The extracellular hemoglobin of the earthworm, Lumbricus terrestris: Determination of subunit stoichiometry

The giant extracellular hemoglobin of the earth-worm, Lumbricus terrestris, has four major 02-bind-ing chains, a, b, and c (forming a disulfide-linked tri-mer) and d (“monomer”). Participation of additional ”linker ” chains L1, L2, and LS is necessary for the assembly of the =3,900+ kDa two-tiered hexagonal structure. We have determined the proportions of linker chains, trimer, and chain d in the hemoglobin by reverse phase high performance liquid chromatog-raphy which resolves all of the components and also permits simultaneous determination of the heme con-tent. The proportions of components were determined by two independent procedures: integration of the ab-sorbance peaks at 220 nm and amino acid analysis of the peak fractions. The results indicate hat the weigh