Modulation of the oxygen affinity of cobalt-porphyrin by globin

We have combined two extreme effects which influence the oxygen affinity to obtain a cobalt-based oxygen carrier with an affinity similar to that of human adult hemoglobin (HbA). The goal was to obtain an oxygen transporter with a lower oxidation rate. Exchange of the heme group (Fe-protoporphyrin IX) in Hb with a cobalt-porphyrin leads to a reduction in oxygen affinity by over a factor of 10, an oxygen affinity too low for use as a blood substitute. At the other extreme, certain globin sequences are known to provide a very high oxygen affinity; for example, Hb Ascaris displays an oxygen affinity 1000 times higher than HbA. We demonstrate here that these opposing effects can be additive, yielding an oxygen affinity similar to that of HbA, but with oxygen binding to a cobalt atom. We have tested the effect of substitution of cobalt-porphyrin for heme in normal HbA, sperm whale (SW) Mb (Mb), and high affinity globins for leghemoglobin, two trematode Hbs: Paramphistomum epiclitum (Pe) and Gastrothylax crumenifer (Gc). As for HbA or SW Mb, the transition from heme to cobalt-porphyrin in the trematode Hbs leads to a large decrease in the oxygen affinity, with oxygen partial pressures for half saturation (P(50)) of 5 and 25 mm Hg at 37 degrees C for cobalt-Pe and cobalt-Gc, respectively. A critical parameter for Hb-based blood substitutes is the autoxidation rate; while both metals oxidize to an inactive state, we observed a decrease in the oxidation rate of over an order of magnitude for cobalt versus iron, for similar oxygen affinities. The time constants for autoxidation at 37 degrees C were 250 and 100 h for Pe and Gc, respectively.     

Allosteric effectors do not alter the oxygen affinity of hemoglobin crystals.

In solution, the oxygen affinity of hemoglobin in the T quaternary structure is decreased in the presence of allosteric effectors such as protons and organic phosphates. To explain these effects, as well as the absence of the Bohr effect and the lower oxygen affinity of T-state hemoglobin in the crystal compared to solution, Rivetti C et al. (1993a, Biochemistry 32:2888-2906) suggested that there are high- and low-affinity subunit conformations of T, associated with broken and unbroken intersubunit salt bridges. In this model, the crystal of T-state hemoglobin has the lowest possible oxygen affinity because the salt bridges remain intact upon oxygenation. Binding of allosteric effectors in the crystal should therefore not influence the oxygen affinity. To test this hypothesis, we used polarized absorption spectroscopy to measure oxygen binding curves of single crystals of hemoglobin in the T quaternary structure in the presence of the "strong" allosteric effectors, inositol hexaphosphate and bezafibrate. In solution, these effectors reduce the oxygen affinity of the T state by 10-30-fold. We find no change in affinity (< 10%) of the crystal. The crystal binding curve, moreover, is noncooperative, which is consistent with the essential feature of the two-state allosteric model of Monod J, Wyman J, and Changeux JP (1965, J Mol Biol 12:88-118) that cooperative binding requires a change in quaternary structure. Noncooperative binding by the crystal is not caused by cooperative interactions being masked by fortuitous compensation from a difference in the affinity of the alpha and beta subunits. This was shown by calculating the separate alpha and beta subunit binding curves from the two sets of polarized optical spectra using geometric factors from the X-ray structures of deoxygenated and fully oxygenated T-state molecules determined by Paoli M et al. (1996, J Mol Biol 256:775-792).     

Oxygen-binding modulation of hemocyanin from the slipper lobster Scyllarides latus

The oxygen-binding properties of hexameric hemocyanin (Hc) from Scyllarides latus were investigated with respect to pH, temperature, and modulating effect exerted by calcium, lactate, and urate. The oxygen affinity decreased at higher temperature, was slightly affected by pH, and was insensitive to lactate. Nevertheless, urate markedly increased Hc-oxygen affinity and its temperature sensitivity, acting as the physiological major positive effector: four urate sites per hexamer with an overall affinity constant of 1 x 10(4) M(-1) were found and the exothermic contribution of their binding was found to be about 30 kJ mol(-1). Calcium ions largely influenced oxygen affinity: their effect, which has an opposite sign at low (0-1 mM) and high (0.1-1 M) concentration ranges, indicates the presence of two independent types of binding sites with high and low affinity, respectively; however, only the former ones seem to be operative in vivo because, at physiological calcium concentrations, they are already saturated and the oxygen affinity is reduced.     

