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

The quaternary structure of Sepia officinalis hemocyanin (Hc) as studied in immunoelectron microscopy with rabbit IgGs and Fab fragments raised agains t functional units (FU) Sec, Sod, See, Sof, Sog, and Soh and fragment Soab. The architecture of immunocomplexes shows that (i) epitopes characteristic of FUs Soc and Sog and of fragment Soab are located in the two external ti ers of FUs, (ii) FUs Soh and Soe or Sod are located in arches. These result s were confirmed using immunocomplexes made up of Sepia He and IgGs or Fab fragments purified from antisera raised against FUs of Octopus vulgaris and Octopus dofleini. Frozen-hydrated immunocomplexes containing one He molecu le and at least one FU-specific Fab fragment were observed in the electron microscope and submitted to image processing. When the He molecule is viewe d along its 5-fold axis (i) anti-Soc Fab fragments project on a radius pass ing through the arch's pillar, (ii) anti-Sof Fabs project slightly out of t he arches, and (iii) anti-Soh Fabs project between neighboring arches. When applied to a recent three-dimensional (3D) reconstruction volume, these re sults allow us to deduce the intramolecular location of five of the eight F Us. For the last three FUs Limited uncertainties remain: (i) Soc can be loc ated in two positions in the external tier of FUs; (ii) Soa and Sob can bot h occupy three positions in the external tiers; and (iii) because of an imm unological cross-reactivity Sod may be located in the wall and Soe in the a rch, 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 b e answered.