A comparative study of the temperature dependence of the oxygen-binding properties of mammalian hemoglobins.

The effect of temperature on the oxygen-binding properties of hemoglobin (Hb) from ruminants, such as ox, reindeer, musk ox, mouflon and egyptian water buffalo is compared to that of human adult Hb (HbA). A striking difference emerges where in the presence of chloride ions and in the absence of 2,3-diphosphoglycerate [Gri(2,3)P2] a strongly reduced exothermic oxygenation process is observed for all ruminant Hb investigated with respect to HbA. Next, in the presence of physiological concentrations of Gri(2,3)P2, HbA displays a less exothermic oxygenation process, with values tending toward those observed in ruminant Hb [where Gri(2,3)P2 is not a physiological effector and for which the addition of Gri(2,3)P2 has essentially no effect on the oxygenation enthalpy]. Different from HbA, the intrinsically less exothermic oxygen binding seems to be independent of the experimental conditions for ruminant Hb, underlying specific structural characteristics which might be responsible for this feature.     

Arctic life adaptation--I. The function of reindeer hemoglobin

1. The functional properties of hemoglobin from the reindeer (Rangifer tarandus tarandus L.) are characterized as a function of pH, temperature and organic phosphate concentration. 2. Alongside overall similarities shared with most vertebrate hemoglobins, hemoglobin from the reindeer shows significant differences with respect to the effect of both organic phosphates and chloride anions. 3. The limited effect of temperature on oxygen binding (delta H = -4 kcal/mol O2) could be regarded as an interesting case of molecular adaptation to extreme environmental conditions.     

Spectroscopic properties of the nitric oxide derivative of ferrous man, horse, and ruminant hemoglobins: a comparative study.

The spectroscopic (EPR and absorbance) properties of the nitric oxide derivative of ferrous man, horse, buffalo, deer, mouflon, musk ox, ox, and reindeer hemoglobin (HbNO) have been investigated in the absence of any allosteric effector at pH 6.5 (in 0.1 M 2-[N-morpholino]ethanesulphonic acid/NaOH chloride-free buffer system), as well as at 100 K and/or 20 degrees C. Man and horse HbNO show spectroscopic properties that are generally taken as typical of the high affinity state of ferrous tetrameric Hb's; on the other hand, the spectroscopic properties of ruminant (i.e., buffalo, deer, mouflon, musk ox, ox, and reindeer) HbNO are characteristic of the low affinity conformation. These results are in keeping with the functional properties of the mammalian Hb's considered and have been related to the peculiar low oxygen affinity of ruminant Hb's.     

The primary structure of hemoglobin from reindeer (Rangifer tarandus tarandus) and its functional implications

The primary structures of alpha- and beta-chains of hemoglobin from reindeer (Rangifer tarandus tarandus) were determined. Comparison of the reindeer hemoglobin sequence with those of human and bovine hemoglobins showed 50 and 29 substitutions per alpha beta dimer, respectively. The influence of replacements on the modulation of hemoglobin oxygen affinity by heterothopic ligands and temperature, as well as their importance on the structure-function relationships in hemoglobin are discussed.     

Arctic life adaptation--II. The function of musk ox (Ovibos muschatos) hemoglobin

1. The hemoglobin system from musk ox (Ovibos muschatos) has been characterized from the functional point of view with special regard to the effect of organic phosphates and temperature. 2. The results are similar to those previously obtained in the case of reindeer and confirm that hemoglobins from arctic animals may display very low enthalpy change for the reaction with oxygen. 3. This finding is considered an example of molecular adaptation of respiratory pigments to extreme environmental conditions.     

Temperature modulation of bovine hemoglobins

The functional properties of hemoglobin from Egyptian water buffalo have been characterized as a function of pH, temperature and chloride concentration. Alongside overall similarities shared with ox and Arctic ruminant hemoglobins, hemoglobin from buffalo shows significant differences with respect to the effect of temperature. The results obtained may suggest that the limited effect of temperature on oxygen binding recently reported for ox hemoglobin could be regarded as an interesting case of a reminiscence of a past glacial age.     

A possible new mechanism of oxygen affinity modulation in mammalian hemoglobins

Bovine red cells do not contain appreciable amounts of 2,3-diphosphoglycerate (2,3-DPG). Bovine hemoglobin, however, has a particular sensitivity to chloride ions and as a result it can attain oxygen affinity values lower than those measured for human hemoglobin in the presence of 2,3-DPG. The interaction of bovine hemoglobin with anions is modulated by the hydrophobic characteristics of the protein. Comparison of the hydropathy plots of primate and ruminant hemoglobins indicates constant regions of opposite hydrophobicity, which have fixed amino acid differences. A model is proposed for explaining the regulation of oxygen affinity by chlorides, as an alternative to the classic modulation by 2,3-DPG.     

