Changes in erythrocyte 2,3 diphosphoglycerate in women following short term maximal exercise.

15 untrained women were subjected to a walking treadmill test to determine the influence of maximal exercise upon synthesis of erythrocyte 2,3 DPG. Although there was a 9.8% increase in the 2,3 DPG content following exercise, there was a concomitant 9.4% increase in the hemoglobin level; therefore, when 2,3 DPG is expressed as a ratio to hemoglobin (See Article), there was no significant change as a result of exercise stress. It was suggested that three additive factors produced during strenuous exercise; decreased pH; increased hemoglobin concentration; and increased CO2 production result in by-product inhibition of 2,3 DPG synthesis. It is concluded that 2,3 DPG does not provide a physiologic benefit in the adaptation of the oxygen transport system to exercise.     

Diphosphoglycerate mutase and 2,3-diphosphoglycerate phosphatase activities of red cells: comparative electrophoretic study

Erythrocyte diphosphoglycerate mutase (EC 2.7.5.4.) and 2,3-diphosphoglycerate phosphatase (EC 3.1.3.13.) activities of normal human adults, and DPG mutase deficient subject as well as of several animal species were subjected to electrophoretic study on starch gel. In U.V. light 2,3-diphosphoglycerate phosphatase activity was revealed as a band of fluorescence decrease on a fluorescent background, by the oxydation of NADH, whereas diphosphoglycerate mutase appeared as a fluorescent zone. It was found that the electrophoretic pattern of both DPG mutase and 2,3-DPG phosphatase activities was different from one species to the other, but that, in each species, 2,3-DPG phosphatase activity showed the same electrophoretic pattern as DPG mutase activity.     

The effect of thyroxine on the 2,3-diphosphoglycerate content of erythrocytes in vivo and in vitro]

After ending a continous treatment with thyroxine the average dropping of the 2,3 DPG level was 0.4 mumol/ml. T4 decreased on the average by 7.6 microgram/ml. One time application of 1 mg thyroxine p.o. led within 24 hours to an increase of the 2,3 DPG level of -chi = 0.2 mumol/ml, the pH in the erythrocytes increased by 0.02 on the average. Blood incubation with thyroxine added in a concentration of -chi = 24 microgram/100 ml showed no increase of 2,3 DPG, pH and phosphate, while there was a significant acidosis and increase of phosphate in the control blood. The lactate production was significantly lower and glucose consumption was significantly higher in the blood with thyroxine.     

What is the best method to remove 2,3-diphosphoglycerate from hemoglobin?

We have compared the efficiency of four methods to remove 2,3-diphosphoglycerate (DPG) from hemoglobin (Hb), comprising dialysis, repeated ultrafiltration, gel filtration, and ion-exchange chromatography. All the methods eventually yielded hemoglobin solutions with a ratio of <0.002 mol of DPG/mol of Hb₄ and identical oxygen-binding properties but differed with respect to duration and versatility. Considering these factors, we found the combination of an ion-retardation resin and a mixed-bed resin to be most satisfactory.     

Phosphorus-31 relaxation times of 2,3-diphosphoglycerate in intact human erythrocytes

The reciprocals of the spin-lattice relaxation times (T₁s) of the 2-P and 3-P nuclei of 2,3-diphosphoglycerate (DPG) increased linearly as percent DPG bound was raised in model hemoglobin solutions. The 2-P T₁ was slightly greater in intact erythrocytes than in model solutions under similar experimental conditions. The change in the 3-P T₁ with cellular deoxygenation was anomalous indicating that this nucleus should not be used to estimate DPG binding inside intact erythrocytes.     

