Kinetics of the equilibration of O2 with Panulirus interruptus hemocyanin subunits a, b and c

The kinetics of the equilibration (25 degrees C) of O2 with the separated a, b and c subunits of Panulirus interruptus hemocyanin (Hc) as monomer and hexamer forms, as well as the native unfractionated abc mix, have been studied at pH values in the range 6.8-9.6, I = 0.100 M (NaCl). Rate constants kon and koff defined by deoxyHc + O2 <=> oxyHc have been determined by temperature-jump and stopped-flow techniques, respectively. The aggregation of monomer forms of different subunits to give hexamer is favoured by low pH and the availability of Ca2+, traces of which (or any other 2 + metals) are effectively removed by complexing with EDTA (5 mM). With or without EDTA, the hexamer form is present at the lower pH values. At the higher pH values with EDTA present a and b but not c give monomer forms. The hexamers are however retained at the higher pH values on addition of Ca2+ (10 mM). Cooperativity is observed for the hexamer forms at pH > 8, where the existence of relaxed (R) and tense (T) forms gives rise to sigmoidal kinetic plots in the determination of koff. Subunit c is different in that it retains its hexamer structure over the whole pH range, and does not display a Bohr effect. Native unfractionated protein is present as a hexamer mix of a, b and c in non-stoichiometric amounts, which has an enhanced Bohr effect as compared to the separated subunits.