Bohr-effect and buffering capacity of hemocyanin from the tarantula E. californicum

A previous report showed that binding of oxygen to the 24-meric hemocyanin from E. californicum does not correlate linearly with the release of protons as known from hemoglobin. However, this unusual complex phenomenological observation could not be explained at that time. Here, I present a full analysis of the thermodynamic coupling between protons and oxygen for the 24-meric tarantula hemocyanin in Ringer-solution based on the Nested-MWC-model. A strategy is presented which allows to reduce the number of free parameters when fitting the model to the data by including explicitly the equilibrium constants for binding of protons to the different conformations. The results show that the Nested-MWC-model presents a good description of the cooperative and allosteric properties of spider hemocyanin also under physiological conditions and additional constraints imposed by proton-binding data. The analysis allowed to estimate the average number of allosteric proton-binding sites per subunit and the corresponding pK for each conformation. Furthermore, an estimate of the number and affinity of proton buffering binding sites could be given. Approximately 80% of all proton-binding sites are non-allosteric buffering binding sites. The results obtained in this study allow to predict the relative contribution of the four different conformations under conditions found in vivo. The conformational distribution indicates that the conformation with the highest proton affinity (tR) might be important for the proton transport in the hemolymph.