Postnatal changes in 2,3-diphosphoglycerate (2,3-DPG) content of calf erythrocytes

In 82 calves, 22 adult cows and 10 fetuses the 2,3-DPG level was determined in the erythrocytes. The lowest level was found in cows (1.13 microM/g haemoglobin, on the average), and in calves aged 35 days or more. In the foetuses the mean 2,3-DPG concentration in the erythrocytes was 4.98 microM/g Hb and it was higher that that in the erythrocytes of cows, the oldest foetuses and calves during the first two days of postnatal life. During 5 weeks of postnatal life the changes taking place in 2,3-DPG concentration could be divided into two periods: period I or the period of increase covering the first 6 days of life, with a characteristic rise in the concentration of this component from 1.37 to 15.80 microM/g Hb, and period II or the period of decrease lasting from the 6th to the 35th day of life. In period II two phases could be discerned, the first phase lasting from the 6th to the 10th day with a steep fall of 2,3-DPG level from 15.80 to 4.58 microM/g Hb, and the second phase from the 10th to the 35th day of life in which the level of 2,3-DPG reached slowly the value found in adult cows. A comparison of oxygen affinity of haemoglobin in calves aged 6 days, which was composed of about 80% of fetal haemoglobin and about 20% of adult haemoglobin, and in adult cows, which contained exclusively adult haemoglobin, showed that the oxygen-binding capacity of haemoglobin was lower in calves.     

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     

Relationship between the major phosphorylated metabolic intermediates and oxygen affinity of whole blood in the loggerhead (Caretta caretta) and the Green Sea Turtle (Chelonia mydas) during development.

(1) 2,3-Diphosphoglyceric acid (2,3-DPG) is present in the erythrocytes (RBC) of the 68-day loggerhead turtle embryo and 44-day green sea turtle embryo at levels of 7.4 and 5.5 μmoles/ml of RBC, representing the major organic phosphate during the latter period of embryonic development. (2) Inositol pentaphosphate (IPP) is absent in the red blood cells of the embryos of both the loggerhead and green sea turtle. (3) Near equimolar amounts of 2,3-DPG and IPP are present in the erythrocytes of the adult loggerhead and green sea turtle. The total concentration of these two organic phosphates is approximately 0.75 μmoles/ml of RBC in the adult of both species. (4) There is a switch from embryonic to adult hemoglobin during development of these two species of turtles; the two embryonic bands have identical electrophoretic mobilities, whereas the two adult bands migrate differently on cellulose acetate at pH 8.6. (5) The whole blood oxygen affinity of the adult loggerhead and green sea turtle is 60.3 and 32.6 Torr, respectively. (6) The stripped adult hemoglobins in these two species of turtles show no change in oxygen affinity upon addition of 2,3-DPG, ATP, or IPP. (7) It therefore appears unlikely that whole blood oxygen affinity is controlled by organic phosphate modulation of hemoglobin function in these species of turtles.     

Haemoglobin Hiroshima and the Mechanism of the Alkaline Bohr Effect

HAEMOGLOBIN Hiroshima is a variant with interesting physiological properties^1,2 discovered in a Japanese family. Its Bohr effect is halved, its oxygen affinity at physiological pH increased about three-fold and haem-haem interaction is somewhat reduced compared with normal haemoglobin. In 0.1 M NaCl solutions initially stripped of phosphate, 2,3-diphospho-glycerate (2,3-DPG) diminishes the oxygen affinity as in haemoglobin A (H. F. Bunn, unpublished results). The amino-acid substitution originally deduced for this abnormal haemoglobin was histidine 143 (H21)β ? aspartic acid¹. It was possible to conceive of a mechanism which accounted for its diminished Bohr effect³, but the normal response of its oxygen affinity to 2,3-DPG was inconsistent with the proposed role of histidine 143 in 2,3-DPG binding by haemoglobin A^4,5. An X-ray crystallographic study of deoxyhaemoglobin Hiroshima has now revealed that the replacement occurs not in position 143 but 146β. This was confirmed by chemical methods and the physiological properties of this haemoglobin are now satisfactorily accounted for. The results support the role of histidine 146β in the alkaline Bohr effect⁶.     