Hemoglobins, XXVIII. Phosphate-protein-interaction, gene expression and function: the genetic and allosteric control of the oxygen affinity of the fetal blood (author's transl)

This work describes possible molecular mechanisms concerning the control of oxygen affinity in fetal blood of mammalia. There is a genetic control of oxygen affinity through a fetal gene: at constant phosphate concentration (Hb less than P2-glycerate) in humans there is a hemoglobin with only five binding sites to 2,3-bisphosphoglycerate, resulting in an increased oxygen affinity. In several species (sheep, cattle, goat) with Met-Leu as the N-terminal group of the beta-chains, the 2,3-bisphosphoglycerate binding sites are deleted in positions beta 1 and beta 2, so that the regulation is phosphate-independent and thus providing a fetal hemoglobin with an increased oxygen affinity. The allosteric control is observed in pigs. In the postembryonal development "adult" hemoglobin with seven contacts (beta-chains) is demonstrated. The increased oxygen affinity is achieved here by a reduced biosynthesis of 2,3-bisphosphoglycerate (Hb greater than P2-glycerate) (Rapoport-Luebering-cycle). The functional control is discussed with respect to the ontogeny of the hemoglobins.     

On the Oxygen Affinity of Bird Blood

In oxygen affinity characteristics bird blood appears to haveseveral features that distinguish it from mammalian blood. Fordomesticated species at least the range of oxygen half saturationvalues is extremely wide. A difference in the shape of the oxygendissociation curve has been recorded by several authors withan increase in sigmoidocity with increasing oxygen saturation.There is evidence that the oxygen affinity determining organicphosphate of bird red blood cells inositol pentaphosphate (IP5)is relatively metabolically inert. This suggests that modulationof blood oxygen affinity is primarily achieved by altering theIP5 hemoglobin interaction rather than varying IP5 levels perse. In contrast to mammals carbon dioxide has no direct effecton whole blood oxygen affinity for some bird species (hen chickgoose) or it may cause the oxygen affinity to increase (pigeonflamingo). Carbon dioxide is a blood oxygen affinity modulatorof some flexibility its effect in both direction and magnitudebeing dependent on the hemoglobin type red cell pH and organicphosphate levels. The physiological significance of these distinguishingfeatures is discussed.     

Oxygen-binding properties of hemoglobins from estivating and active African lungfish.

The oxygen-binding characteristics and the multiplicity of the stripped hemoglobiin from active lungfish Protopterus amphibius, are the same as in specimens that have been estivating for about 30 months, showing that alteration in the hemoglobin molecules is not involved in the earlier reported increase in oxygen affinity of whole blood during estivation (Johansen et al., '76). At pH 7.0 and 26 degrees C the hemolysates show a high oxygen affinity (P50 = 3.1 Torr), a Bohr factor (delta log P50/delta pH) of - 0.33, and a cooperativity coefficient (n) of 1.7. Between 15 and 26 degrees C, the apparent heat of oxygenation (delta H) is - 8.6 Kcal-mole-1 at pH 7.0, corresponding with data for other fish. A low sensitivity of oxygen affinity to urea appears to be adaptive to the high urea concentrations in estivating lungfish. The salt sensitivity is, however, similar to human hemoglobin. The hemoglobin consists of two major (electrophoretically anodal) components, which differ slightly in oxygen affinity but are both sensitive to pH and nucleoside triphosphates (NTP). Guanosine triphosphate (GTP), the major erythrocytic organic phosphate, however, depresses the oxygen affinity of the composite and separated hemoglobins more effectively than ATP suggesting that GTP is the primary modulator of oxygen affinity. Comparative measurements reveal only one major hemoglobin component in P. annectens which has a markedly lower oxygen affinity and phosphate sensitivity than P. amphibius hemoglobins and thus seems less pliable to phosphate-mediated variation in oxygen affinity. The data are discussed in relation to the hemoglobin systems of other fish.     

