SUBUNITS COMPOSITION AND ALLOSTERIC CONTROL IN CARCINUS-AESTUARII HEMOCYANIN

Carcinus aestuarii hemocyanin (Hc) exists in two aggregation forms at pH 7.5 and 20 mM Ca2+: 24S accounting for 90% of total hemocyanin and 16S accounting for 10%. Removal of metal cations by EDTA at neutral pH causes the complete dissociation of 24S hemocyanin into two different 16S. At pH 9.2, 24S hemocyanin dissociates into a pH stable 16S and a 5S component, The 5S component consists of three monomeric fractions named CaeSS1 (10%), CaeSS2 (50%) and CaeSS3 (40%): the latter fraction consisting of two isoforms. The fractions CaeSS1, CaeSS2 and CaeSS3 h ave been studied as far as their reassociation properties to form hexa mers are concerned. We investigated the oxygen-binding properties of t he native form (24S). the mixture of the two 16S forms, the pH-stable 16S alone and of purified subunit fractions to define the role of each species on the expression of the allosteric behaviour of the 24S aggr egate. Thr analysis of O-2-binding data reveals that 24S-Hc call be we ll described by the modified Monod Wyman and Changeaux-model (nested M WC-model), while the half-molecules (TGS) bind oxygen according to the simple MWC-model. The two hexameric 16S within the dodecameric 24S he mocyanin can he regarded as nested allosteric units. They behave as be ing functionally coupled in the T-states (IT and rT). In the R-states (tR and rR) the two half-molecules seem to be functionally uncoupled s ince they have the same values of oxygen binding constants as deduced for isolated 16S hexamers.