Non-heme protein in the giant extracellular hemoglobin of the earthworm Lumbricus terrestris.

The protein/heme mass ratio for the extracellular hemoglobin of the earthworm Lumbricus terrestris has been redetermined. We find a value of 19,000 g of protein per mol of heme. Four major, heme-containing chains (a, b, c, and d), present in equal proportions, have a total molecular mass, with four hemes, of 69,664 Da based on their sequences. The intact hemoglobin comprises 12 subunits that form a two-layered hexagonal structure of about 3.8 MDa. This value, together with our determination of the protein/heme ratio, requires that 4 abcd units are present in each 1/12th subunit and that 192 heme-containing chains are present in the hemoglobin molecule. Our data indicate that approximately 2200 g of non-heme protein is present for each mole of heme-containing chain, or about 35,200 g per 1/12th subunit. This conclusion is consistent with the observation that chains of 31-37 kDa are present. On this basis the intact molecule would have 12 non-heme chains and 204 chains in all to give a total molecular mass of 3.77 MDa, close to that observed.