Model of the pH‐Dependence of the Concentrations of Complexes Involving Metabolites, Haemoglobin and Magnesium Ions in the Human Erythrocyte

Abstract

The rate of glucose consumption and the concentrations of glycolytic intermediates in human erythrocytes have long been known to be pH sensitive. Despite the extensive literature on modelling erythrocyte metabolism, no model developed so far can adequately describe all of these pH-dependent changes. None of these models have included all the significant association reactions between metabolites, Hb and Mg2+ that will influence metabolism. As part of a larger enterprise to develop a detailed model of erythrocyte glycolysis, we present a sub-model which predicts, as a function of pH and oxygenation state, the concentrations of free and Mg2+-bound metabolites that are substrates, co-factors and effectors of glycolysis. This model shows that pH changes around physiological values can cause large changes in the distribution of metabolites between free, bound and Mg2+-complexed forms, based on binding interactions alone ; in oxygenated cells, at pH 7.2 -7.6, many glycolytic intermediates undergo changes in concentration of 50-100 %. The model also predicts intracellular concentrations of free Mg2+ in erythrocytes to be 0.4 mM and 0.64 mM in oxygenated and deoxygenated cells, respectively, assuming a total magnesium concentration of 3 mM (~8 8 % of the total magnesium usually found in erythrocytes). This is in close agreement with the values found by Flatman [Flatman, P. W. (1980) J. Physiol. 300,[19][20][21][22][23][24][25][26][27][28][29][30] Hexokinase has a high 'flux control coefficient' in human erythrocyte glycolysis, so the dependence of its rate on the pH and oxygenation state of haemoglobin is important. With a low oxygen tension and an intracellular pH of 7.34, the major inhibitor of its activity (2,3-bisphosphoglycerate) is 85 % bound to either haemoglobin or MgZf, and the maximum possible flux of substrate via it would be 2.05 mmol L erythrocytes-' h-'. However, if the haemoglobin were saturated with oxygen, and the pH were 7.2, it was calculated that the maximum rate would be 1.48 mmol L erythrocytes-' h-'; this is primarily due to a doubling of the free 2,3-bisphosphoglycerate concentration. However, the full extent of the inhibition is counteracted because the concentration of the Mg2+-2,3-bisphosphoglycerate would be approximately doubled. Many other similar comparisons are possible with this new model, which highlights the complex network of interactions between haemoglobin, Mg", H' and the metabolites as substrates and effectors of the glycolytic reactions.Keywords: erythrocyte glycolysis ; regulation of metabolism ; pH effect; magnesium ions ; computer simulation.Many metabolically significant ions, such as ATP, 2,3-bisphosphoglycerate [Gra(2,3)P2] and other phosphorylated metabolites in human erythrocytes, bind reversibly with haemoglobin (Hb) and Mg2+. These interactions significantly affect the free and bound concentrations of metabolites within the cells and thus significantly affect the activity of many enzymes (Gupta et al., 1978b;Hamasaki and Rose, 1974;Gerber et al., 1973;Bunn et al., 1971)...

Simultaneous Determination of Low Free Mg2+ and pH in Human Sickle Cells using 31P NMR Spectroscopy

“…Ϫ1 was in excellent agreement to that calculated by Mulquiney and Kuchel (37), which approximated to 8.0 ϫ 10 4 M Ϫ1 when adjusted for pH and ionic strength.…”

“…The total erythrocyte magnesium concentrations were then calculated by subtracting the plasma total magnesium from the whole blood measurement. Concentrations are presented per liter of cell water, assuming a 95% packing efficiency and 70% cellular water content for normal cells, 66% for whole sickle blood, and 65 and 62% for medium and dense sickle cells, respectively (37). The hematocrits were determined after centrifugation at 13,000 ϫ g for 10 min in a microhematocrit centrifuge using a Hawksley microhematocrit reader, with all measurements made in triplicate.…”

Impact of biomineralization processes on the Mg content of foraminiferal shells: A biological perspective

“…In solution, various protonated forms of ATP constitute a complex multiple-equilibrium mixture, with each species having different affinities for Mg. Therefore decrease in intracellular pH will reduce the Mg 2+ buffer capacity of ATP [Mulquiney and Kuchel, 1997].…”

Free magnesium-ion concentration in erythrocytes by31P NMR: the effect of metabolite–haemoglobin interactions

“…(5). According to binding analysis, 39 human erythrocytes, fully oxygenated at pH 7.2, have ATP partitioned in the following ratios: free ATP, 0.07; HbATP, 0.10; MgATP, 0.72; HbMgATP, 0.11. From these data it can be calculated that the standard 31 P NMR method will overestimate the true concentration of free Mg 2+ by 4.4%.…”