A study of the subunit structure of the extracellular hemoglobin of Lumbricus terrestris.

The dissociation of the extracellular hemoglobin of the earthworm Lumbricus terrestris was investigated. At neutral pH, its sz00,~ = 58.9 f 0.2 S and D:o,w = 1.61 f 0.05 X 1O-7 cm2 s-l corresponded to a molecular weight of 3.23 f 0.18 x 106. A minimum molecular weight of 24,000 per heme group was determined from the iron and heme contents, 0.221 f 0.011 weight % and 2.78 =k 0.14 weight %, respectively. Gel filtration in sodium dodecyl sulfate and acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed the presence of two major fragments possessing molecular weights of 52,000 f 2,000 and 12,000 f 1,000. The larger of the two subunits dissociated upon sodium dodecyl sulfate electrophoresis in mercaptoethanol into three bands corresponding to molecular weights of 14,000 i 1,000, 16,000 f 1,000, and 19,000 f 1,000. Sodium dodecyl sulfate electrophoresis of Lumbricus hemoglobin in the presence of mercaptoethanol provided a reproducible pattern of six components, four bands with molecular weights of 12,000, 14,000, 16,000, and 19,000, respectively, and two bands with molecular weights of about 31,000 and 36,000. Partial amino acid compositions of the electrophoretically isolated subunits suggested the presence of six different polypeptide chains in earthworm hemoglobin. It is probable that not all of the polypeptide chains in the native molecule are involved in heme binding.