Binding of beta-naphthyl triphosphate to human adult hemoglobin accompanying deoxygenation. Investigated by simultaneous measurements of fluorescence, absorbance and partial pressure of oxygen

Binding of a fluorescent allosteric effector, beta-naphthyl triphosphate (beta-NapP3), to human adult hemoglobin (HbA) at various levels of oxygen saturation were investigated by simultaneous measurements of fluorescence, absorbance and oxygen partial pressure. Amounts of beta-NapP3 bound to HbA were easily estimated from the fluorescence intensities of HbA solutions, because it was previously proved that the fluorescence of beta-NapP3 bound to HbA is completely quenched. Exchange reactions of the above fluorescent allosteric effector with 2,3-bisphosphoglycerate (DPG) were also examined at various levels of oxygen saturation. It was found that beta-NapP3 binds to deoxyHbA tetramer in the molar ratio of 2:1, and that one of the two beta-NapP3 competes with DPG. It was also found that beta-NapP3 binds to completely oxygenated HbA tetramer in the molar ratio of 1:1, and that the bound beta-NapP3 was not released by adding DPG. The binding affinity of beta-NapP3 for the noncompetitive site of completely oxygenated HbA, to which DPG does not bind, was smaller than that for the noncompetitive site of deoxyHbA, to which DPG also does not bind. Furthermore, the correlations between oxygen bindings by HbA and the bindings of beta-NapP3 to HbA in the intermediate stages of deoxygenation were investigated. It was revealed that HbA as a tetramer exists in three conformational states rather than simple two states as Monod, Wyman, and Changeux had proposed.     

The effect of 2,3-diphosphoglycerate on the solubility of deoxyhemoglobin S

Although highly charged polyanions, such as inositol hexaphosphate, have been clearly shown to decrease the solubility of deoxyhemoglobin S, the effect of 2,3-diphosphoglycerate (DPG), the endogenous allosteric effector within the red cell, has been more controversial. In this work we have compared the effect of DPG on the solubility of native deoxyhemoglobin S and a derivative in which the DPG binding site is blocked by cross-linking the two beta 82 lysine residues. At pH 6.6 and 30 degrees C the solubility of deoxyhemoglobin S was found to be decreased by 15% (i.e., from 18.8 to 16.0 g/dl) in the presence of saturating concentrations of DPG. Under the same conditions DPG had no effect on the solubility of the cross-linked derivative. This result establishes unequivocally that the binding of DPG within the beta cleft directly facilitates the polymerization of deoxyhemoglobin S. Under physiological conditions, the solubility of deoxyhemoglobin S was found to be decreased by 6% in the presence of an equimolar concentration of DPG. A solubility decrease of this magnitude is sufficient to enhance the tendency of SS cells to sickle and may exacerbate the clinical symptoms of sickle cell disease.     

Levels of cyclic-2,3-diphosphoglycerate in Methanobacterium thermoautotrophicum during phosphate limitation.

Batch-grown Methanobacterium thermoautotrophicum cells grew nonexponentially in the absence of exogenous Pi until intracellular cyclic-2,3-diphosphoglycerate (cyclic DPG) had fallen below 2 mumol/g (dry weight), the limit of detection. Growth resumed immediately upon transfer to medium containing Pi Cyclic DPG levels were also below detection in Pi-limited chemostat cultures operating at a dilution rate of 0.173 h-1 (4-h doubling time), with reservoir Pi concentrations below 200 microM. At this dilution rate, the Pi concentration in the culture was 4 microM. An H2-limited steady state was achieved with 400 microM Pi in the inflowing medium (67 microM in the culture). The cyclic DPG content of these cells was 72 to 74 mumol/g, about one-third the amount in batch-grown cells. The specific growth rate accelerated immediately to 0.36 h-1 (1.9-h doubling time) under washout conditions at high dilution rate. The cellular content of cyclic DPG declined over a 2-h period, and then increased rapidly as the Pi level in the medium approached 200 microM. Expansion of the cyclic DPG pool coincided with a marked increase in Pi assimilation. These results indicated that M. thermoautotrophicum accumulated cyclic DPG only when Pi and H2 were readily available.     

