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.     

Effect of 2,3-diphosphoglycerate concentration on the alkaline fragility of phosphofructokinase-deficient canine erythrocytes

1. Erythrocytes in whole blood samples from dogs with phosphofructokinase (PFK) deficiency had lower 2,3-diphosphoglycerate (2,3-DPG) concentrations, higher ATP concentrations, and were more alkaline fragile than normal canine erythrocytes. 2. Reticulocytes from a PFK-deficient dog contained nearly three times the ATP concentration of normal canine erythrocytes, and had 2,3-DPG concentrations similar to normal canine erythrocytes. 3. PFK-deficient reticulocytes are not alkaline fragile. 4. The erythrocyte 2,3-DPG concentration in whole blood samples from PFK-deficient dogs was increased to normal by in vitro incubation with dihydroxyacetone, pyruvate and phosphate. This incubation resulted in only a slight increase in ATP concentration. 5. The alkaline fragility of these 2,3-DPG replenished PFK-deficient erythrocytes was normal. 6. Findings in this study indicate that the increased alkaline fragility of canine PFK-deficient erythrocytes is the result of decreased intracellular 2,3-DPG concentration.     

Control of glycolysis in magnesium deficiency: studies on intact red cells and hemolysates]

The behaviour of glycolytic flux and glycolytic metabolic concentrations was studied under conditions of magnesium deficiency. The Mg-deficiency was produced in whole animals (rats) by feeding a diet almost completely free of Mg and in hemolysates of men by the addition of a chelating agent. The results show that the decrease of the free Mg-level is diminished by partial destruction of ATP and 2,3-DPG. The analysis of the control strength of the overall flux leads to the conclusion that the decrease of the glycolytic rate is caused by an inhibition of the hexokinase-phosphofructokinase-control system. The decrease of the MgATP-Complex and free Mg++-level explains the diminished phosphorylation of glucose by the hexokinase. The ATP-inhibition of the phosphofructokinase is amplified by a small increase of free ATP-concentration and a simultaneous decrease of the Fru-6P-level. The increase of the PEP-level is caused by the diminished free Mg++ and MgATP-complex and does not demonstrate a larger control strength of the pyruvate kinase.     

Effects of training on erythrocyte 2,3-diphosphoglycerate in normal men

The erythrocyte 2,3-diphosphoglycerate concentration (2,3-DPG) and the activity of red cell hexokinase, pyruvate kinase, glucose-6 phosphate dehydrogenase and glutathione reductase were studied in 27 normal volunteers before and after 2 and 4 months of physical endurance training. The 4 months of training increased maximal oxygen uptake and physical working capacity (PWC130) by 16% (p less than 0.001) and 29% (p less than 0.001) respectively. Resting heart rate was decreased (p less than 0.001) by 11 beats.min-1. With 2 months of training the erythrocyte 2,3-DPG concentration increased by 9% (p less than 0.001); with 4 months training the increase was only 4% (p less than 0.05). The training-induced increase in red cell 2,3-DPG was not accompanied by enhanced activity of erythrocyte hexokinase, pyruvate kinase, glucose-6 phosphate dehydrogenase or glutathione reductase. It is concluded that the rise in red cell 2,3-DPG induced by physical endurance training is not due to activation of red cell glycolytic enzymes or the enzymes involved in the pentose-phosphate cycle.     

Effect of intracellular magnesium and oxygen tension on K+-Cl- cotransport in normal and sickle human red cells.

In red cells from normal individuals (HbA cells), the K+-Cl- cotransporter (KCC) is inactivated by low O2 tension whilst in those from sickle cell patients (HbS cells), it remains fully active. Changes in free intracellular [Mg2+] have been proposed as a mechanism. In HbA cells, KCC activity was stimulated by Mg2+ depletion and inhibited by Mg2+ loading but the effect of O2 was independent of Mg2+. At all [Mg2+]is, the transporter was stimulated in oxygenated cells, minimally active in deoxygenated ones. By contrast, the stimulatory effects of O2 was abolished by inhibitors of protein (de)phosphorylation. HbS cells had elevated KCC activity, which was of similar magnitude in oxygenated and deoxygenated cells, regardless of Mg2+ clamping. In deoxygenated cells, the antisickling agent dimethyl adipimidate inhibited sickling, Psickle and KCC. Results indicate a role for protein phosphorylation in O2 dependence of KCC, with different activities of the relevant enzymes in HbA and HbS cells, probably dependent on Hb.     

Refinement and evaluation of a model of Mg2+ buffering in human red cells.

