Rapana thomasiana hemocyanin (RtH): dissociation and reassociation behavior of two isoforms, RtH1 and RtH2

Rapana thomasiana hemocyanin (RtH) is a mixture of two hemocyanin isoforms, termed RtH1 and RtH2. The two subunit types, purified by ion exchange chro matography, were used for macromolecular reassociation studies. In vitro re association was achieved with Tris-saline stabilizing buffer at pH 7.4, con taining 100 mM calcium and magnesium chloride at 4 degreesC. The relatively slow progress of reassociation was monitored, and the different oligomeric forms of RtH1 and RtH2 were studied by transmission electron microscopy, u sing samples negatively stained with 1% (w/v) uranyl acetate or 5% (w/v) am monium molybdate containing 1% (w/v) trehalose at pH 7.0. The two subunits reassociate to produce characteristic didecamers, oligomeric and polymeric forms depending on the dissociated material and the reassociation condition s (i.e. divalent ion concentration, duration). In contrast to the didecamer s of the freshly isolated RtH preparations, RtH1 and RtH2 show after 2 week s' reassociation a clear tendency to generate multidecameric structures. Th e behavior of the native RtH1 and RtH2 during reassociation in the presence of 100 mM calcium and magnesium chloride corresponds to the reported commo n oligomerization characteristics of KLH1/HtH1 and KLH2/HtH2, respectively. It is important to note that during the reassociation of the RtH isoforms: (I) no smaller diameter tubular polymers (ca. 25-27 nm) were formed from t he subunits as well as from the decamers; (II) multidecamers with one or mo re 'nucleating' didecamers, were detected in addition to the multidecamers, composed of didecamers with associated decamers at one or both ends. (C) 2 001 Elsevier Science Ltd. All rights reserved.