Organic phosphates in the red blood cells of fish

Fish are dependent on aerobic metabolism. They respond to changes in oxygen availability by a wide spectrum of compensatory and respiratory adjustments to safeguard tissue oxygenation. Such adjustments are directed to facilitate both oxygen uptake at the gas exchange surfaces and oxygen unloading to tissues. The importance of erythrocytic organic phosphates as regards oxygen transfer has been recognised since 1967 when the 'dramatic' effect of 2,3DPG on human haemoglobin was first reported. The present review examines the appearance of all the major erythrocytic organic phosphates during the evolutionary radiation of fish. In addition, it provides examples illustrating qualitative and quantitative ontogenetic changes of organic phosphates in the red blood cell of several fish species and describes their effects on oxygen affinities. The interaction of the organic phosphates with haemoglobins and divalent cations are also examined. Of particular interest is the regulation of erythrocytic organic phosphates according to both environmental and physiological conditions.     

Effect of organic phosphates on methemoglobin reduction by ascorbic acid.

The rate of methemoglobin reduction by ascorbic acid was accelerated in the presence of ATP,2,3-diphosphoglycerate (2,3-DPG), and inositol hexaphosphate (IHP). The acceleration was as much as three times, four times, and ten times in the presence of ATP, 2.3-DPG, and IHP at pH 7.0, respectively. The changes of the concentrations of methemoglobin and ascorbic acid during the methemoglobin reduction were determined, and the reaction was found to proceed stoichiometrically in the presence of IHP. The reduction rate of methemoglobin by ascorbic acid was compared at different concentrations of organic phosphates (ATP,2,3-DPG, and IHP) at various pH values (6.3, 7.0, 7.7). From the changes in the reduction rate under different concentrations of organic phosphates, the dissociation constants of ATP, 2,3-DPG, and IHP to methemoglobin could be determined and were estimated to be 3.3 X 10(-4) M, 2 X 10(-3) M, and 8 X 10(-6) M at pH 7.0, respectively. On the basis of these results, the acceleration mechanism of methemoglobin reduction by ascorbic acid due to the presence of organic phosphates was described. The physiological role of 2,3-DPG in human red cells was discussed in relation to the reduction of methemoglobin by ascorbic acid.     

Effects of whole body UV-irradiation on oxygen delivery from the erythrocyte

In 16 healthy caucasian volunteers (mean age: 22.2 years) the influence of whole body UV-irradiation on the oxygen transport properties of erythrocytes was investigated. Four hours after irradiation with UV (using the minimal erythema dose, MED) no variation of haemoglobin concentration, hematocrit, mean corpuscular haemoglobin concentration, pH or standard bicarbonate could be found, whereas inorganic plasma phosphate (Pi), calcium, the intraerythrocytic 2,3-diphosphoglycerate (2,3-DPG), the activity of erythrocytic phosphofructokinase (PFK) and pyruvatekinase (PK) increased significantly. The half saturation tension of oxygen (P50-value) tended to increase. The increase of Pi causes--via a stimulation of the glycolytic pathway--an increase in 2,3-DPG concentration and thus results in a shift of the oxygen dissociation curve. It is therefore possible to enhance tissue oxygenation by whole body UV-irradiation.     

The deoxygenation kinetic properties of human fetal hemoglobin: effect of 2,3-diphosphoglycerate

The deoxygenation kinetics of isolated adult and fetal hemoglobin are measured. The results demonstrate that significant functional differences exist between the two tetrameric hemoglobins. It is pointed out that these functional differences closely parallel the differences in similar properties of beta and gamma chains. It is also shown that 2,3-diphosphoglycerate (2,3-DPG) has no significant effect on the deoxygenation rate of fetal hemoglobin. This result appears to be consistent with the reported weaker binding of 2,3-DPG to the oxygen linked groups of fetal hemoglobin.     

Decreased level of 2,3-diphosphoglycerate and alteration of structural integrity in erythrocytes infected with Plasmodium falciparum in vitro

2,3-Diphosphoglycerate (2,3-DPG), an intracellular metabolite of glycolytic pathway is known to affect the oxygen binding capacity of haemoglobin and mechanical properties of the red blood cells. 2,3-DPG levels have been reported to be elevated during anaemic conditions including visceral leishmaniasis. 2,3-DPG activity in P. falciparum infected red blood cells, particularly in cells infected with different stages of the parasite and its relationship with structural integrity of the cells is not known. Chloroquine sensitive and resistant strains of P. falciparum were cultured in vitro and synchronized cultures of ring, trophozoite and schizont stage rich cells along with the uninfected control erythrocytes were assayed for 2,3-DPG activity and osmotic fragility. It was observed that in both the strains, in infected erythrocytes the 2,3-DPG activity gradually decreased and osmotic fragility gradually increased as the parasite matured from ring to schizont stage. The decrease in 2,3-DPG may probably be due to increased pyruvate kinase activity of parasite origin, which has been shown in erythrocytes infected with several species of Plasmodium. The absence of compensatory increase in 2,3-DPG in P. falciparum infected erythrocytes may aggravate hypoxia due to anaemia in malaria and probably may contribute to hypoxia in cerebral malaria. As 2,3-DPG was not found to be increased in erythrocytes parasitized with P. falciparum, the increased osmotic fragility observed in these cells is not due to increased 2,3-DPG as has been suggested in visceral leishmaniasis.     

