The hemocyanin of the shamefaced crab Calappa granulata: structural-functional characterization

Arthropod hemocyanins (Hcs) transport and store oxygen and are composed of six subunits, or multiples thereof depending on the species. Calappa granulata He is found as a mixture of dodecamers (95%) and hexamers (5%). Removal of calcium ions and alkaline pH induce an incomplete partially reversible dissociation of dodecameric He. Two-dimensional electrophoretic pattern of dissociated He indicated a large heterogeneity in He subunit: most differences are likely to be explained by post-translational modifications. Dodecameric He showed a large Bohr effect (Delta log P-50/Delta pH = -0.95) and a normal cooperativity (h(50) values = 2.7 +/- 0.2) in the presence of 10 mM CaCl2. The hexameric molecule displayed lower Bohr effect and cooperativity than the dodecamer. Lactate effect on the oxygen affinity (Delta log P-50 = 0.55) and the increase of lactate concentrations in animals kept in emersion were related to the increased oxygen requirements that occur during hypoxia in vivo. Calcium affects oxygen affinity only at high concentrations: this He appeared to lack the calcium high-affinity binding sites found in other species. The effect of temperature on both oxygen affinity and cooperativity was measured in the absence and presence of 10 mM lactate, allowing calculation of the exothermic contribution of lactate binding (Delta H = -25 kJ mol(-1)).