Intramolecular localization of the functional units of Sepia officinalis hemocyanin by immunoelectron microscopy

Abstract The quaternary structure of Sepia officinalis hemocyanin (Hc) as studied in immunoelectron microscopy with rabbit IgGs and Fab fragments raised against functional units (FU) So c, So d, So e, So f, So g, and So h and fragment So ab. The architecture of immunocomplexes shows that (i) epitopes characteristic of FUs So c and So g and of fragment So ab are located in the two external tiers of FUs, (ii) FUs So h and So e or So d are located in arches. These results were confirmed using immunocomplexes made up of Sepia Hc and IgGs or Fab fragments purified from antisera raised against FUs of Octopus vulgaris and Octopus dofleini. Frozen-hydrated immunocomplexes containing one Hc molecule and at least one FU-specific Fab fragment were observed in the electron microscope and submitted to image processing. When the Hc molecule is viewed along its 5-fold axis (i) anti- So c Fab fragments project on a radius passing through the arch’s pillar, (ii) anti- So f Fabs project slightly out of the arches, and (iii) anti- So h Fabs project between neighboring arches. When applied to a recent three-dimensional (3D) reconstruction volume, these results allow us to deduce the intramolecular location of five of the eight FUs. For the last three FUs limited uncertainties remain: (i) So c can be located in two positions in the external tier of FUs; (ii) So a and So b can both occupy three positions in the external tiers; and (iii) because of an immunological cross-reactivity So d may be located in the wall and So e in the arch, or vice versa . An analysis of the quaternary structure considering the possible locations of the 80 FUs and postulating a single type of subunit shows that 80 possibilities of paths still exist for the polypeptide chain. To solve definitely these 80 possibilities only five questions remain to be answered.