Dissociation and Oxygen‐Binding Behaviour of β‐Hemocyanin from Helox pomatia

The dissociation behaviour of Helix pomatiaβ- hemocyanin as influenced by pH, calcium-iopn concentraion and ionic strength, and the oxygen vinging properiotes were investigated. The dissociation behaviour of β-hemocyuanin and α-hemocyanin differs distinctly in a number of properties: (a) they show a different response to high salt concentration, (b) the stabilizing influence of calcium ions on the whole molecules of β-hemocyanin is smaller than that on α-hemocyanin, (c) half molecules of β-hemocyanina are stablized to a large extent in teh presence of calcium ions and (d) at low ionic strength no peak separation between one-tenth and one-twentieth molecules of β-hemocyanin is observed in the ultracentrifuge, which indicates an equilibrium between the species. The oxygen binding behaviour of β-hemocyanin shows the typical features of proteins with a large hnumber of binding sites. The Hill plot is characterized by a high value of the Hill coefficient (maxm nH=7), coupled to a low value for the interatrion free energy per site. The oxygen binding behaviour can bacisally be described by a mocdified tewo-state allosteric model with the variation, imposed by teh expeimental data, taht KT is pH-dependent (negative Bohr effect), while KR is pH-indepenmdent. Regulation of the cooperativity by teh alloosteric effector H+ is mainly due to changes in the allosteric equilibrium constant. In order to describe the cooperativity as a function of pH, the minimum number of interacting binding sites has to be varied, indicating that the size of the ‘functional constellation’ is variavle. This finding may have important implications on the relationship between the so-called ‘structural’ and ‘functional constellation’. A comparison fo the oxygen binding properties of β-and α-hemocyanin shows that these proteins are complementary in their behaviour, with respect to the responses to changes in H+ anmd Ca2+ concentrations. This may be of importance for the animal in order ito meet specific physiological and enviromental requirements.