Functional and computer modelling studies of haemoglobin from horse. The haemoglobin system of the Sardinian wild dwarf horse.

A study was made of the haemoglobin (Hb) system from the Sardinian dwarf horse (Equus caballus jara), one of the last surviving wild horse species in Europe. The oxygen binding properties of the whole haemolysate and of the four different horse Hbs, separated by ion-exchange chromatography, were studied with special regard to the effect of chloride, 2,3-diphosphoglycerate and lactate. Results indicate that no significant functional differences exist between the four Hb components of horse haemolysate. Moreover, the molecular basis of the intrinsically low oxygen affinity and of the weak interaction of horse Hb with 2,3-diphosphoglycerate is discussed in the light of the primary structure of the molecule and of the results of a computer modelling approach. On these bases, it is suggested that the A1 (Thr-->Ser) and A2 (Pro-->Gly) substitutions observed in the beta chains from horse Hb may be responsible for the displacement of the A helix that is known to be a key structural feature of those Hbs that display an altered interaction with 2,3-diphosphoglycerate as compared with human Hb.     

Effect of long-term training and acute physical exercise on red cell 2,3-diphosphoglycerate

A statistically significant 10% increase (p less than 0.005) in mean red cell 2,3-diphosphoglycerate (2,3-DPG) concentration, concomitantly with a mean 16% increase (p less than 0.001) in the predicted maximal oxygen uptake (VO2max) was observed in 29 recruits, who were studied during 6 months of physical training in military service. The increase in 2,3-DPG was higher, the lower the initial 2,3-DPG and VO2max levels. The mean initial 2,3-DPG level was higher in the subjects with a higher initial VO2max. A strenuous but highly aerobic 21-km marching exercise elicited a mean 9% increase (p less than 0.005) in red cell 2,3-DPG concentration. A significantly greater response of 2,3-DPG to marching exercise was observed in subjects with a lower pre-test VO2max than in those with a higher pre-test VO2max. During another more competitive march 2,3-DPG remained almost unchanged and was associated with a tendency towards a negative correlation with the acccompanying lactate response (r = -0.60, p less than 0.05). Red cell 2,3-DPG response to a standardized exercise is considered to be a suitable indicator for evaluating the effect of training on an individual.     

Adaptation to Hypoxia by Increased HbO<Subscript>2</Subscript> Affinity and Decreased Red Cell ATP Concentration

THERE is a decrease in the O₂ affinity of mammalian haemoglobin (Hb) as the levels of 2,3-DPG or ATP are increased, which is explained by an allosteric effect on the HbO₂ binding^1,2. Similar observations on amphibians³ and fish⁴, which have molar ratios of ATP to Hb similar to those of DPG to Hb in mammals, suggest that red cell organic phosphates modulate Hb function in all vertebrates. The adaptation of mammals to various hypoxic stresses involves reduced HbO₂ affinity^5–9, the attendant increase in O₂ “unloading” capacity being mediated by an increase in the concentration of red cell 2,3-DPG. We have found the opposite response in hypoxic fish and suggest that an increased O₂ affinity results in increased O₂ transport for the fish.     

Comparison of carbonic anhydrase activity and other haematological values of two Eutherian and three Australian marsupial mammals

1. Carbonic anhydrase activity and 2,3-diphosphoglycerate (2,3-DPG) concentration were determined in whole blood from humans (Homo sapiens), rabbits (Oryctolagus cuniculus), eastern grey kangaroos (Macropus giganteus), pademelons (Thylogale billardierii) and brush-tailed possums (Trichosurus vulpecula). 2. Marsupial blood carbonic anhydrase activity increased as species body size decreased. 3. T. billardierii haemoglobin was found to have a polymorphism which may be the same (beta 2 = histidine or glutamine) as that of M. giganteus. 4. The concentration of 2,3-DPG int e red cells of T. billardierii was approximately equal to that of the haemoglobin tetramer. Levels of 2,3-DPG in the other species were similar to those previously reported.     

