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

Native Paralithodes camtschaticae hemocyanin is found as a mixture of dodecamers (24S; 80%) and hexamers (16S; 20%). Removal of Ca21 ions by dialysis against EDTA-containing buffer solution at neutral pH induces complete dissociation of the 24S form into the 16S form. Under these conditions, a further increase in pH to 9.2 produces complete dissociation of the hexamers into monomers (5S). In both cases, the dissociation process is reversible. The dodecamer (24S) is composed of two different hexamers which can be discriminated only by ion-exchange chromatography in the presence of Ca21 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 investigated. Native hemocyanin binds O2 co-operatively (nH ˆ 3) and with low affinity (p50 < 103 Torr). The two monomeric fractions, ParcI and ParcII, are not co-operative and the affinity is twice that of the native protein ( p50 < 65 and 52 Torr). Oxygen-binding measurements of native hemocyanin carried out at different pH 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.