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 mea sured using pulsed-field gradient (PFG)-P-31 nuclear magnetic resonanc e spectroscopy in solutions containing 2.7-5.0 mM hemoglobin (Hb) and a range of DPG concentrations. The dependence of the measured values o f D on the fraction of the total DPG in the sample that is bound to Hb enabled the estimation of the dissociation constants (K-d) of complex es of DPG with carbonmonoxygenated, oxygenated, and deoxygenated Hb; t he values of K-d (mM), measured at 25 degrees C, pH 6.9 and in 100 mM bis Tris/50 mM KCI, were 1.98 +/- 0.26, 1.8 +/- 0.5 and 0.39 +/- 0.26, respectively. In intact erythrocytes the apparent diffusion coefficie nt, D-app, of DPG was larger in oxygenated and carbonmonoxygenated cel ls (6.17 +/- 0.20 x 10(-11) m(2)s(-1)) than in deoxygenated cells (4.1 0 +/- 0.23 X 10(-11) m(2)s(-1)). Changes in intracellular DPG concentr ation (5-55 mM) in erythrocytes, brought about by incubation in a medi um containing inosine and pyruvate, did not result in significant chan ges in the value of D-app; this result supports the hypothesis that DP G binds to other sites in the erythrocyte. Monte Carte simulations of diffusion in biconcave discs were used to test the adequacy of the val ues of K-d estimated in solution to describe the binding of DPG to Hb in oxygenated and deoxygenated erythrocytes. The results of the simula tions implied that the value of K-d estimated for deoxygenated Hb-DPG was greater than expected from the experiments involving intact erythr ocytes. This difference is surmised to be at least partly due to the d ifficulty of measuring D at low-ligand concentrations. Notwithstanding this shortcoming, the PFG method appears to be suitable for probing i nteractions between macromolecules and ligands when the K-d is in the millimolar range. It is one of the few techniques available in which t hese interactions can be studied in intact cells. In addition, the Mon te Carlo simulations of the diffusion experiments highlighted importan t differences between theory and experiment relating to the nature of molecular motion inside the cells.