Chloride-Dependent Organic Phosphate Sensitivity of the Oxygenation Reaction in Crocodilian Hemoglobins

We show that crocodilian hemoglobins (Hbs), which previously have been considered to be model pigments lacking allosteric interaction with organic phosphate esters, do exhibit oxylabile ATP and 2,3-diphosphoglycerate (DPG) binding that decreases O2 affinity and increases pH sensitivity (Bohr effect), in the absence of Cl- and at the low Cl- concentrations that may occur in crocodilian plasma during 'post-ingestive alkaline tides'. Hbs from different species vary in their phosphate sensitivities. In Alligator mississippiensis Hb, O2 affinity shows greater ATP than DPG sensitivity at low [cofactor]/[Hb] ratios. In Paleosuchus palpebrosus Hb, where even a high Cl- concentration (0.1 mol l-1) does not completely suppress the phosphate effects, the opposite is true, whereas both ATP and DPG exert similar effects in Caiman crocodilus Hb. Lactate, at concentrations that may occur after intensive exercise, similarly depresses Hb O2-affinity, indicating an O2 demand/O2 supply feedback regulation. Curiously, inositol hexaphosphate (IHP), a potent allosteric effector in other vertebrate Hbs, has no effect on A. mississippiensis and P. palpebrosus Hb and only small effects on C. crocodilus Hb, presumably because of steric hindrance at the binding site. The molecular mechanisms underlying the observations (particularly the implication of ss82-Lys, i.e. the lysine residue at position 82 of the ss-chains that binds phosphate and Cl-) are considered. Their physiological significance requires further study.