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The amino-acid sequence and the oxygen-binding properties of the two haemoglobins of the Antarctic seabird south polar skua have been investigated. The two haemoglobins showed peculiar functional features, which were probably acquired to meet special needs in relation to the extreme environmental conditions. Both haemoglobins showed a weak alkaline Bohr effect which, during prolonged flight, may protect against sudden and uncontrolled stripping of oxygen in response to acidosis. We suggest that a weak Bohr effect in birds may reflect adaptation to extreme life conditions. The values of heat of oxygenation suggest different functional roles of the two haemoglobins. The experimental evidence suggests that both haemoglobins may bind phosphate at two distinct binding sites. In fact, analysis of the molecular models revealed that an additional phosphate binding site, formed by residues NA1α, G6α and HC3α, is located between the two α chains. This additional site may act as an entry/leaving site, thus increasing the probability of capturing phosphate and transferring it to the main binding site located between the two β chains by means of a site–site migratory mechanism, thereby favouring the release of oxygen. It is suggested that most haemoglobins possess an additional phosphate binding site, having such a role in oxygen transport. [ABSTRACT FROM AUTHOR]

Concerning the number and type of the hemoglobin components, the moray Muraena helena is characterized by three different phenotypes whose frequencies are nearly identical. Thus, the cathodal component is present in all individuals, whereas one or both of two amodal components may be present in the same phenotype. These components have been separated by chromatography. The oxygen binding properties of the purified hemoglobin components have been studied in the absence and presence of saturating concentrations of ATP or GTP and as a function of pH. The cathodal component shows an intrinsic O2 affinity four times higher than that of both anodal components, a very small Bohr effect and a significant decrease in O2 affinity upon addition of ATP and GTP (three and four times respectively with respect to stripped conditions), the latter being more effective than the former over the entire pH range examined. The anodal components do not appear functionally distinguishable and show the presence of an enhanced Bohr effect (Root effect) that is under the strict control of nucleotide triphosphates ATP, GTP, which, unlike in the cathodic component, exert the same effect on oxygen affinity. The complete sequence of the β chains of the cathodal and of one of the anodal components have been determined. The possible molecular basis of these different functional characteristics are discussed in the light of the globin sequence and of those amino acid residues which are known to be responsible of hemoglobin functional behaviour. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 rnyo-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]. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Human hemoglobin, reacted at th four amino termini with 4-isothiocyanotobenzenesulphonic 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 β of the α chains; α2β2ICBS and α2ICBSβ2. All of the modified tetramers show as reduced oxygen affinity and reduced cooperativity; furthermore the oxygen affinity of the Hb-ICBS and α2β2ICBS is unaffected by 2,4-biphosphoglycerate while the oxygen affinity of α2ICBSβ2 is decreased in the presence of this organic phosphate. The oxygen affinity of Hb-ICBS and α2ICBSβ2 is independent of chloride concentration, while the α2β2ICBS hybrid shows a reduced response to this anion. The tetrames α2ICBSβ2 and α2ICBSβ2ICBS 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 α2β2ICBS 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 satisfactory 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. [ABSTRACT FROM AUTHOR]

The crystallization properties of the hemolysate and of the purified hemoglobin components from Wistar rat have been studied under various experimental conditions. The results suggest the existence of hemoglobins in the crystalline state even within the red blood cells. However the functional properties of rat erythrocytes are identical to those of whole hemotysate in solution. [ABSTRACT FROM AUTHOR]

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(β93)-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 β93, 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. [ABSTRACT FROM AUTHOR]

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 D-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. [ABSTRACT FROM AUTHOR]

The functional properties of human hemoglobin and of isolated α- and β-chains with free SH groups blocked by 2-chloromercuri-4-nitrophenol, 4-chloromercuri-2-nitrophenol and p-chloromercuribenzoate have been determined. The oxygen affinity of all the hemoglobin derivatives is slightly higher than that of the untreated protein; the shape of the oxygen equilibrium curve is pH dependent (except for p-chloromercuribenzoate reacted hemoglobin). The alkaline Bohr effect is similar to that of normal hemoglobin, but in the acid range it appears to be modified to a different extent in the various cases. Thus the properties of the modified hemoglobins appear to depend on the details of structure of the organic mercurial reacted with the β93-SH group. The results with isolated β-chains show that the presence of the organic mercurials on F9(93), but not on G14(112) is associated with a decrease in oxygen affinity and a large increase in the oxygen dissociation velocity constant. [ABSTRACT FROM AUTHOR]

The ligand-binding kinetics of the isolated component from trout (Salmo irideus) blood has been extensively investigated ; it appears to be fully consistent with their equilibrium behaviour, described previously. At alkaline pH (&assymp; 7.5), in a region where the Root effect is not operative, comparison of the kinetics of the various components (Hb trout I, II and IV) shows considerable similarities between them. For Hb trout I (and II) the time course of the reaction with both O2 and CO is practically pH independent. On the other hand Hb trout IV, characterized by a Root effect, displays a strong dependence on pH of the kinetics of ligand binding. Thus in the pH region where the Root effect becomes operative there is a large change of the character, as well as of the speed, of the kinetics with both O2 and CO. The Root effect characteristic of Hb trout IV appears to originate, from a kinetic point of view, in a pH-dependent change of both the “on” and “off” constants, although at least for O2 the effect of pH on the “off” kinetic constants is much more marked. [ABSTRACT FROM AUTHOR]

This paper reports some physico-chemical and functional properties of two of the isolated hemoglobin components from trout (Salmo irideus) blood (Hb trout I and Hb trout IV). Both hemoglobins in the oxygenated state are tetrameric at pH between 7 and 7.8 and protein concentration above &assymp; 0.1 mg/ml. The tetramer is extremely stable towards dissociation into subunits and the stability constants, estimated by differential gel filtration, are between 10 and 50 times larger than that of human hemoglobin in phosphate buffer. A very small degree of dissociation is also suggested by the very small extent of hybrid formation even in concentrated KI or triethanolamine. The dichroic properties of both Hb trout I and Hb trout IV show distinct features in the wavelength region between 300 and 200 nm. Hb trout IV resembles human hemoglobin in the shape of the CO spectrum and in the values of the reduced ellipticity (θ); furthermore upon deoxygenation the value of θ at 222 nm becomes more negative by about 50%. On the ether hand Hb trout I shows a different circular dichroism spectrum and in particular the ellipticity at 222 nm is lower than that of Hb trout IV. Moreover the spectrum is insensitive to the presence of heme ligands. Hb trout IV binds human haptoglobin (Hp) as shown by quenching of the fluorescence of the tryptophanyl residues of Hp upon addition of hemoglobin. The apparent stoichiometry of complex formation corresponds to two Hb tetramers per Hp molecule (while for human hemoglobin the stoichiometry corresponds to &assymp; one tetramer per Hp). A clear cut effect of Hp binding is observed on the kinetics of O2 and especially CO combination as studied by flash photolysis. The effect of several third components on the O2 equilibrium of both Hb trout I and IV has been investigated. In general the effect of adding ions or other molecules to a solution of lib trout I is very minor. On the other hand Hb trout IV is more susceptible to changes in solvent composition. Of particular importance is the action of ATP, the physiological organic phosphate, which is without effect on Hb trout I, while it has a clear effect on Hb trout IV. The addition of ATP shifts the Root effect, the characteristic property of Hb trout IV, to higher pH values, thereby acting as one of the allosteric effectors. [ABSTRACT FROM AUTHOR]