The extracellular hemoglobin of the earthworm, Lumbricus terrestris. Oxygenation properties of isolated chains, trimer, and a reassociated product.

The extracellular hemoglobin of the earthworm Lumbricus terrestris has a two-tiered hexagonal structure that can be dissociated into 1/12 subunits. The Hb contains four major kinds of oxygen-binding chains, a, b, c, and d, of which a-c form a disulfide-linked trimer. Additional non-heme chains are necessary for the assembly of the intact 3800-kDa molecule of approximately 200 subunits. Oxygen equilibria have been measured for chains c and d, the abc trimer, the partially reassembled product of addition of chain d to the trimer, and the intact molecule. The results show that oxygenation of the trimer but not the isolated c or d subunits is modulated by both pH and Ca2+ ions. Cooperativity of oxygen binding by the trimer is low (Hill coefficient approximately 1.3). However, addition of chain d results in a substantial decrease in oxygen affinity and a large increase in cooperativity so that the oxygen equilibrium becomes indistinguishable from that of the intact native molecule at pH 6.8. Light-scattering data show that the smallest observed trimeric abc unit is the dimer (abc)2 at pH 6.8. Analysis of the major sedimentation velocity boundary of the product of the abc unit and chain d in the CO form in the absence of calcium surprisingly can be accounted for entirely in terms of a nondissociating dimer, (abc)2, and chain d. The data for the CO form in the presence of calcium are best fitted in terms of (abc)2.d. Although both subunits c and d also form dimers, oxygen binding by subunit c, but not d, is highly cooperative. These observations, taken together, suggest that the two dimers (abc)2 and d2 are likely to be the major participants in forming the primary functional unit, (abcd)2, which at pH 7.4 is partially dissociated when in the CO form. Subunit d is clearly necessary for the formation of a cooperative unit. The hypothesis that (abcd)2 is a primary functional unit is consistent with a stoichiometry of 2 (abcd)2 units per 1/12 subunit or 24 such units in each molecule of Hb which would contain, in all, 192 heme-containing chains.