The oxygen-binding modulation of hemocyanin from the Southern spiny lobster <Emphasis Type="Italic">Palinurus gilchristi</Emphasis>

Arthropod hemocyanins transport and store oxygen and are composed of six subunits, or multiples thereof depending on the species. Palinurus gilchristi hemocyanin is found only as 1 × 6-mers, as normally occurs in spiny lobsters. An alkaline pH and removal of calcium ions induce a wholly reversible dissociation into monomers. The oxygen-binding properties of 1 × 6-meric hemocyanin from P. gilchristi were investigated with respect to pH and modulating effect exerted by calcium, lactate and urate. The oxygen affinity was highly affected by pH in the presence of calcium ions, while in its absence the Bohr coefficient became 60% lower. The protein is insensitive to lactate, but affected by urate which markedly increased hemocyanin–oxygen affinity, acting as the physiological major positive effector. Calcium ions decrease oxygen affinity at low concentration range (0–1 mM), while as concentration becomes higher than 100 mM, the oxygen affinity increases, indicating the presence of two independent types of calcium-binding sites with high and low affinity, respectively. The previous hypothesis, that the presence of high-affinity binding sites in addition to low affinity ones could be a characteristic feature of Palinuran hemocyanins, has been tested by analyzing, with respect to calcium–hemocyanin interaction, three other species belonging to Palinura.     

Maternal-Fetal Oxygen Transfer in Lower Vertebrates

There exists a difference in oxygen affinity between fetal andmaternal bloods in almost all vertebrates examined and thisdifference in affinities probably facilitates oxygen transferto the fetus. It is likely that the high oxygen affinity offetal blood represents a biochemical pre-adaptation from anancestral oviparous embryo for oxygen uptake in a relativelyhypoxic environment. In most cases, the maternal-fetal differencein blood oxygen affinities is due to the characteristics ofthe fetal red cell and not due to any changes in the adult redcell during pregnancy. These characteristics are based on thepresence of a unique fetal hemoglobin with an intrinsicallyhigh affinity for oxygen or on the absence of high red cellconcentrations of organic phosphates—allosteric modulatorsof hemoglobin function. However, in several species of snake,representing different families, it appears that pregnancy isassociated with apronounced decrease in the oxygen affinityof the adult red cell. This suggests that the blood of the pregnantfemale is better able to unload oxygen to the fetus than couldthe blood of thenonpregnant adult. The maternal-fetal differencein blood oxygen affinities in these species isprobably due tothe characteristics of the fetal red cell as well as to thechange in the affinity of the adult cell during pregnancy. Nonetheless,although the magnitude of the pregnancy-associated change inoxygen affinity of the adult cell in these snakes suggests thatit is physiologically significant, the actual significance remainsto be determined.     

Functional characterization of the single hemoglobin of the migratory bird Ciconia ciconia

Hemolysate from white stork displayed a single hemoglobin component, thus resulting into two bands and two globin peaks in dissociating PAGE and reversed phase-HPLC, respectively. Stripped hemoglobin showed an oxygen affinity higher than that of human HbA, a small Bohr effect, and a cooperative oxygen binding. A small decrease of oxygen affinity, of the same extent in all the pH range examined, was observed by addition of chloride, thus indicating an unusual chloride-independent Bohr effect (DeltalogP50/Deltalog pH=-0.24). Saturating amounts of inositol hexakisphosphate, largely decreased hemoglobin-oxygen affinity (DeltalogP(50)=1.17 at pH 7.0), and increased the extent of its Bohr effect (DeltalogP50/DeltalogpH=-0.45). The phosphate binding curve allowed to measure a very high overall binding constant (K=1.18 x 10(5) M(-1)). The effect of temperature on the oxygen affinity was measured, and the enthalpy change of oxygenation resulted almost independent on pH. Structural-functional relationships are discussed by considering some amino acid residues situated at alpha1/beta1 and alpha1/beta2 interfaces, such as alpha38 and alpha89 positions. The presence of only one hemoglobin component, a rare event among birds, and its functional properties have been related to the physiological oxygen requirements of this soaring migrant bird and to its technique of flight during migration.     

