Dissociation kinetics of hemocyanin from Octopus vulgaris

The native form of hemocyanin (Hc) from Octopus vulgaris can be completely dissociated, at alkaline pH and in the presence of EDTA, from 49S decamers to 11S monomers. The kinetics of this process was studied, using a Bio-Logic stopped flow system, by following the time dependence of the 450-nm light intensity, scattered at 90°, in the 7.9–8.8 pH range. All experimental traces were best fitted by a sum of three exponential decay functions. We then tried to best fit these decay functions with a series of kinetic models, the best of them resulting in one whose dissociation of decamers to monomers takes place in three consecutive and irreversible steps, with a highly cooperative step concerning dissociation of octamers to dimers, which appears to be the only intermediate species. This model was preferred over several others, not only for the best norm value but also for the best accordance between each calculated and experimental kinetic parameter (rate constants and amplitudes). Although other more complex models may be considered, our best fit model represents the simplest one, which is able to describe the observed dissociation kinetics.