ANIONIC BINDING-SITE AND 2,3-DPG EFFECT IN BOVINE HEMOGLOBIN

It is generally believed that bovine hemoglobin (BvHb) interacts weakl y with 2,3-diphosphoglycerate (2,3-DPG) in a chloride-free media and n ot at all in the presence of physiological concentrations of chloride (100 mM). This lack of interaction has raised several questions at bot h structural and evolutionary levels. Results obtained in this study v ia P-31 nuclear magnetic resonance (NMR) show that, even in the presen ce of 100 mM chloride ions, 2,3-DPG does, in fact, interact with bovin e deoxy-Hb. This spectroscopic observation has been confirmed by oxyge n binding experiments, which have also shown that, under certain condi tions, chloride and 2,3-DPG may display a synergistic effect in modify ing the oxygen affinity of bovine hemoglobin. It could be that this sy nergistic effect has its structural basis in a conformational modifica tion induced by 2,3-DPG, possibly causing extra chloride anions to app roach the positive charges which constitute the anion binding site. An other possibility, not necessarily an alternative, is the additional c hloride binding site recently identified [Fronticelli, C., Sanna, M. T ., Perez-Alvarado, G. C., Karavitis, M., Lu, A.-L., and Brinnigar, W. S. (1995) J. Biol. Chem 270, 30588-30592] involving lysine beta 76 tha t in bovine Hb substitutes for the alanine residue present in human he moglobin. All of-these findings are in agreement with the very low ent halpy of oxygenation that characterizes bovine Hb when both chloride a nd 2,3-DPG are present in concomitance. The results reported here clea rly show that bovine hemoglobin does react with 2,3-DPG and is functio nally affected by this organic phosphate. Hence, the very low intraery throcytic concentration of 2,3-DPG (0.5 mM) in adult bovine red blood cells is the result of metabolic adaptation which cannot be explained solely by the different amino acid sequence at the level of the 2,3-DP G binding site.