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Biomedical subjects

B Giardina

Publications and source records attributed to B Giardina.

At least 217 records · Page 12Linked to original sources
PubMed1981

Purification and functional properties of the hemoglobin components from the rat (Wistar).

Homogeneous components of Wistar rat hemoglobin have been isolated and characterized from the molecular and functional point of view. The O2 equilibrium behaviour of the three main components (HbII, HbIII, HbIVA) has been investigated as a function of pH and organic phosphate concentration. The ligand-binding kinetics of the isolated components have been also studied and are fully consistent with their equilibrium behaviour. It should be remarked that the choice of the system was governed largely by the ability of rat hemoglobins to crystallize very quickly. This almost unique molecular property together with the complete reversibility of the process may allow information to be obtained on the thermodynamics of ligand-linked phase changes.

Animals↗
PubMed1981

Functional properties of hemoglobins from Triturus cristatus.

The functional properties of hemoglobins from Triturus cristatus carnifex have been characterized both from an equilibrium and a kinetic point of view. A special attention has been given to the effect of organic phosphates in view of their role in the modulation of the hemoglobin function. Under stripped conditions newt hemoglobins display a negative Bohr effect which becomes much less pronounced in the presence of 0.2 M inorganic phosphates. The effect of organic phosphates involves not only a drop in the O2 affinity but also a large change in the shape of the O2 Bohr effect which becomes markedly positive in the presence of myo-inositol hexakis(phosphate). The experimental data reported make it possible to give a complete picture of the functional behaviour of newt hemoglobins, making a contribution to clarifying the dependence of the sign of the Bohr effect on the acclimatization temperature of the animals that has been previously reported [Morpurgo, G., Battaglia, P. A. and Leggio, T. (1970) Nature (Lond.) 225, 76--77].

Adenosine Triphosphate↗
PubMed1980

Temperature perturbation of the allosteric equilibrium in trout hemoglobin.

The carbon monoxide binding kinetics of the isolated trout Hb I has been investigated by flash photolysis at various temperatures, from approximately 20 degrees to 72 degrees C. The time course of recombination has been quantitatively analyzed with a simple two-state allosteric model, making use of the thermodynamic data previously obtained. These new experiments and their analysis show that a simple two-state kinetic model is adequate, in the case of trout Hb I, to describe quantitatively the time course of CO binding at all temperatures. Moreover, we show that temperature can be used to perturb the quaternary conformational equilibrium, the high affinity state of the molecule (R) being progressively more populated at higher temperatures.

Allosteric Regulation↗
PubMed1978

The effect of 2-methoxy-5-nitrotropone on the oxygen affinity of human erythrocytes and hemoglobin.

Human hemoglobin reacted with 2-methoxy-5-nitrotropone at pH 7.4 undergoes modification of the four N-terminal amino groups. The modified protein shows increased oxygen affinity with complete abolition of the effect of K-glycerate 2, 3-bisphosphate. The Bohr effect is abolished in the acid range and drastically reduced at alkaline pH values. Changes in the kinetics of ligand reactions parallel the oxygen equilibrium results. Cooperative effects are still present. Human erythrocytes treated with 2-methoxy-5-nitrotropone show increased oxygen affinity and some decrease in methemoglobin reductase efficiency but no change in resistance to hemolysis.

Erythrocytes↗
PubMed1978

A comparative approach to protein- and ligand-dependence of the Root effect for fish haemoglobins.

Ligand-binding equilibria, kinetics and (13)C n.m.r. spectra of bound (13)CO, of the haemoglobins from two fishes that are very distant on the evolutionary scale, i.e. the fourth haemoglobin component from Salmo irideus and the single component from Osteoglossum bicirrhosum, were studied. The C-terminal sequence was also determined for the haemoglobin from Osteoglossum. The results show that (i) the C-terminal residues of both chains are not directly responsible for the characteristic heterotropic effect known as Root effect, since for both fish haemoglobins these residues are identical with those of human haemoglobins. (ii) In all haemoglobins characterized by the Root effect a dependence of the (13)CO n.m.r. resonances on pH is observed. However, the extent of the shift(s) depends on the particular protein, and is probably the result of a combination of both tertiary and quaternary conformational changes. (iii) Both haemoglobins from trout and Osteoglossum manifest a functional heterogeneity between the two types of chains in the tetramer, which increases with proton activity. For CO, the effect is very small for trout haemoglobin IV, and very marked for Osteoglossum haemoglobin; for O(2) strongly heterogeneous binding curves were obtained at approx. pH6.2 with both haemoglobins. (iv) Estimations of the relative values of the affinity constants for the alpha and beta chains in the tetramer were obtained for both haemoglobins from (13)CO n.m.r. spectra at low fractional saturation. On the basis of these findings the molecular mechanism underlying the Root effect is discussed.

Amino Acid Sequence↗
PubMed1978

Properties of hemoglobin G. Ferrara (beta57(E1) Asn replaced by Lys).