An expanded two-state allosteric model for interactions of human hemoglobin A with nonsaturating concentrations of 2,3-diphosphoglycerate

Oxygen binding curves (OEC) for red cell suspensions have a biphasic shape and reduced n50 values when the concentration of 2,3-diphosphoglycerate (DPG) is lowered by aging or experimental procedures. The mechanism for the abnormal shape of the OEC has been related to variations in the activity of free DPG. DPG binds to tetrameric Hb at a single site, and in red cells its normal concentration is equivalent to that of tetrameric Hb. This equivalence renders the oxygen affinity of Hb and the shape of the OEC very sensitive to small changes in the activity of DPG. The OEC for stripped Hb solutions in the presence of nonsaturating concentrations of DPG also exhibit a biphasic shape but with much larger changes in the n values than observed for red cells. Upon addition of chloride, a known competitor of DPG binding to Hb, the shape of the OEC becomes similar to that of red cell suspensions with the same DPG/Hb ratio. Studies on Hb solutions in the presence of varying concentrations of DPG, but without chloride, have revealed that the cofactor shifts the entire OEC to the right, including both its upper and lower asymptotes. This finding indicates that DPG lowers the intrinsic oxygen affinity for both the T and R states. Theoretical considerations leading to a successful modeling of OEC obtained under varying conditions of DPG and chloride require an expanded two-state allosteric model in which allowance is made for DPG-dependent variations in the dissociation constants of oxygen for both the T and R conformations.     

Biochemical parameters to assess viability of blood stored for transfusional use

Blood collected in citrate-phosphate-dextrose-adenine (CPDA-1) containing transfusional bags, was weekly tested throughout a 35 day period. Biochemical assays included plasmatic glucose, electrolytes, free Hb, acid-base balance and hemogasanalysis. Intraerythrocytic ATP and 2,3 DPG were also determined. Results show that an almost total 2,3 DPG depletion occurs during the first three weeks, whereas intracellular ATP are about 50% of the initial values, at the same time. Lowering of pH is also maximal at the third week. pCO2 variation pattern is biphasic: an early increase due to HCO3- titration by lactic acid arising from red cells glycolysis, followed by a decrease probably due to plastic bag permeability to CO2 itself. The percentage of O2Hb also rises during blood storage: this might be the combined result of increase in pO2 and decrease in 2,3 DPG content. A rise of free Hb was obtained; extracellular K+ levels underwent a sixfold increase in 35 days. The mechanism of relative variations of these parameters and the gas transport are discussed. Some of these parameters might be used as routine controls to asses viability and functional status of stored red cells for transfusional use.     

Identification of a Major Locus Contributing to Erythrocyte 2,3-Diphosphoglycerate Variability in Hooded (Long-Evans) Rats

The erythrocyte glycolytic intermediate 2,3-diphosphoglycerate (DPG) and adenosine triphosphate (ATP) play an important role in oxygen transport and delivery by binding to hemoglobin (Hb) and reducing its affinity for oxygen. Considerable quantitative variability in the levels of DPG and ATP exists in human populations and in a population of hooded (Long-Evans) rats we have studied. This paper presents the results of studies on the genetic component of DPG-level variation in an outbred population of hooded rats. Beginning with about 100 rats, a two-way selection experiment was initiated. Pairs of rats with the highest DPG levels were mated to produce a High-DPG rat strain and animals with the lowest DPG levels were mated to produce a Low-DPG strain. Mean DPG levels responded rapidly to selection and, from generation 3 on, the differences between strain means were highly significant. Ten High-DPG strain rats were intercrossed with 10 Low-DPG strain rats of generation 10 to produce an F₁ generation in which the DPG levels were almost as high as those of High-DPG animals. This indicates partial dominance of High-DPG alleles. The F₂ DPG-level distribution showed two distinct subpopulations. The high DPG subpopulation contained three times as many animals as the low DPG subpopulation. From these results and the statistical analyses performed, it was concluded that the DPG differences between strains were due to an allelic difference at one major locus, the allele carried by the High-DPG strain showing partial dominance over the allele carried by the Low-DPG strain. It appears that this locus may also effect ATP levels to a large extent and is polymorphic in hooded rat populations. Identification of this locus gives us a useful tool for studies of the physiological effects of DPG variability, as well as providing an example of a major gene effect in a quantitatively varying trait.     