The total Mg2+ content of human red cells ([Mg]T,i) is partitioned between free and bound forms. The main cytoplasmic Mg2+ buffers are ATP and 2,3 bisphosphoglycerate. Haemoglobin binds free ATP and bisphosphoglycerate, preferentially in the deoxygenated state. Thus, the free ionized Mg2+ concentration ([Mg2+]i) oscillates with the oxy-deoxy condition of the cells. The binding reactions are also modulated by the pH changes that accompany the oxygenation-deoxygenation transitions. The complex interactions between Mg2+, its ligands and Hb can be encoded in a set of equilibrium equations representing all the known binding reactions of the system. To develop a comprehensive understanding of the Mg2+ homeostasis of intact red cells it is necessary to correct and refine the equations and parameters of the model by systematic comparisons between model predictions and measured cytoplasmic Mg2+ buffering curves under a variety of experimental conditions. Earlier models largely underestimated total Mg2+ binding in intact cells. We carried out experiments in which [Mg]T,i and [Mg2+]i were controlled over a wide range ([Mg]T,i between 0.1 and 23 mM) by the use of the ionophore A23187, under diverse metabolic conditions, and the results were used to interpret the adjustments required for good model fits. By the inclusion of low-affinity Mg2+ binding to ATP and bisphosphoglycerate, and also binding of Mg2+ to haemoglobin (four ions per tetramer) with an apparent dissociation constant of 45 mM we were able to realistically model, for the first time, all the experimentally observed changes in [Mg2+]i in human red cells under diverse metabolic conditions.     

Effect of irradiation and/or leucocyte filtration on RBC storage lesions

Red blood cell (RBC) storage lesions have been shown to be associated with some adverse reactions; numerous studies have focused on the lesions caused by storage, and few data on the RBC storage lesions caused by prestorage treatments of leucocyte filtration and irradiation. In this study, we examined the changes related with the RBC storage lesions, including 2,3-diphosphatidylglyceric acid (2,3-DPG), pH, free hemoglobin (Hb), supernatant free K+ and Na+ concentration, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH). Along with the increasing storage time, decreases in 2, 3-DPG levels, pH and Na+ concentration, increases in K+ and free Hb concentrations, and significant morphological changes in RBC in all groups were found. The changes in the groups of irradiation, leucocyte filtration and the combined irradiation and leucocyte filtration were more significant than those in the untreated group. Meanwhile, the MCV levels of the three treated groups were significantly lower than those in the untreated group, while the MCH variations were significantly higher. Our results suggest that irradiation and leucocyte filtration before storage may aggravate blood storage lesions.     

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.     

Biochemical mechanisms of red blood cell 2,3-diphosphoglycerate increase at high altitude

Red blood cell 2,3 diphosphoglycerate (2,3-DPG) levels increase after ascent to high altitude. Studies were undertaken to identify the biochemical mechanisms responsible for eliciting the 2,3-DPG response in several types of subjects. These included (1) short-term exposure to 3400 m in ten subjects; (2) exposure to 4300 m in an additional ten subjects; (3) studies in 28 high-altitude normal residents of 3100 m; and (4) studies in 28 high-altitude residents with chronic mountain polycythemia. Controls were 41 residents of 240 m. Regression analysis identified the glycolytic variables, termed “key variables,” on which variation in 2,3-DPG levels was dependent (P < .05). Key variables common to the short-term studies were glucose-6-phosphate, phosphoenolpyruvate, and the ratio of the levels of adenosine diphosphate to adenosine triphosphate. The positions of these key variables in the glycolytic pathway and their mean levels suggest erythrocyte hexokinase and pyruvate kinase activation as possible enzymatic mechanisms. Key variables unique to the 3400 m study suggested phosphofructokinase activation also acted to increase 2,3-DPG levels. 2,3-DPG levels in the normal 3100 m residents were not different from low-altitude values, and 2,3-DPG levels in these samples did not appear to be dependent on any of the glycolytic variables examined. Among the high-altitude residents with polycythemia, higher 2,3-DPG levels were dependent on glucose-6-phosphate, fructose diphosphate, dihydroxyacetone phosphate, and the ratio of adenosine diphosphate to adenosine triphosphate levels. The positions of these variables in the glycolytic pathway and their mean levels suggested activation of the hexokinase and phosphofructokinase enzymes.     

Adaptation of red cell enzymes and intermediates in metabolic disorders.

