Hemocyanins in Spiders, XIII. Kinetics of Oxygen Dissociation from Individual Subunitsof EurypelmaandCupienniusHemocyanin

Abstract

The 5 monomeric components of Eurypelma [E. californicum] hemocyanin show homogeneous O2 dissociation kinetics with very similar rates of O2 dissociation. At pH 9.6 the apparent rate constants for O2 dissociation are about 10 s-1; at pH 7.5 rates of about 13 s-1 are observed. The heterodimer of the Eurypelma system has heterogeneous kinetic behavior. The dominant 1st-order rate constant for O2 dissociation is similar to that of the monomeric components at pH 9.6, but is insensitive to pH. The initial 10-20% of the reaction shows a faster rate, suggestive of lower O2 affinity. Artificial homodimers, corresponding to subunits bb or cc, behave like the bc heterodimer. Functional differentiation between the monomers of Eurypelma hemocyanin was detectable only in the presence of 1.5 M KCl. Subunit d of the Eurypelma system appears to be unique in that it exhibits a pH-linked decrease in rate of O2 dissociation in the presence of high salt concentrations. A mixture of monomers of Cupiennius [C. salei] hemocyanin had a rate of O2 dissociation of 5.4 s-1 at pH 9.6. The dominant rate observed for the dimeric component of Cupiennius hemocyanin is higher, about 10.4 s-1. As for the Eurypelma dimers, the Cupiennius dimer shows heterogeneous O2 dissociation kinetics. As in other hemocyanins, the dissociation rate constant is the determining factor in establishing the O2 equilibrium properties of Eurypelma and Cupiennius hemocyanin. Very little functional heterogeneity or pH sensitivity is shown by the isolated monomeric components. The dimers behave different from monomers. The subunit interactions which are possible in the oligomers must be the major contributor to the homotropic and heterotropic allosteric effects exhibited by the intact molecules. The functional equivalence of the subunits of Eurypelma hemocyanin in O2 binding is in sharp contrast to their chemical and immunological differences and their distinct roles in reassembly of the native oligomers.