Hemoglobin function in stored blood, XVII. Maintenance of red cell 2,3 DPG (function) and ATP (viability) for six weeks in ACD or CPD-adenine-inosine-methylene blue.

Blood preservatives containing adenine for six week storage have been prepared with inosine and methylene blue at various pH levels in order to maintain, 23-DPG levels for immediate oxygen transport upon transfusion. In one experiment, the adverse effect of a high pH on ATP maintenance was demonstrated in the presence of methylene blue and inosine. In this and other experiments it was clear that ATP was better maintained in low pH preservatives and DPG better maintained in higher pH preservatives. However, 2,3-DPG levels were kept from falling with CPD-adenine-inosine over a wide range of pH values. A CPD-adenine-inosine preservative at a pH 5.8 maintained normal DPG levels for three weeks of storage. A similar preservative but with a pH of 6.6 maintained normal DPG levels for 35 days of storage. It is suggested that if all blood bank units are going to have normal DPG levels for optimal oxygen transport at the time of transfusion then a CPD preservative with a higher pH and/or metabolic nutrients and regulators such as inosine or methylene blue would be required.     

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.     

Simultaneous determination of low free Mg2+ and pH in human sickle cells using 31P NMR spectroscopy

The concentrations of free magnesium, [Mg(2+)](free), [H(+)], and [ATP] are important in the dehydration of red blood cells from patients with sickle cell anemia, but they are not easily measured. Consequently, we have developed a rapid, noninvasive NMR spectroscopic method using the phosphorus chemical shifts of ATP and 2,3-diphosphoglycerate (DPG) to determine [Mg(2+)](free) and pH(i) simultaneously in fully oxygenated whole blood. The method employs theoretical equations expressing the observed chemical shift as a function of pH, K(+), and [Mg(2+)](free), over a pH range of 5.75-8.5 and [Mg(2+)](free) range 0-5 mm. The equations were adjusted to allow for the binding of hemoglobin to ATP and DPG, which required knowledge of the intracellular concentrations of ATP, DPG, K(+), and hemoglobin. Normal oxygenated whole blood (n = 33) had a pH(i) of 7.20 +/- 0.02, a [Mg(2+)](free) of 0.41 +/- 0.03 mm, and [DPG] of 7.69 +/- 0.47 mm. Under the same conditions, whole sickle blood (n = 9) had normal [ATP] but significantly lower pH(i) (7.10 +/- 0.03) and [Mg(2+)](free) (0.32 +/- 0.05 mm) than normal red cells, whereas [DPG] (10.8 +/- 1.2 mm) was significantly higher. Because total magnesium was normal in sickle cells, the lower [Mg(2+)](free) could be attributed to increased [DPG] and therefore greater magnesium binding capacity of sickle cells.     

Effect of Salmonella minnesota R595 LPS on the dipalmitoylphosphatidyl-ethanolamine (DPPE)-dipalmitoylglycerol (DPG)-water model membrane system

The effect of Salmonella minnesota R595 lipopolysaccharide (LPS) on model membrane consisting of a mixture of fully hydrated lipids (dipalmitoylphosphatidylethanolamine (DPPE) and dipalmitoylglycerol (DPG)) was investigated by differential scanning calorimetry (DSC), small-angle X-ray scattering (SAXS) and freeze–fracture methods. The DPPE–DPG/water system forms a multilamellar arrangement in the gel phase which transforms into a mixture of inverted hexagonal and cubic structures. By the presence of LPS the thermotropic behaviour of the system was affected significantly only at its high concentration (1:1 mol/mol LPS/DPPE–DPG) in the gel phase, while above the chain melting transition the ratio of the inverted cubic and the hexagonal structures was changed and at the 1:1 mol/mol LPS/DPPE–DPG ratio a complex and amorphous phase was formed. The structural parameters of the inverted hexagonal and cubic phases are modified by the temperature and also by the LPS concentration, as deduced from the characteristic SAXS curves. Summarizing the effects of the LPS molecules on the DPPE–DPG/water vesicle system a schematic phase diagram was constructed.     