The metabolic activity of the red cell glycolytic pathway hexose monophosphate shunt (HMP) with dependent glutathione system was studied in patients with hyperthyroidism (n = 10), hyperlipoproteinemia (n = 16), hypoglycemia (n = 25) and hyperglycemia (n = 23). In uncontrolled diabetics and patients with hyperthyroidism the mean value of glucose phosphate isomerase (GPI), glucose-6-phosphate dehydrogenase (G-6-PD), glutathione reductase (GR) was increased, whereas these enzyme activities were reduced in patients with hypoglycemia. Apart from a few values of hexokinase (HK) which were lower than normal the results in hyperlipoproteinemia patients remained essentially unchanged, including the intermediates such as 2,3-diphosphoglycerate (2,3-DPG), adenosine triphosphate (ATP) and reduced glutathione (GSH). While increased rates of 2,3-DPG and ATP in hypoglycemia patients were obtained, these substrates were markedly reduced in diabetics.     

Regulation of red cell 2,3-DPG and Hb-O2-affinity during acute exercise

Reports from the literature and our own data on red cell 2,3-DPG and its importance for unloading O2 from Hb to the tissues during exhaustive exercise are contradictory. We investigated red cell metabolism during incremental bicycle ergometry of various durations. Furthermore changes in blood composition occurring during exercise were simulated under in vitro conditions. The effect of a moderate (11.2 mmol X l-1 lactate, pH = 7.127) and severe (18 mmol X l-1 lactate, pH = 6.943) lactacidosis on red cell 2,3-DPG concentration was compared with the effect of similar acidosis induced by HCl. Our data indicate that the concentration of 2,3-DPG in red cells depends on the degree of lactacidosis, but not on the duration of exercise. During moderate lactacidosis red cell 2,3-DPG remains unchanged. This can be explained by an interruption of red cell glycolysis on the PK and GAP-DH step caused by a lactate and pyruvate influx into the erythrocyte, as well as an intraerythrocytic acidosis and a drop in the NAD/NADH ratio. During severe lactacidosis and HCL-induced acidosis a decrease in 2,3-DPG due to an inhibition of 2,3-DPGmutase and other glycolytic enzymes can be found. Mathematical correction of the observed P-50 value for the decrease in 2,3-DPG occurring during severe lactacidosis showed that a decrease in Hb-O2-affinity during strenuous exercise depends on the degree of lactacidosis and temperature elevation.     

Red cell 2,3-DPG, ATP, and mean cell volume in highly trained athletes. Effect of long-term submaximal exercise

20 male elite long distance runners were compared to a control group of blood donors to determine the effect of training on red blood cells. The acute effects of exercise on red cells were investigated in 11 of the runners following a race of 15-30 km. The runners had elevated resting values of red cell 2,3-DPG (P less than 0.05) and mean cell volume (P less than 0.01); blood Hb and ATP were not different from concentrations in the control group. The red cell status of the athletes may be explained by an increased proportion of young erythrocytes in runners. No statistically significant changes in red cell 2,3-DPG, ATP, mean cell volume or blood Hb were found post exercise.     

Regulatory properties of human erythrocyte hexokinase during cell ageing

Human red blood cell hexokinase exists in multiple molecular forms with different isoelectric points but similar kinetic and regulatory properties. All three major isoenzymes (HK Ia, Ib, and Ic) are inhibited competitively with respect to Mg.ATP by glucose 6-phosphate (Ki = 15 microM), glucose 1,6-diphosphate (Ki - 22 microM), 2,3-diphosphoglycerate (Ki = 4 mM), ATP (Ki = 1.5 mM), and reduced glutathione (Ki = 3 mM). All these compounds are present in the human erythrocyte at concentrations able to modify the hexokinase reaction velocity. However, the oxygenation state of hemoglobin significantly modifies their free concentrations and the formation of the Mg complexes. The calculated rate of glucose phosphorylation, in the presence of the mentioned compounds, is practically identical to the measured rate of glucose utilization by intact erythrocytes (1.43 +/- 0.15 mumol h-1 ml red blood cells-1). Hexokinase in young red blood cells is fivefold higher when compared with the old ones, but the concentration of many inhibitors of the enzyme is also cell age-dependent. Glucose 6-phosphate, glucose 1,6-diphosphate, 2,3-diphosphoglycerate, ATP, and Mg all decay during cell ageing but at different rates. The free concentrations and the hemoglobin and Mg complexes of both ATP and 2,3-diphosphoglycerate with hemoglobin in the oxy and deoxy forms have been calculated. This information was utilized in the calculation of glucose phosphorylation rate during cell ageing. The results obtained agree with the measured glycolytic rates and suggest that the decay of hexokinase during cell ageing could play a critical role in the process of cell senescence and destruction.     

