Stoichiometry of Subunits and Heme Content of Hemoglobin from the Earthworm Lumbricus terrestris*

The extracellular hemoglobin (Hb) of the earthworm, Lumbricus terrestris, has four major O2-binding chains, a, b, c (forming a disulfide-linked trimer), and d (“mon-omer”). Additional structural chains, “linkers, ” are re-quired for the assembly of the;200-polypeptide mole-cule. The proportion of linker chains had been reported to be one-third of the total mass on the basis of densi-tometry of Coomassie Blue-stained SDS-gels. Reverse-phase high performance liquid chromatography (HPLC), however, gave 16.3 % linkers on the basis of both 220-nm absorbance and amino acid analysis (Ownby, D