Hemocyanin from the Australian freshwater crayfish Cherax destructor. Oxygen binding studies of major components

Publisher Website
Google Scholar
Abstract
O binding curves were obtained for unfractionated hemocyanin from C. destructor and its major components, the 25S and 17S forms. The binding was characterized by positive cooperativity at pH 7.8 with a P50 of .apprx. 4 mmHg and a Hill coefficient, nH, of .apprx. 3. There was no evidence of concentration dependence of the binding curves in the range 0.6-6 mg/mL, a finding which excludes a dynamic equilibrium between polymeric forms of different O affinity as a source of the cooperative binding. A positive Bohr effect operates between pH 6.8 and pH 7.8 and removal of Ca ions from the 25S and 17S aggregates markedly reduces their affinities for O. Cooperativity is retained in these circumstances though nH drops to about 2.5 in the case of the 25S and 2.0 in the case of the 17S form. The 2 major monomers M1 and M2, from which the 25S and 17S complexes are constructed, may be reconstituted into the hexamers (M1)6 and M2)6. These show O binding behavior perfectly consistent with that expected of native hexamers as studied in the 17S fraction, a mixed population of hexamers. The monomer M1 also can be studied in monomeric form and showed indistinguishable O2 binding at pH 7.8 and pH 10, the curve being a rectangular hyperbola as expected. The O binding curve of the single subunit hexamer (M1)6 was fitted adequately by a polynomial expression of order 6 as required for a molecule with 6 binding sites. Further interpretation in terms of a particular binding model was not attempted because available knowledge of the structures of arthropod hemocyanin aggregates and their O binding sites does not justify it.