Purification and regulatory properties of pigeon erythrocyte pyruvate kinase

1. Pigeon erythrocyte pyruvate kinase (PK) was purified 22,000 fold by successive column chromatography on Sephadex DEAE A-50 and Red Agarose. The resulting enzyme preparation had a specific activity of 815.3 U/mg protein and an overall yield of 18.5%. 2. The molecular weight, as determined by gel filtration on Sephadex G-200 was 152,000. 3. Isoelectric focusing in the pH range of 3-10 showed that pigeon erythrocyte contained at least 3 PK isozymes with isoelectric points of 5, 5.7 and 6. 4. The variation of activity of PK at various ADP and phosphoenolpyruvate (PEP) concentrations was studied. The Km values for ADP and PEP were 0.40 and 0.46 mM respectively. 5. The enzyme was activated by FDP, and inhibited by ATP, highly phosphorylated inositol derivatives and 2,3-DPG: 6. It was activated by K+ and Mg2+ ions. 7. Phosphorylated hexoses and Pi stimulated the activity of PK. 8. The regulatory role of PK of pigeon erythrocytes, which lack the typical 2,3-DPG bypass, is discussed.     

The effect of increasing glucose in CPD on the quality of stored blood

Changes in quality of blood units containing one and a half or double amounts of glucose, stored at +4 degrees C for three weeks were analysed. An experimental preservative containing glucose and fructose (1 : 1) was also used. No other additives (purine or purine-nucleoside) were applied. A standard CPD preservative of the National Inst. of Haematology and Blood Transfusion was used as control. The pH, plasma free haemoglobin, K+ content, red blood cell (RBC) ATP and 2,3-DPG content, and RBC fragility index were determined in each sample. Increase of glucose concentration, the addition of fructose had a beneficial effect on blood pH, and on plasma free haemoglobin and K+ concentration. 150% glucose improved the 2,3-DPG maintenance in stored blood.     

Organic phosphate binding to hemoglobin in intact human erythrocytes determined by 31P nuclear magnetic resonance spectroscopy.

Phosphorus nuclear magnetic resonance (31P NMR) spectroscopy was used to estimate the percent of 2,3-diphosphoglycerate and ATP bound to hemoglobin in intact human erythrocytes at 37 degrees C. Binding was assessed by comparing the chemical shifts (delta) of 2,3-diphosphoglycerate and of ATP observed in intact cells with the delta values of these organic phosphates determined in model solutions closely simulating intracellular conditions, in which percent binding was directly evaluated by membrane ultrafiltration. The results showed that the percent of bound 2,3-diphosphoglycerate in intact cells varied with pH, the state of oxygenation, and 2,3-diphosphoglycerate concentration. The values ranged from 33% in cells incubated with glucose in air at an intracellular pH of 7.2 to 100% in cells incubated with inosine in N2 at a pH of 6.75. At the same 2,3-diphosphoglycerate concentration, a greater percentage of the compound appeared to be bound in erythrocytes than in the closely simulated model system. ATP was not significantly bound to hemoglobin under any condition examined, but appeared to be strongly complexed to Mg2+ inside the erythrocyte. The binding percentages for both 2,3-diphosphoglycerate and ATP in intact cells estimated by 31P NMR spectroscopy were lower than those calculated by others from individual association constants determined for the binding of different ligands to hemoglobin.     

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.     

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.     

24-hour study of thawed erythrocytes. Value of a protective solution

We have previously shown that thawed RBC concentrate can be stored at +4 degrees C during 9 days if resuspended in a synthetic medium: ESOC. We now report the in vitro evolution of thawed RBC stored with or without protective medium during the 24 hours legal time-limit. (Formula: see text) We show that without protection, the ATP and 2,3-DPG levels remain acceptable, but spontaneous or caused hemolysis is high. The level of free Hb is soon over the legal limit. The addition of our protective medium enhances ATP and hemolysis is strongly reduced. We conclude that a protective medium should be added to all thawed RBC concentrates.     

Effect of temperature and chloride on steady-state inhibition of angiotensin I-converting enzyme by enalaprilat and ramiprilat.

Recent work has provided new evidence that ATP is the major constituent of the low-Mr iron pool in the reticulocyte. The interaction of the iron complex of ATP with mitochondria was investigated in the present experiments. When ATP-Fe3+ was incubated with mitochondria, Fe3+, free of ATP, bound with high affinity to Fe3+ receptors on the mitochondria. The binding was saturable and reversible. Iron which was complexed to PPi, nitrilotriacetate, citrate, ADP and GTP also showed saturable binding to mitochondria; Fe3+ complexed to AMP bound non-specifically, as did Fe2+/ascorbate complexed to AMP bound non-specifically, as did Fe2+/ascorbate and Fe2+/dithionite.