Kinetic control of co-operativity in the oxygen binding of Panulirus interruptus hemocyanin

Abstract

The kinetics of the oxygen reaction of Panulirus interruptus hemocyanin have been studied at pH 9.6 under conditions where the protein exists in the undissociated, co-operative state and in the dissociated, non-co-operative state. Temperature-jump relaxation measurements of the undissociated protein at high oxygen saturation levels show one relaxation process which has been assigned to the high oxygen affinity (R) state, the on and off kinetic constants being 3.1 × 10 7 m −1 s −1 and 60 s −1, respectively. Stopped-flow measurements of the oxygen dissociation reaction show (1) an autocatalytic time-course of the reaction at pH 9.6 and (2) an increase in the overall oxygen dissociation rate constant, as the pH is decreased from 9.6 to 7.0. Temperature-jump relaxation measurements of the dissociated protein show one relaxation process characterized by a very high oxygen dissociation rate constant (1500 s −1) and a combination constant which is of the same order of magnitude as reported for undissociated protein ( k on = 4.6 × 10 7 m −1 s −1 ). The behaviour of dissociated protein can be considered as characteristic of the low oxygen affinity (T) state. The results presented in this paper, together with data available for other hemocyanins as well as hemoglobins, lead to the conclusion that respiratory proteins show a common feature in the kinetic control of co-operative oxygen binding: the stability of the oxygen-protein complex is largely determined by the value of the dissociation rate constant, the oxygen combination process very often appearing to be diffusion controlled.