Allostery in Callinectes sapidus hemocyanin: cooperative oxygen binding and interactions with L-lactate, calcium, and protons.

: Allosteric interactions of Callinectes sapidus hemocyanin have been investigated by generation of precision oxygen-binding curves using a modified Imai apparatus and their subsequent analysis using numerical methods. The Monod-Wyman-Changeux (MWC) model, the MWC model extended to include hybrid R3T3 states, and the phenomenological Adair equation were fit to the experimental data. The hybrid MWC model provided a quantitatively better fit to the data than did the MWC model. The fits of the hybrid model and the Adair equation were statistically similar, supporting the adequacy of the hybrid model as a description of cooperative oxygen binding by C. sapidus hemocyanin. The hybrid model is also supported by an apparent negative cooperativity at the fourth oxygen-binding step, a feature that is not possible with the conventional MWC model. The oxygen-binding curves obtained at various concentrations of three allosteric effectors, Ca, protons, and L-lactate, were analyzed in terms of the hybrid MWC model. All three effectors altered both the affinities of the model states and the equilibrium constants between the states. L-Lactate, which increases affinity and decreases cooperativity, increased the affinity of both the R and T states as well as preferentially stabilizing the hybrid and R states. Protons, which decrease affinity and cooperativity, act by reducing the affinity of both the R and T states and stabilizing the R state. Ca ions, which increase both affinity and cooperativity, act by increasing the affinity of the R state and preferentially stabilizing the T state.