Studies on avian erythrocyte metabolism. Effect of organic phosphates on oxygen affinity of embryonic and adult-type hemoglobins of the chick embryo.

The effects of 2,3 diphosphoglyceric acid (2,3-DPG), adenosine triphosphate (ATP), and inositol hexaphosphate (IHP) on the oxygen affinity of whole “stripped” hemoglobin (WSH), hemoglobin H (Hb-H), hemoglobin A (Hb-A) and hemoglobin D (Hb-D) isolated from 18-day chick embryo blood have been determined. The effect of the three organic phosphates upon the oxygen dissociation curves is similar and the following order of decreasing oxygen affinity of the organic phosphates was observed for each hemoglobin: 2,3-DPG < ATP < IHP. 2,3-DPG appears to have a slightly greater effect upon the P₅₀ of Hb-H than upon that of either of the two adult-type hemoglobins. However, this effect seems insufficient to suggest a preferential interaction of 2,3-DPG with Hb-H which would account for either the large amounts of 2,3-DPG in the erythrocytes of embryos or the higher oxygen affinity of the whole blood. The effects of the organic phosphates upon the Hill constant of the purified hemoglobins are variable. It is concluded that since the distribution of hemoglobins H, A, and D in the erythrocytes during the developmental period from 18-day embryos to 6-day chicks remains fairly constant, the previously described progressive decrease in oxygen affinity of the whole blood during this period results from changes in the total amount and distribution of the intraerythrocytic organic phosphates.²     

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.     

Spectroscopic and kinetic aspects of Elephas maximus hemoglobin

In comparison with myoglobin and human and Glycera dibranchiata hemoglobins, the heme distal side amino acid exchanges within the heme environment of elephant tetrameric hemoglobin (Hbe) only slightly affect the electronic and ESR spectra of Hbe(III) and Hbe(II) derivatives, several of which were prepared and characterized by optical and ESR spectroscopy. Addition of 2,3-bisphosphoglycerate [Gri(2,3)P2] or inositol hexakisphosphate to Hbe(II)NO causes tension in the Fe-N(proximal His) bond, although the behaviour differs in detail from that of HbA(II)NO. There are two equilibrium states of Hbe having significantly different kinetics for the Hbe(III)----Hbe(II) reaction of Hbe(III)NO. This autoreduction occurs in the form of two parallel processes, which collapse into one intermediate rate in the presence of Gri(2,3)P2. The temperature dependences of the rates enable deduction of delta H0 and delta S0 for the linked equilibrium, and yield linear Eyring plots for Hbe(III)NO, from which activation parameters were estimated on the basis of a previously described mechanism.     

Evolution of ruminant hemoglobins. Thermodynamic divergence of ox and buffalo hemoglobins.

The ligand-binding properties of hemoglobins from two homozygote phenotypes (AA and BB) of water buffalo (Bubalus bubalis) have been characterized by equilibrium and kinetic techniques. In the case of the BB phenotype, the two constituent hemoglobins have been purified and separately analysed. Buffalo hemoglobins display the reduced sensitivity to organic phosphates characteristic of ruminant hemoglobins, their physiological effector probably being the chloride ion. In contrast to the other known hemoglobins from ruminants, all the hemoglobins from the water buffalo display a significant temperature sensitivity, the delta H for oxygen binding in the presence of physiological effectors approaching that of human hemoglobin (delta H = -30.5 kJ/mol O2). This discrepancy with the other ruminant hemoglobins (e.g. ox, delta H = -10.4 kJ/mol O2), whose primary structure is very similar to that of buffalo, hemoglobins might be correlated to the different habitat and phylogenetic history of the two subfamilies (Bos and Bubalus) of Bovidae.     

The sensitivity of hemoglobin oxygen affinity to diphosphoglycerate and the characteristic pH of methemoglobin.

