Hemocyanin from the Australian freshwater crayfish Cherax destructor. Oxygen binding studies of major components.
Oxygen binding curves have been obtained for unfractionated hemocyanin from Cherax destructor and it major components, the 25S and 17S forms. In all cases the binding was characterized by positive cooperativity at pH 7.8 with a P50 of approximately 4 mmHg and a Hill coefficient, nH, of approximately 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 oxygen affinity as a source of the cooperative binding. A positive Bohr effect operates between pH 6.8 and pH 7.8 and removal of calcium ions from the 25S and 17S aggregates markedly reduces their affinities for oxygen. 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 two 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 oxygen binding behavior perfectly consistent with that expected of native hexamers as studied in the 17S fraction, a mixed population of hexamers. The monomer M1 can also be studied in monomeric form and was found to show indistinguishable oxygen binding at pH 7.8 and pH 10, the curve being a rectangular hyperbola as expected. The oxygen 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 six 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 oxygen binding sites does not yet justify it.
D000494:Allosteric Regulation; D000818:Animals; D003400:Astacoidea / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006433:Hemocyanins / Q000378:metabolism*; D010100:Oxygen / Q000097:blood*; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding
D010446:Peptide Fragments; D006433:Hemocyanins; D010100:Oxygen