Hemocyanin subunit organization of the gastropod Rapana thomasiana

RtH1 and RtH2, the two hemocyanin isoforms of the prosobranch gastropod Rap ana thomasiana, have been purified by anion-exchange chromatography and stu died by SDS-PAGE and immunoelectrophoresis. Both subunit types are built up of eight functional units (FUs). Under reducing conditions subunit RtH2 sp lits into two fragments, RtH2-a-f and RtH2-gh, suggesting the presence of a disulfide bridge between FU2-f and FU2-g. By proteolytic cleavage of the s ubunits into three-, two-, and single-FU fragments, purification of fragmen ts by HPLC, N-terminal sequencing of the peptides, and crossed-line immunoe lectrophoresis, FUs-a-h of RtH2 and FU-a, FU-d, FU-e, and FU-f of RtH1 were identified and correlated to the eight-FUs pattern of immunoelectrophoresi s. FU-a, FU-e, and FU-f of RtH1 and RtH2 are very closely related immunolog ically. RtH1 and RtH2 both correspond immunologically to KLH2, one of the t wo hemocyanin isoforms of the prosobranch gastropod Megathura crenulata. (C ) 1999 Academic Press.