Effect of nitric oxide on the transport and release of oxygen in fetal blood

It is well known that nitric oxide (NO), the most important vasodilator agent, plays an important role in lowering vascular resistance in the human umbilical-placental circulation and that its deficiency is related to the pathogenesis of pre-eclamptic disorder. Besides it has recently been demonstrated that human hemoglobin (HbA) is able to transport nitric oxide, as S-nitrosohemoglobin (SNO-Hb), from the arterial to the venous blood. In the present study we examine the functional properties of the adult and fetal nitrosated hemoglobins to see if the double transport of oxygen and NO may influence the fetal oxygenation and the relation between maternal and fetal blood. Our results show that S-nitrosation significantly increases the oxygen affinity of the adult Hb (HbA) with respect to native protein (no-nitrosated) while the functional properties of HbF are less influenced. The oxygen affinity modification, found for SNO-HbA, was ascribed to the nitrosation of cysteine beta 93: really, the same residue is also present in the gamma chains of fetal hemoglobin, while the increase of affinity is less evidenced; hence, it is probable that the 39 aminoacidic substitutions between beta and gamma chains allay the effects of S-nitrosation. As regards the physiological modulators (protons, chloride ions, 2,3-diphosphoglyceric acid, and temperature), they influence the oxygen affinity of the two hemoglobins S-nitrosated, in equal mode with respect to the native forms determining the same variation on the oxygen affinity. Hence, our results evidence the fact that the NO release by SNO-HbA "in vivo" would be limited to regions of extremely low oxygen tension (such as hypoxic regions), while in fetus, SNO-HbF would unload nitric oxide and oxygen at pressure values close to normal.     

Functional aspects of hemoglobin evolution in the mammals

Summary Comparative studies of red cell 2,3 Diphosphoglycerate (DPG) and its effect on hemoglobin oxygen affinity from a taxonomically diverse set of mammals indicate two anomalous groups: members of the superfamilies Bovoidea (Actiodactyla) and Feloidea (Carnivora). In both taxa all of the individuals assayed had very low or unmeasurable quantities of DPG and red cell lysates with little, if any, DPG effect as measured by the change in oxygen affinity in the absence and presence of the phosphate. However, in both groups compensatory changes have occurred in hemoglobin structure and function so as to reduce the native oxygen affinity and thus cause them to resemble the hemoglobins of DPG-utilizing mammals as they occur in the setting of the red cell. We conclude that this parallelism of function is the result of convergent evolution.     

Nitric oxide formation from the reaction of nitrite with carp and rabbit hemoglobin at intermediate oxygen saturations

The nitrite reductase activity of deoxyhemoglobin has received much recent interest because the nitric oxide produced in this reaction may participate in blood flow regulation during hypoxia. The present study used spectral deconvolution to characterize the reaction of nitrite with carp and rabbit hemoglobin at different constant oxygen tensions that generate the full range of physiological relevant oxygen saturations. Carp is a hypoxia-tolerant species with very high hemoglobin oxygen affinity, and the high R-state character and low redox potential of the hemoglobin is hypothesized to promote NO generation from nitrite. The reaction of nitrite with deoxyhemoglobin leads to a 1 : 1 formation of nitrosylhemoglobin and methemoglobin in both species. At intermediate oxygen saturations, the reaction with deoxyhemoglobin is clearly favored over that with oxyhemoglobin, and the oxyhemoglobin reaction and its autocatalysis are inhibited by nitrosylhemoglobin from the deoxyhemoglobin reaction. The production of NO and nitrosylhemoglobin is faster and higher in carp hemoglobin with high O(2) affinity than in rabbit hemoglobin with lower O(2) affinity, and it correlates inversely with oxygen saturation. In carp, NO formation remains substantial even at high oxygen saturations. When oxygen affinity is decreased by T-state stabilization of carp hemoglobin with ATP, the reaction rates decrease and NO production is lowered, but the deoxyhemoglobin reaction continues to dominate. The data show that the reaction of nitrite with hemoglobin is dynamically influenced by oxygen affinity and the allosteric equilibrium between the T and R states, and that a high O(2) affinity increases the nitrite reductase capability of hemoglobin.     

