Zinc as modulator of oxygenation function and stabilizer of quaternary structure in earthworm hemoglobin

Blood of the earthworm Pheretima hilgendorfi contains 10.7 mM Ca, 2.0 mM Mg, 75.5 mM Na, 5.9 mM K, 0.9 mM Zn and 0.3 mM Mn. Some of these cations cannot be removed completely from the blood by dialysis, and 36 atoms of Ca, 1-3 atoms of Mg and 1-2 atoms of Zn per 164 atoms of Fe were retained in purified preparation of the hemoglobin (Hb). At physiological pH, oxygen affinity and the Hill coefficient at half saturation (n1/2 value) of the Hb increased in the presence of 100 mM of Ca, Mg or Na. These effects were in the order of Ca > Mg > Na. At physiological concentration, however, the effect of each of these three cations on oxygenation was rather small. On the other hand, Zn gave a remarkable effect at less than 1 mM. This suggests a possibility that Zn acts as a primal modulator for the oxygenation function of the Hb in vivo. Oxygenation data at various pH values in the presence of each cation strongly suggest that Zn binds to a site different from those for the other three cations. Zn at a concentration of only 1 mM protected considerably the dissociation of the whole molecule to smaller components at alkaline pH and Zn thus contributes to the stabilization of the quaternary structure of the Hb.