Structure and function of Hb Saint-Jacques (alpha 2 beta 2 140 (H18) Ala----Thr): a new high-oxygen-affinity variant with altered bisphosphoglycerate binding

A low P50 value in a fresh red blood cell suspension was discovered in a polycythemic patient (Hb 19 g X dl-1). Routine acid and alkaline electrophoreses of the hemolysate were identical to normal hemolysate. Isoelectrofocusing (pH gradient 6-8) did not reveal any abnormal band whether performed with the fully liganded or deoxygenated samples. Precise analyses of the oxygen dissociation curves of the propositus' red cells demonstrated a biphasic Hill plot, a normal Bohr effect and low interaction with 2,3-bisphosphoglycerate (2,3-DPG). Studies on the unfractionated hemolysate confirmed these observations and the inhibition of the effect of organic phosphates. Structural studies were carried out on the mixture of beta A + beta X chains and revealed the presence of two beta Tp14 peptides. Sequencing the abnormal beta Tp14 peptide showed the substitution Ala----Thr of the beta 140 (H18) residue. This new variant was named Hb Saint-Jacques. Examination of the three dimensional model of HbAo indicates that the substitution beta 140 (H18) Ala----Thr induces van der Waals interactions with the nearby lysine-82 (EF6) and leucine-81 (EF5) and a displacement of the EF corner of the beta chains. This is likely to change the normal position of the lysine-82 (EF6), a major anionic binding site in the central cavity between the two beta chains. Functional studies confirm the interpretation of a steric hindrance inhibiting the binding of large organic phosphates to Hb Saint-Jacques.     

Age-related changes in glycolysis and levels of modulators of oxygen transport function of hemoglobin in human erythrocytes

The glycolysis rate and the amount of the acid-transport hemoglobin function modulators (diphosphoglycerate (2,3-DPG), ATP, glutathione, chlorides) in human erythrocytes are studied under the effect of age changes. It is shown that the glycolysis rate and the content of ATP decrease under ageing by 16% and 21%, respectively. The concentration of 2,3-DPG lowers insignificantly (by 8%). At the same time it is established that the amount of reduced glutathione in erythrocytes of middle-aged and old people enhances by 25-29% and that of chlorine ions by 23-24%. The revealed changes in the concentration of modulators of the hemoglobin affinity to oxygen with ageing are, probably, one of reasons of the oxyhemoglobin dissociation increase.     

Erythrocyte 2,3-DPG,ATP and oxygen affinity in hemodialysis patients

Patients on a chronic hemodialysis regimen were studied with respect to their erythrocyte adaptation to anemia. Erythrocyte 2,3-diphosphoglycerate (DPG) concentration was suboptimal compared with that of anemic patients who were not uremic. In uremic patients erythrocyte 2,3-DPG correlated poorly with hemoglobin level but more strongly with plasma pH. Differences between observed levels of erythrocyte 2,3-DPG and the values predicted using data from other anemic patients also correlated with pH. Gradual correction of plasma pH with oral sodium bicarbonate resulted in a substantial increase in erythrocyte 2,3-DPG and a decrease in oxygen affinity. Therefore, maintenance of normal pH in uremic subjects may improve tissue oxygenation. On the other hand, the rapid correction of acidosis during dialysis resulted in increased oxygen affinity. This response was due to the direct effect of pH on oxygen affinity in the absence of a significant change in erythrocyte 2,3-DPG or adenosine triphosphate (ATP) during hemodialysis. Erythrocyte ATP but not 2,3-DPG correlated with serum inorganic phosphate in uremic subjects. A 21% reduction of serum phosphate produced by ingestion of aluminum hydroxide gel had no significant effect on these variables.     

Structure-function relationships in the low-affinity mutant haemoglobin Aalborg (Gly74 (E18)beta----Arg).

Haemoglobin Aalborg (Gly74 (E18)beta----Arg) has a reduced oxygen affinity, in both the absence and the presence of organic phosphates; it has a raised affinity for organic phosphates, and it is moderately unstable. By contrast, haemoglobin Shepherds Bush (Gly74 (E18)beta----Asp) has an increased oxygen affinity in both the absence and the presence of organic phosphates, a diminished affinity for organic phosphates and is also unstable. We have determined the crystal structure of deoxyhaemoglobin Aalborg at 2.8 A resolution and compared it to the structures of deoxy- and oxyhaemoglobin A and of deoxyhaemoglobin Shepherds Bush. The guanidinium group of Arg74(E18)beta protrudes from the haem pocket and donates hydrogen bonds to the E and F helices. The carboxylate group of Asp74(E18)beta forms a hydrogen bond only with residue EF6 and is partially buried, which may be why haemoglobin Shepherds Bush appears to be more unstable than haemoglobin Aalborg. To discover why the latter has a low oxygen affinity, we superimposed the B, G and H helices of haemoglobin A, whose conformation is known to be unaffected by ligand binding, on those of haemoglobin Aalborg. This also brought helices E and the haems into superposition, but revealed a shift of the F helix of deoxyhaemoglobin Aalborg towards the EF-corner. This shift is opposite to that which occurs on ligand binding and on transition to the quaternary oxy-structure, and is linked to an increased tilt of the proximal histidine residue away from the haem axis. Since the relative positions of helices E and F and of the haem group are thought to be the main determinants of the changes in oxygen affinity, the shift of helix F may account for the reduced oxygen affinity of haemoglobin Aalborg. The shift may be due to a combination of steric and electrostatic effects introduced by the arginine residue's side-chain. The effects of the arginine and aspartate substitutions at position E18 beta on the 2,3-diphosphoglycerate affinity are equal and opposite. They can be quantitatively accounted for by the electrostatic attraction or repulsion by the oppositely charged side-chains.     