Thermodynamics of 2,3-diphosphoglycerate association with human oxy- and deoxyhemoglobin

The association of 2,3-diphosphoglycerate with oxy- and deoxyhemoglobin was studied by means of ultrafiltration and microcalorimetry. It was found that in addition to parameters that are known to influence the binding of 2,3-diphosphoglycerate to both species of hemoglobin (such as pH, temperature and concentration of competing anion), the association is also strongly dependent on the hemoglobin concentration. The difference between the apparent association constants for the formation of the complex of the organic phosphate with oxy- and deoxyhemoglobin is relatively small. At pH 7.3, 25° C and 0.154 M chloride this difference is only 0.6 kcal/mole of free energy favoring the Hb·DPG complex. This free energy difference increases with decreasing pH but is not strongly affected by hemoglobin concentration. The enthalpy change for the formation of the 2,3-diphosphoglycerate complex with deoxyhemoglobin is 8–10 kcal/mole more exothermic than the complex with oxyhemoglobin.     

Erythrocyte phosphofructokinase in rat strains with genetically determined differences in 2,3-diphosphoglycerate levels

We have studied the erythrocyte enzyme phosphofructokinase (PFK) from two strains of Long-Evans rats with genetically determined differences in erythrocyte 2,3-diphosphoglycerate (DPG) levels. The DPG difference is due to two alleles at one locus. With one probable exception, the genotype at this locus is always associated with the hemoglobin (Hb) electrophoretic phenotype, due to a polymorphism at the ^III-globin locus. The enzyme PFK has been implicated in the DPG difference because glycolytic intermediate levels suggest that this enzyme has a higher in vivo activity in High-DPG strain rats, although the total PFK activity does not differ. We report here that partially purified erythrocyte PFK from Low-DPG strain cells is inhibited significantly more at physiological levels of DPG (P<0.01) than PFK from High-DPG strain erythrocytes. Citrate and adenosine triphosphate also inhibit the Low-DPG enzyme more than the High-DPG enzyme. Therefore, a structurally different PFK, with a greater sensitivity to inhibitors, may explain the lower DPG and ATP levels observed in Low-DPG strain animals. These data support a two-locus (Hb and PFK) hypothesis and provide a gene marker to study the underlying genetic and physiologic relationships of these loci.This investigation was supported in part by Grant AM 14898, National Research Service Award 5 F 32 AM 05418, and Biochemical Research Support Grant 5 S07 RR 05551 from the National Institutes of Health.     

Association in Long-Evans hooded rats of red cell 2,3-diphosphoglycerate levels with hemoglobin types

Two sublines of commercially available Long-Evans hooded rats have been developed by genetic selection. These sublines have widely differing levels of erythrocyte 2,3-diphosphoglycerate (DPG) due to different alleles at a single genetic locus. In the present work, it is shown that rats from the commercial population are also polymorphic at a hemoglobin locus, probably involving two alleles of the ^III-globin chain locus. Particular hemoglobin types have been found to be strongly associated with certain DPG types, not only in the high-DPG and low-DPG lines but also in the commercial population. Two explanations for this association are considered. One is a single-locus hypothesis, with hemoglobin allelic variation causing DPG variation, and the other is a two-locus hypothesis, with marked linkage disequilibrium.This work was supported by a Michigan Heart Association Grant, by the Meyers Foundation, and by NIH Training Grant (5 T01-GM-0071).     

