Hemocyanins in Spiders, XII. Dissociation and Reassociation ofEurypelmaHemocyanin

Abstract

The dissociation and reassembly of E. californicum hemocyanin was studied under various conditions of pH, ionic strength and protein, Ca and Mg concentrations. The hemolymph concentrations of Ca and Mg were 4 and 0.9 mM, rspectively. Eurypelma hemocyanin does not dissociate upon dilution down to 0.04 mg/ml. At physiological pH, phosphate causes partial dissociation. Dissociation at alkaline pH requires 2 days for completion and is dependent on hemocyanin concentration. Reassociation, starting with the total dissociated subunits, yielded 37 S, 16 S and untreated 5 S material. The yield of 37 S particles after 1 day was 75%, but rose slowly to 84% after 2 wk. Raising the Ca concentration above 1 mM resulted in an increase of 16 S particles (hexamers). High Mg concentrations inferfered with ordered reassembly. 37 S, native-like particles are readily formed in the absence of both Ca and Mg. Reassociation is strongly favored if ionic strength is increased. The significance of the 16 S reassociation product was studied by repeated dissociation and rassociation and analysis of subunit composition. The 37 S reassociate contained the whole set of subunits, their quantitative proportions being identical compared to native 37 S hemocyanin. The 16 S material was lacking the dimeric subunit bc4 ( = 4D). This hexameric product arises by incorrect reassociation of monomeric subunits. P50 and nH values were recorded for the reassociated hemocyanins. The 37 S component had a higher affinity and lower cooperativity than native hemocyanin, possibly due to aging of the subunits.