31P-NMR measurements of ATP, ADP, 2,3-diphosphoglycerate and Mg2+ in human erythrocytes

Absolute 31P-NMR measurements of ATP, ADP and 2,3-diphosphoglycerate (2,3-DPG) in oxygenated and partly deoxygenated human erythrocytes, compared to measurements by standard assays after acid extraction, show that ATP is only 65% NMR visible, ADP measured by NMR is unexpectedly 400% higher than the enzymatic measurement and 2,3-DPG is fully NMR visible, regardless of the degree of oxygenation. These results show that binding to hemoglobin is unlikely to cause the decreased visibility of ATP in human erythrocytes as deoxyhemoglobin binds the phosphorylated metabolites more tightly than oxyhemoglobin. The high ADP visibility is unexplained. The levels of free Mg2+ [( Mg2+]free) in human erythrocytes are 225 mumol/l at an oxygen saturation of 98.6% and instead of the expected increase, the level decreased to 196 mumol/l at an oxygen saturation of 38.1% based on the separation between the alpha- and beta-ATP peaks. [Mg2+]free in the erythrocytes decreased to 104 mumol/l at a high 2,3-DPG concentration of 25.4 mmol/l red blood cells (RBC) and a normal ATP concentration of 2.05 mmol/l RBC. By increasing the ATP concentration to 3.57 mmol/l RBC, and with a high 2,3-DPG concentration of 24.7 mmol/l RBC, the 31P-NMR measured [Mg2+]free decreased to 61 mumol/l. These results indicate, that the 31P-NMR determined [Mg2+]free in human erythrocytes, based solely on the separation of the alpha- and beta-ATP peaks, does not give a true measure of intracellular free Mg2+ changes with different oxygen saturation levels. Furthermore the measurement is influenced by the concentration of the Mg2+ binding metabolites ATP and 2,3-DPG. Failure to take these factors into account when interpreting 31P-NMR data from human erythrocytes may explain some discrepancies in the literature regarding [Mg2+]free.     

Diurnal changes of the 2,3-diphosphoglycerate concentration in human red cells and the influence of posture.

The 2,3-diphosphoglycerate (2,3-DPG) concentration, oxygen half saturation pressure at pH 7.4 (P50), pH in plasma and red cells, and mean corpuscular hemoglobin concentration (MCHC) of venous blood were determined during unrestricted daily activity (series I) throughout 24 hrs as well as during prolonged bed rest until noon (series II). In series I almost synchronous dirunal behavior of P50 2,3-DPG, and plasma pH as well as red cell pH became significantly apparent with highest values in the afternoon. The [2,3-DPG] yielded most pronounced alterations, which made up to 13.5% of the average day value. During prolonged recumbency the [2,3-DPG] showed a nonsignificant tendency to decline; the P50 remained unchanged throughout that period. The possible reason for the missing [2,3-DPG] increase is a reduced change of red cell pH in series II. An influence of a posture dependent aldosterone secretion either directly on the 2,3-DPG metabloism of indirectly via mediating the red cell pH and thus ruling the formation of this organic PHOSPHORIS COMPOUND IS DISCUSSED.     

Comparative effects of phosphoenolpyruvate on selected mammalian erythrocytes

1. Incubation of human, rat, cow, sheep, dog, rabbit and monkey erythrocytes with phosphoenolpyruvate (PEP) resulted in increased intracellular 2,3-diphosphoglycerate (2,3-DPG). 2. Physiologic temperature (37 degrees C) and a pH less than 6.5 were required for transport and metabolism of PEP in rat and monkey erythrocytes. 3. Although erythrocytes from all species (except pig) exhibited PEP transport and metabolism, hemoglobin oxygen affinity (HOA) was affected only in species whose hemoglobins are sensitive to 2,3-DPG. 4. These results suggest that the effect of PEP incubation on HOA is mediated through 2,3-DPG.