Pressure increases oxygen affinity of whole blood and erythrocyte suspensions

Effect of hydrostatic pressure (HP) on whole blood (WB) or erythrocyte suspension hemoglobin (Hb) O2 affinity has been studied using newly developed techniques. O2 partial pressure at which hemoglobin is half-saturated with O2 (P50) measurements were made at 5 HP (1, 26, 51, 76, and 126 ATA) on thin films of human WB or erythrocytes at 37 degrees C. CO2 partial pressure of WB was either 28 or 57 Torr (film pH 7.51 or 7.31). HP increased affinity of erythrocytes and WB. For erythrocytes in tris(hydroxymethyl)aminomethane buffer, the ratio (r) of P50 (1 ATA)/P50 (51 ATA) was 1.089 (P less than 0.01) at pH 7.0. WB P50 decreased with HP at a rate of -3.3 X 10(-2) Torr X atm-1; change in P50 at higher HP vs. 1 ATA was highly significant (P less than 0.01). No effect of HP was seen on the CO2 Bohr coefficient. Inert gas choice, N2 vs. helium (He), had no effect. Measurement of decrease of P50 with HP at 76 ATA in hemolyzed WB gave an r of 1.15, as great or greater than that found in WB, indicates that Donnan equilibrium alteration is not involved. No effect of HP was found in WB on the ratio of P50 of erythrocytes with normal (5 mmol/l erythrocytes) 2,3-diphosphoglycerate (DPG) to P50 of erythrocytes with less than 5% of normal DPG; i.e., no effect of pressure was seen on the independent influence of DPG on P50. WB measurements of Hb O2 uptake under simulated physiological conditions are characterized by a net decrease in partial molal volume on oxygenation of 30-35 ml/mol Hb4.     

Hemoglobin affinity for 2,3-bisphosphoglycerate in solutions and intact erythrocytes: studies using pulsed-field gradient nuclear magnetic resonance and Monte Carlo simulations.

The diffusion coefficient (D) of 2,3-bisphosphoglycerate (DPG) was measured using pulsed-field gradient (PFG)-31P nuclear magnetic resonance spectroscopy in solutions containing 2.7-5.0 mM hemoglobin (Hb) and a range of DPG concentrations. The dependence of the measured values of D on the fraction of the total DPG in the sample that is bound to Hb enabled the estimation of the dissociation constants (Kd) of complexes of DPG with carbonmonoxygenated, oxygenated, and deoxygenated Hb; the values of Kd (mM), measured at 25 degrees C, pH 6.9 and in 100 mM bis Tris/50 mM KCl, were 1.98 +/- 0.26, 1.8 +/- 0.5 and 0.39 +/- 0.26, respectively. In intact erythrocytes the apparent diffusion coefficient, Dapp, of DPG was larger in oxygenated and carbonmonoxygenated cells (6.17 +/- 0.20 x 10(-11) m2s-1) than in deoxygenated cells (4.10 +/- 0.23 x 10(-11) m2s-1). Changes in intracellular DPG concentration (5-55 mM) in erythrocytes, brought about by incubation in a medium containing inosine and pyruvate, did not result in significant changes in the value of Dapp; this result supports the hypothesis that DPG binds to other sites in the erythrocyte. Monte Carlo simulations of diffusion in biconcave discs were used to test the adequacy of the values of Kd estimated in solution to describe the binding of DPG to Hb in oxygenated and deoxygenated erythrocytes. The results of the simulations implied that the value of Kd estimated for deoxygenated Hb-DPG was greater than expected from the experiments involving intact erythrocytes. This difference is surmised to be at least partly due to the difficulty of measuring D at low-ligand concentrations. Notwithstanding this shortcoming, the PFG method appears to be suitable for probing interactions between macromolecules and ligands when the Kd is in the millimolar range. It is one of the few techniques available in which these interactions can be studied in intact cells. In addition, the Monte Carlo simulations of the diffusion experiments highlighted important differences between theory and experiment relating to the nature of molecular motion inside the cells.