Immunological correlates between multiple isolated subunits of Androctonus australis and Limulus polyphemus hemocyanins: An evolutionary approach

Immunological cross-reactivities between isolated subunits of the scorpion Androctonus australis (Aa) and of the horseshoe crab Limulus polyphemus (Lp) hemocyanins were studied using subunit-specific antibodies prepared through immunoadsorption to pure immobilized subunits. Rocket immunoelectrophoreses of the various subunits of both hemocyanins were carried out at constant antigen concentration against the various subunit-specific antibody preparations. Then the data were analyzed through factorial correspondence analysis and compared to the respective intramolecular locations of the subunits in both hemocyanins. The results show that the dimeric subunits located in the central part of each (4 × 6)meric structure ( Aa whole molecule and Lp half molecule) were strongly preserved. In addition, the (8 × 6)mer-forming subunit of Lp hemocyanin ( LpIV) and the subunit occupying the same intramolecular position in Aa hemocyanin ( Aa5A) were also strongly preserved. Besides the strong antigenic relatedness, less pronounced crossed immunoprecipitations or no precipitation at all were observed between subunits with homologous positions suggesting a minor structural and/or functional roles for these subunits. All the antigen-antibody combinations leading to an absence of immunoprecipitation were screened for the presence of soluble immunocomplexes by radioimmunological tests. In all cases, soluble immunocomplexes were observed. These results suggest the following evolution scenario. First, the central dimeric subunits, responsible of the dodecamer aggregation ( Aa3C and 5B and LpV and VI) were already differentiated when Merostomata diverged from Arachnida. Second, the differentiation of the (8 × 6)mer-forming subunit occurred in the Merostomata ramification in a preserved subunit already possessing a functional advantage. Third, the differentiation of subunits Aa3A and Aa3E recently occurred in the scorpion ramification.