Hemoglobin G. Ferrara is an abnormal human hemoglobin in which an asparagine residue is replaced by a lysyl residue at position beta57 (beta57 Asn replaced by Lys). Oxygen equilibria show that cooperativity and alkaline Bohr effect are maintained to normal levels while the acid Bohr effect appears increased; in addition, a smaller effect of diphosphoglycerate is also observed. Flash photolysis experiments performed as a function of protein concentration show that the fraction of quickly reacting form is always higher than that of human hemoglobin A. This fact, together with the increase of the oxygen affinity observed at acid pH values, may be related to an enhanced dissociation of the molecule into dimers. Several attempts to isolate the native chains by treatment of the protein with p-chloromercuribenzoate were unsuccessful due to the great instability of the isolated variant beta-chains, which precipitated completely during incubation with p-chloromercuribenzoate. Therefore, although the substitution is on the surface of the molecule, there are several properties of hemoglobin G. beta Ferrara which are clearly different from hemoglobin A.

Diphosphoglyceric Acids↗
PubMed1978

Properties of trout hemoglobins reconstituted with unnatural hemes.

Native globins isolated from trout hemoglobin compoents I and IV have been reconstituted with proto-, meso-, and deuteroheme, and the spectral and functional properties of the reconstituted hemoglobins have been investigated. Equilibrium and kinetic studies allow the following conclusions. (a) The properties of the proto-reconstituted hemoglobins are very similar, or indistinguishable, from those of the native Hb's I and IV. (B) The CO binding kinetics for both proteins were found to be consistent with the equilibrium data: the overall association rate constant increases (and the autocatalytic character of the reaction decreases) in the order proto, meso, deutero. (c) A marked pH dependence of both ligand affinity and cooperativity is maintained in the reconstituted Hb's IV: at pH 6 the fractional saturation with oxygen in air (Root effect) is lower for proto- than for meso- and deutero-Hb IV. The results obtained, including partial photodissociation experiments at different pH values, can be considered, to a first approximation, consistent with the basic features of a simple two-states model.

Allosteric Regulation↗
PubMed1978

Single cell observations of gas reactions and shape changes in normal and sickling erythrocytes.

Microspectrophotometry has been applied to single red blood cells to reinvestigate the linked processes of diffusion of gases inside the erythrocyte and their combination with hemoglobin. The experiments took advantage of the photosensitivity of the cabron monoxide derivative of hemoglobin, which allows ligand release from the CO-saturated red cells under strong illumination and recombination when the light is switched off. The photochemical method was also used to study the kinetics of sickling on ligand removal in single erythrocytes of Hb S carriers. The results give new information on the mechanism of the sickling process.

Anemia, Sickle Cell↗
PubMed1978

A macromolecular transducer as illustrated by trout hemoglobin IV.

Oxygen binding by trout Hb IV has been investigated as a function of pH up to 10 atmospheres (1 MPa) of pure O2. The results bring out an extreme proton-oxygen linkage, which gives rise to a Root effect. They are discussed in relation to the function of the hemoglobin as an oxygen pump. The system is of special interest as providing a prototype of a macromolecule acting as a transducer by coupling two allosterically linked reactions.

Allosteric Regulation↗
PubMed1977

Properties of trout hemoglobin covalently bound to a solid matrix.

This paper reports the ligand binding properties of the major hemoglobin component from trout (Salmo irideus) covalently bound to a solid matrix (Sepharose or Sephadex). A comparison between the functional properties of this protein in solution and of the protein-matrix complex shows significant changes although the basic properties of the molecule are maintained on covalent binding to Sepharose (or Sephadex). Thus the Root effect, characteristic of Hb trout IV, is still present while the heme-heme interactions are, on the average, smaller in the matrix bound protein as compared to the soluble form. No differences in the O2 binding properties were observed when the protein was coupled to the resin, as the ligand bound or as the ligand free derivative. Although an unequivocal interpretation of the data is made difficult by the lack of information on the number and identity of the groups involved in the coupling, the main changes in the protein functional properties may be related to the chemical modifications "per se" more than to the immobilization imposed to the macromolecule by coupling to the matrix. Structural changes which mainly involve perturbation of the tertiary structure of the molecule may qualitatively rationalize the data.

Animals↗
PubMed1977

Physicochemical and functional properties of Perinereis cultrifera (Grübe) erythrocruorin.

Perinereis erythrocruorin has the following physicochemical properties: So20,w = 55S, corresponding to a molecular weight around 2.7-10(6); minimum molecular weight (on the basis of the heme content) 23 700 +/- 500; isoelectric point 5.1; alpha-helix content approximately 40%. At alkaline pH values in the oxygenated form the 55-S molecules dissociate into subunits with a weight average sedimentation coefficient of 3S, corresponding to a molecular weight approximately 35 000. Deoxygenation of partially dissociated samples promotes association of the 3-S subunits into a 9S component. The functional properties of Perinereis erythrocruorin are characterized by a low cooperativity in oxygen binding (n 1/2 = 1.5) at neutral pH. Cooperativity increases reversibly towards both the acid and alkaline pH range, irrespective of changes in molecular weight. This finding, taken together with the ultracentrifuge results, suggests that a subunit may represent the functional unit of the protein. The pH dependence of the oxygen affinity can be accounted for in terms of a single oxygen linked group with a pK of 8.

Allosteric Site↗