Amphitrite ornata erythrocruorin II. Molecular controls of function

Abstract

In the marine terebellid worm Amphitrite ornata the vascular fluid contains a high molecular weight erythrocruorin, while cells of the coelom contain a monomeric hemoglobin. The structural integrity of the erythrocruorin molecule is known to be dependent on the presence of a minimal concentration of divalent cations (1–3 mM) in the medium. The functional properties of Amphitrite erythrocruorin are also affected by cations. The oxygen affinity tends to increase with increasing cation concentration and the degree of cooperative interactions, expressed in the kinetics and equilibria of ligand binding, goes through a maximum. Maximal Hill coefficients of 3–4 are observed with 50 mM CaCl₂, 50 mM MgCl₂ or 1 M NaCl in measurements at the physiological pH of 7.75. Only 2 mM CaCl₂ is required for maximal cooperativity at pH 8.5. This suggests partial deprotonation of the cation binding site at high pH. It is somewhat unusual that pH effects on cooperativity are reversible, since this is not a common feature of the giant erythrocruorin molecules. The oxygen binding experiments revealed a marked effect of divalent cations of Amphitrite erythrocruorin at high pH and cation concentration. Above pH 8.5, at 50 mM CaCl₂ and 12°C, the erythrocruorin will form a polymer upon deoxygenation. This polymerization is readily reversible by bringing the temperature from 12 to 20°C or by oxygenation. Under physiological conditions of pH and cation concentration and at 12°C, the ervthrocruorin and the monomeric coelomic hemoglobin require a similar oxygen pressure for half saturation. However, the allosteric regulation of function is absent for the coelomic protein.