Effect of polyanionic polymers on haemoglobin oxygen-binding properties: application to the synthesis of covalent conjugates with low oxygen affinity

Polyanionic water-soluble polymers containing sulphate, phosphate and polycarboxylate groups were synthesized. These compounds, when simply added to haemoglobin solutions, were shown to lower the affinity of the protein for oxygen. Their influence on oxygen affinity was regarded as the result of a specific interaction of the polymer anionic groups inside the 2,3-diphosphoglycerate-binding site of deoxyhaemoglobin. On the other hand, these polymers were linked to deoxyhaemoglobin to give covalent conjugates also exhibiting an oxygen affinity lower than that of free haemoglobin in the presence of 2,3-diphosphoglycerate, its natural effector, which means that after fixation, the polyanionic polymers are still acting as effectors.     

Effect of Cl- and H+ on the oxygen binding properties of glutaraldehyde-polymerized bovine hemoglobin-based blood substitutes

Bovine hemoglobin was cross-linked with glutaraldehyde, resulting in high oxygen affinity polymeric hemoglobin dispersions of varying molecular weight distributions. High oxygen affinity acellular oxygen carriers were designed in order to exhibit oxygen release profiles closer to that of human red blood cells (RBCs), without exhibiting the inherent increased vasoactivity that occurs with low oxygen affinity acellular oxygen carriers (1, 2). Oxygen dissociation curves were measured for polymerized hemoglobin dispersions at various pH values (7.0, 7.4, and 8.0) and chloride ion concentrations. Unmodified hemoglobin showed an increase in oxygen affinity with increased chloride ion concentration and a decrease in oxygen affinity with increased pH, as was previously demonstrated in the literature (3). For glutaraldehyde-polymerized hemoglobin dispersions, the ability of the oxygen affinity to respond to changes in Bohr H+ and Cl- concentration was weakened. However, at acidic physiological pH (pH = 7), the Bohr effect was still present at high Cl- concentrations. Thus, the Bohr effect maintained some dependency on the Cl- concentration.     

pH effects on the binding of oxygen to non-vertebrate monomeric hemoglobins. A linked function model

Monomeric invertabrate hemoglobins with high oxygen affinity usually contain a tyrosine in the distal region of the heme. This feature has stimulated investigations revealing that one of the properties resulting from the presence of the distal tyrosines is a decreased off rate on the binding of oxygen, thus developing the high affinity. Despite that fact that the pK value of the tyrosine group differs significantly from the groups it replaces little attention has been paid to the pH dependence of the binding of oxygen to the high affinity hemoglobins. Such a pH dependence has been reported on two of the monomeric hemoglobins with relatively low oxygen affinity and one monomeric hemoglobin of intermediate affinity. The pH data of these hemoglobins has been analysed with a linked function model involving the hydrogen ion. pK values required for the low-affinity hemoglobins vary from 4.5 to 7.5. When applied to the high-affinity hemoglobins, the linked function model provides reasonable values for the binding parameters. These pK values vary from 3.0 to 9.0.     

