Molecular heterogeneity of the hemocyanin isolated from the king crab Paralithodes camtschaticae

Native Paralithodes camtschaticae hemocyanin is found as a mixture of dodec amers (24S; 80%) and hexamers (16S; 20%). Removal of Ca2+ ions by dialysis against EDTA-containing buffer solution at neutral pH induces complete diss ociation of the 24S form into the 16S form. Under these conditions, a furth er increase in pH to 9.2 produces complete dissociation of the hexamers int o monomers (5S). In both cases, the dissociation process is reversible. The dodecamer (24S) is composed of two different hexamers which can be discrim inated only by ion-exchange chromatography in the presence of Ca2+ ions. At alkaline pH and in the presence of EDTA, two major monomeric fractions can be separated by ion-exchange chromatography: ParcI (60%) and ParcII (40%). The reassociation properties of the two fractions were studied separately to define their ability to form hexamers and dodecamers. The oxygen-binding properties of the different aggregation states were inve stigated. Native hemocyanin binds O-2 co-operatively (n(H) = 3) and with lo w affinity (p(50) approximate to 103 Torr). The two monomeric fractions, Pa rcI and ParcII, are not co-operative End the affinity is twice that of the native protein (p(50) approximate to 65 and 52 Torr). Oxygen-binding measurements of native hemocyanin carried out at different p H values indicate a strong positive Bohr effect within the pH range 6.5-8.0 and an increase in oxygen affinity at pH below 6.5.