Dimerization of native Limulus polyphemus hemocyanin in the presence of calcium ions and sucrose

Abstract

In the presence of both sucrose and millimolar concentrations of calcium, native Limulus polyphemus hemocyanin, having a sedimentation coefficient of ~ 60S, forms a dimer with a sedimentation coefficient of ~ 90S. Dimer formation was observed by sucrose gradient ultracentrifugation and gel filtration. It occurs at pH 7.0 but not at pH 8.5 or 6.0, and it is dependent on the calcium concentration. Treatment with EDTA to remove calcium reverses the dimerization process. The effect of sucrose may be due to its known preferential exclusion from the vicinity of proteins [15]. Such preferential exclusion would favor the formation of dimers if the excluded volume of the protein were to decrease on dimerization. Formation of dimer is accompanied by the binding of calcium to hemocyanin, as determined by analysis of gradient fractions by atomic absorption spectroscopy. In addition, stoichiometric amounts of europium (III), known to occupy the calcium binding sites of hemocyanin, also promotes dimer formation. Dimer formation is not induced by 100 mM sodium chloride. The observation of a 90S form of Limulus hemocyanin indicates that there is nothing inherent in the structure of the 60S molecule that prevents further aggregation.