The sensitivity of the oxygen affinity of a hemoglobin to 2,3-diphosphoglyceric acid concentration has been defined as the change in log1/2O2 (deltalogp1/2O2) which results from saturating the hemoglobin with 2,3-diphosphoglyceric acid. The sensitivity varies from one hemoglobin species to another and is linearly rated to the difference in the logarithm of the binding constants of 2,3-diphosphoglyceric acid to deoxy- and oxyhemoglobin, the characteristic pH (pHch), and inversely proportional to the magnitude of the alkaline Bohr effect measured in a saturating amount of 2,3-diphosphoglyceric acid. Its magnitude is higher in large animals than in small animals and varies linearly with the charged amino acid composition of the hemoglobin. The charged amino acid residues must have been selected for in mammals with high metabolic needs and against in animals with low metabolic needs. Variability in the effect of 2,3-diphosphoglyceric acid on the oxygen transport in the different animal hemoglobins must therefore be the result of a positive Darwinian Selection of the charged amino acid residues in their hemoglobins. Furthermore, all the charged groups and not those at the binding site alone, affect the 2,3-diphosphoglyceric acid binding constant of a hemoglobin.     

Methyl acetyl phosphate as a covalent probe for anion-binding sites in human and bovine hemoglobins

An allosteric modulator of oxygen release in human erythrocytes is 2,3-diphosphoglycerate, but bovine erythrocytes apparently utilize chloride for this purpose since they contain little, if any, 2,3-diphosphoglycerate. In order to identify the sites to which these anions bind, the site-specific acetylating agent, methyl acetyl phosphate, has been employed to compete with these allosteric modulators and to mimic their effects on hemoglobin function. With human hemoglobin A, methyl acetyl phosphate competes with 2,3-diphosphoglycerate and acetylates only Val-1(beta), Lys-82(beta), and Lys-144(beta) within or near the cleft that binds this organic phosphate (Ueno, H., Pospischil, M. A., Manning, J. M., and Kluger, R. (1986) Arch Biochem. Biophys. 244, 795). With bovine hemoglobin, the acetylation is competitive with chloride ion. The sites of acetylation in oxy bovine hemoglobin are Met-1(beta) and Lys-81(beta) and for deoxy bovine hemoglobin, they are Val-1(alpha) and Lys-81(beta). Thus, these sites are expected to be involved in the binding of chloride to bovine hemoglobin. Treatment of either human or bovine hemoglobins with methyl acetyl phosphate under anaerobic conditions leads to a lowering of their oxygen affinity and hence the covalent modifier has the same effect on hemoglobin function as the non-covalent regulators, 2,3-diphosphoglycerate and chloride. The Hill's coefficient of hemoglobin is unaffected by treatment with methyl acetyl phosphate. Under aerobic conditions, specifically acetylated bovine hemoglobin also has a lowered oxygen affinity, and human hemoglobin A shows a slight change in its oxygen affinity. In general, bovine hemoglobin is more responsive than human hemoglobin to both chloride and methyl acetyl phosphate; the latter agent results in a permanent covalent labeling of the protein. Therefore, the results support the idea that methyl acetyl phosphate may be a useful probe for deciphering the sites of binding of anions to proteins.     

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.     

Some functional and structural properties of Bufus paracnemis and Pipa pipae hemoglobins

1. Hemoglobin from terrestrial and an aquatic amphibia Bufo paracnemis and Pipa pipae respectively both living in the same region (Belém, Pará) were compared. 2. The number of hemolysate components were determined by starch-gel electrophoresis and CMC-chromatography. P. pipae hemoglobin presented 4 components and B. paracnemis 2, all of anodic mobility. 3. Functional properties of the hemoglobins (oxygen affinity. Bohr effect, carbon monoxide equilibrium) were determined and compared. 4. The differences found in the functional properties were correlated with the different habitat: aquatic or terrestrial for the amphibia. 5. The study of the oxygen functional properties of the hemoglobin showed in the stripped proteins of P. pipae in the presence of ATP, an oxygen affinity (P50 = 7.24 mmHg) that of B. paracnemis at the same pH (P50 = 15.84 mmHg). At high pH the P50 values are different being 15.84 mmHg for the terrestrial frog and 5 mmHg for P. pipae haemoglobin both at pH 8. In addition Bohr effect was noted only in P. pipae hemolysate in the presence of ATP. 6. The CO-equilibrium affinity constant in the presence of ATP are similar in both frogs, of about, log C50 = -6.9. The ratio Pco/Po2 for B. paracnemis hemoglobin was about 300 whereas for that of P. pipae was about 100 only. 7. The kinetic study of reactive sulfhydryl groups in both frog hemoglobin with 4-PDS (specific SH group reagent was used and shown high pseudo-first order constant for B. paracnemis hemoglobin) (k' = 0.46/min) either in presence or not of ATP, compared for that of P. pipae where values were K' = 0.003/min in the stripped protein and k' = 0.014/min in the presence of ATP. 8. Denaturation kinetic studies of the hemoglobin with sodium benzoate was performed and the results were compared with that of Rana catesbeiana, i.e. the pseudo-first order of the hemoglobin denaturation reaction are low for the aquatic P. pipae adult although for the B. paracnemis high molecular resistant was also noted. For P. pipae hemoglobin, nevertheless with ATP such resistance becomes higher. This does not occur with B. paracnemis hemoglobins.     

