Haliotis tuberculata hemocyanin (HtN): analysis of oligomeric stability ofHtH1 and HtH2, and comparison with keyhole limpet hemocyanin KLH1 and KLH2

The multimeric/higher oligomeric states of the two isoforms of Haliotis tub erculata hemocyanin (HtH1 and HtH2) have been assessed by transmission elec tron microscopy (TEM) of negatively stained specimens, for comparison with previously published structural data from keyhole limpet hemocyanin (KLH1 a nd KLH2) [see Harris, J.R., Gebauer, W., Guderian, F.U., Markl, J., 1997a. Keyhole limpet hemocyanin (KLH), I: Reassociation from Immucothel followed by separation of KLH1 and KLH2. Micron, 28, 31-41; Harris, J.R., Gebauer, W ., Sohngen, S.M., Nermut, M.V., Markl, J., 1997b. Keyhole limpet hemocyanin (KLH). II: Characteristic reassociation properties of purified KLH1 and KL H2. Micron, 28, 43-56; Harris, J.R., Gebauer, W., Adrian, M., Markl, J., 19 98. Keyhole limpet hemocyanin (KLH): Slow in vitro reassociation of KLH1 an d KLH2 from Immucothel. Micron, 29, 329-339]. In purified samples of both H tH isoforms, the hollow cylindrical ca 8 MDa didecamer predominates togethe r with a small number of decamers, but tri- and longer multidecamers are de tectable only in the HtH2. The stability of the two HtH isoforms under vary ing ionic conditions have been monitored, thereby enabling conditions for t he production of stable decamers to be established. The ability of these de camers to reform multimers in the presence of 10 and 100 mM concentrations of calcium and magnesium ions in Tris-HCl buffer (pH 7.4), and also of indi vidual HtH1 and HtH2 subunits (produced by pH 9.6 dissociation in glycine-N aOH buffer), to reassociate in the presence of calcium and magnesium ions, has been assessed. For the HtH1 decamers, the predominant multimeric produc t is the didecamer at 10 and 100 mM calcium and magnesium concentrations, w hereas for the HtH2 decamers, large numbers of multidecamers are produced, with the reaction proceeding more completely at the higher calcium and magn esium concentration. With the HtH1 subunit, reassociation in the presence o f 10 and 100 mM calcium and magnesium ions yielded an almost 100% conversio n into didecamers, whereas the HtH2 subunit produced a mixture containing l arge numbers of short multidecamers and relatively few didecamers, together with a considerable number of smaller diameter helical/tubular polymers. T he association properties of the HtH1 and HtH2 decamers, and the subunit re association, firmly indicate the integrity and structural competency of the protein under the experimental conditions used. Data on the association of KLH2 decamers is also presented, which together with previously published data on the association KLH1 decamers and the reassociation of KLH1 and KLH 2 subunits, enables a detailed comparison of the two hemocyanin isoforms fr om both molluscan species to be made. Biochemical manipulation of the oligo mer states and the subunit reassociation of molluscan hemocyanins can usefu lly be assessed by the study of negatively stained TEM specimens. (C) 2000 Elsevier Science Ltd. All rights reserved.