Plant hemoglobins: important players at the crossroads between oxygen and nitric oxide

Plant hemoglobins constitute a diverse group of hemeproteins and evolutionarily belong to three different classes. Class 1 hemoglobins possess an extremely high affinity to oxygen and their main function consists in scavenging of nitric oxide (NO) at very low oxygen levels. Class 2 hemoglobins have a lower oxygen affinity and they facilitate oxygen supply to developing tissues. Symbiotic hemoglobins in nodules have mostly evolved from class 2 hemoglobins. Class 3 hemoglobins are truncated and represent a clade with a very low similarity to class 1 and 2 hemoglobins. They may regulate oxygen delivery at high O₂ concentrations. Depending on their physical properties, hemoglobins belong either to hexacoordinate non-symbiotic or pentacoordinate symbiotic groups. Plant hemoglobins are plausible targets for improving resistance to multiple stresses.     

Binding affinity and biological activity of oxygen and sulfur isosteres at melatonin receptors as a function of their hydrogen bonding capability

Analogues of melatonin (1) and of N-acetyl 5-ethoxytryptamine (3) in which the oxygen atoms are replaced by sulfur have been prepared and tested against human and amphibian melatonin receptors. All sulfur analogues show a decreased binding affinity at human MT1 and MT2 receptors and a reduced potency as melatonin agonists on the Xenopus melanophore assay. The 5-methoxy oxygen of melatonin is significantly more important for receptor binding than the amide oxygen. N-Acetyl 5-ethoxytryptamine shows a decrease in both binding affinity and potency in comparison with melatonin. In this series, replacing either the ethoxy or amide oxygen by sulfur has a similar but smaller effect on both binding affinity and potency. Using K(B)(H) values from Abraham's equations we have assessed the possibility of estimating EC50 values for sulfur isosteres from the EC50 values of their oxygen analogues.     

Unusual oxygen binding behavior of a 24-meric crustacean hemocyanin

Hemocyanins from Crustacea usually are found as 1 × 6 or 2 × 6-meric assemblies. An exception is the hemocyanin isolated from thalassinidean shrimps where the main component is a 24-meric structure. Our analysis of oxygen binding data of the thalassinidean shrimp Upogebia pusilla based on a three-state MWC-model revealed that despite the 24-meric structure the functional properties can be described very well based on the hexamer as allosteric unit. In contrast to the hemocyanins from other thalassinidean shrimps the oxygen affinity of hemocyanin from U. pusilla is increased upon addition of l-lactate. A particular feature of this hemocyanin seems to be that l-lactate already enhances oxygen affinity under resting conditions which possibly compensates the rather low intrinsic affinity observed in absence of l-lactate. The fast rate of oxygen dissociation might indicate that in this hemocyanin a higher cooperativity is less important than a fast response of saturation level to changes in oxygen concentration.     

异常血红蛋白HbQueens及Hb广州—杭州的携氧功能及78—180K的穆斯堡尔谱测定

本实验通过对HbQueens[α_(34)B_(15)Leu→Arg]及Hb广州-杭州[α_(64)E_(13)Asp→Gly]的氧亲合力及它们在78-180K范围的穆斯堡尔谱测定,得到氧亲合力大小顺序为HbQueens>Hb广州-杭州>HbA,由穆斯堡尔谱结果的计算所得的结构参数表明中心Fe~(2+)与卟啉环平面的距离为HbQueens相似文献   

Haemoglobin Rahere (beta Lys-Thr): A new high affinity haemoglobin associated with decreased 2, 3-diphosphoglycerate binding and relative polycythaemia.

A new haemoglobin with increased oxygen affinity, beta82 (EF6) lysine leads to threonine (Hb Rahere), was found during the investigation of a patient who was found to have a raised haemoglobin concentration after a routine blood count. The substitution affects one of the 2, 3-diphosphoglycerate binding sites, resulting in an increased affinity for oxygen, but both the haem-haem interaction and the alkaline Bohr effect are normal in the haemolysate. This variant had the same mobility as haemoglobin A on electrophoresis at alkaline pH but was detected by measuring the whole blood oxygen affinity; it could be separated from haemoglobin A, however, by electrophoresis in agar at acid pH. The raised haemoglobin concentration was mainly due to a reduction in plasma volume (a relative polycythaemia) and was associated with a persistently raised white blood count. This case emphasises the need to measure the oxygen affinity of haemoglobin in all patients with absolute or relative polycythaemia when some obvious cause is not evident.