Interaction of zinc and hemoglobin: binding of zinc and the oxygen affinity.

Stripped human hemoglobin was shown to have a high apparent zinc association constant of 1.3 X 10(7) M-1 with a stoichiometry of one zinc for every two hemes. The saturation of this site produces a dramatic 3.7-fold increase in the oxygen affinity. The effect of zinc on the oxygen affinity is interrelated with the interaction of 2,3-diphosphoglyceric acid (2,3-DPG) and hemoglobin. Thus, a smaller zinc effect is observed in the presence of added 2,3-DPG. Information about the location of the zinc-binding site responsible for the increased oxygen affinity has been obtained by comparing the binding of zinc to various hemoglobins. Blocking the beta93 sulfhydryl group decreases the apparent zinc association constant by an order of magnitude. The substitution of histidine-beta143 in hemoglobin Abruzzo [beta143 (H21) His leads to Arg] and hemoglobin Little Rock [beta143 (H21) His leads to Gln] decreases the apparent zinc association constant by two orders of magnitude. The substitution of histidine-beta143 by other amino acids and the reaction of the beta93 sulfhydryl group are known to produce dramatic increases in the oxygen affinity. The binding of zinc to one or both of these amino acids can, therefore, explain the zinc-induced increase in the oxygen affinity.     

Gas exchange properties of goat hemoglobins A and C

Hypoxic or anemic goats with the A hemoglobin genotype switch to the production of hemoglobin C, resulting in a reduced blood oxygen affinity. However, the physiologic consequences of this switch are not clear. We therefore studied the gas exchange properties of the two hemoglobin types. We found that purified hemoglobins A and C have very similar oxygen affinities and H+ Bohr effects, but in the presence of CO2, the affinity of hemoglobin C is substantially less than that of hemoglobin A. That this is not a nonspecific ionic effect is suggested by identical effects of NaCl on O2 binding to the two proteins and by a 2-fold higher capacity of hemoglobin C to bind CO2. The data can be explained by a class of CO2 binding sites in the beta C chain whose affinity is much higher than that of either of the primary sites or of those in Hb A. Our results suggest that in hemoglobin C-containing red cells CO2 acts as a potent allosteric effector, analogous to the role played by 2,3-diphosphoglycerate in human red blood cells. Goat hemoglobin C may have advantages over hemoglobins A or B in O2 transport under hypoxic conditions or in anemia.     

The hemoglobin system of the brown moray Gymnothorax unicolor: structure/function relationships.

The Gymnothorax unicolor hemoglobin system is characterized by two components, called cathodic and anodic on the basis of their isoelectric point, which were separated by ion-exchange chromatography. The oxygen-binding properties of the purified components were studied in the absence and presence of chloride and/or GTP or ATP in the pH range 6.5-8.0. Stripped cathodic hemoglobin showed a small reverse Bohr effect, high oxygen affinity, and low co-operativity; the addition of chloride only caused a small decrease in oxygen affinity. In the presence of GTP or ATP, the oxygen affinity was dramatically reduced, the co-operativity increased, and the reverse Bohr effect abolished. Stripped anodic hemoglobin is characterized by both low oxygen affinity and co-operativity, and displayed a normal Bohr effect; the addition of chloride increased co-operativity, whereas ATP and GTP significantly modulated oxygen affinity at acidic pH values, enhancing the Bohr effect and giving rise to the Root effect. The complete amino-acid sequences of the alpha and beta chains of both hemoglobins were established; the molecular basis of the functional properties of the hemoglobins is discussed in the light of the primary structure and compared with those of other fish hemoglobins.     

Hemoglobins from bats (Myotis myotis and Rousettus aegyptiacus): a possible example of molecular adaptation to different physiological requirements

The functional properties of the hemoglobin systems from two different species of bat i.e. Rousettus aegyptiacus and Myotis myotis have been studied as a function of chloride, polyphosphates, pH and temperature. Apart from overall similarities shared with most mammalian hemoglobins, the two systems show significant differences with respect to the effect of chloride and temperature sensitivity. These findings have been related to the different physiological needs of the two species.