Bracelet protein: a quaternary structure proposed for the giant extracellular hemoglobin of Lumbricus terrestris.

Abstract

The complete dissociation of the hexagonal bilayer structure of Lumbricus terrestris hemoglobin (3900 kDa) at neutral pH, in the presence of urea, guanidine hydrochloride, sodium perchlorate, potassium thiocyanate, sodium phosphotungstate, and sodium phosphomolybdate, followed by gel filtration at neutral pH on Sephacryl S-200 or Superose 6, produced two fragments, II (65 kDa) and III (17 kDa); NaDodSO4/polyacrylamide gel electrophoresis showed that peak II consisted of subunits D1 (31 kDa, chain V), D2 (37 kDa, chain VI), and T (50 kDa, disulfide-bonded trimer of chains II, III, and IV) and that peak II consisted of subunit M (16 kDa, chain I). When dissociation was incomplete, two additional peaks were present, peak Ia eluting at the same volume as the whole hemoglobin and peak Ib (200 kDa). Scanning transmission electron micrographs of peak Ia showed it to consist of whole molecules and of incomplete hexagonal bilayer structures, missing an apparent 1/12th. Peak Ib contained all four subunits but was usually deficient in subunits D1 and D2, was not always in equilibrium with the whole molecule, and could be dissociated further into II and III. The patterns of dissociation observed at neutral pH were very similar to those observed previously at alkaline pH and at acid pH and appear to be incompatible with the generally accepted multimeric model of Lumbricus hemoglobin subunit structure. A model is proposed in which it is postulated that the stoichiometries of some of the subunits need not be constant and that subunits D1 and D2 either form a "bracelet" decorated with complexes of T and M subunits or serve as "linkers" between the latter, to provide the appearance of a two-tiered hexagonal structure. Additional support for the proposed model comes from observations that the fragment II obtained subsequent to dissociation at pH 4, in sodium phosphotungstate, in sodium perchlorate, and in potassium thiocyanate was found to be in equilibrium with a hexagonal bilayer structure IaR(II), whose dimensions were approximately equal to 20% smaller than those of the native hemoglobin.