PubMed1982

The effect of pH and D-glycerate 2,3-bisphosphate on the O2 equilibrium of normal and SH(beta 93)-modified human hemoglobin.

The present paper reports data for the effect of pH and D-glycerate 2,3-bisphosphate (D-glycerate-2,3-P2) on the oxygen equilibrium of normal and SH(beta 93)-modified human hemoglobin. At sufficiently high D-glycerate-2,3-P2 concentrations, both oxy and deoxy forms of HbA are saturated with the organic phosphate at all pH values between 6 and 9. Furthermore the difference in the affinity for D-glycerate-2,3-P2 between deoxy and oxy hemoglobin remains constant with pH, implying that the pK values of the Bohr effect groups in deoxy and oxyhemoglobin are not affected by the presence of D-glycerate-2,3-P2 on the hemoglobin molecule. In the hemoglobins modified in position beta 93, the difference in affinity between deoxy and oxy hemoglobin for D-glycerate-2,3-P2 decreases with decrease in pH due mainly to a decrease in the affinity of the deoxy form for D-glycerate-2,3-P2. The effect of the chemical modification appears to be primarily a change in pK of a Bohr group in deoxy hemoglobin which is especially pronounced in the presence of phosphates.

2,3-Diphosphoglycerate↗
PubMed1982

Kinetics of reactions within single erythrocytes: studies by microspectrophotometry.

Single cell spectroscopy has been applied to red blood cells along three different lines of research: a) distribution of single hemoglobin components among different erythrocytes, b) chemical reaction with ligands and diffusion in the red blood cell c) dynamics of hemoglobin S aggregation in single red blood cell. The results reported here show, in general, the great possibilities of using single-cell spectroscopy in investigating complex integrated systems.

Anemia, Sickle Cell↗