PAGE, A.J.*; MCNEILL, J.M.; VANDERGON, T.L.; RIGGS, C.K.; RIGGS, A.F.: Cerebratulus lacteus Body Wall and Neural Hemoglobins Associate to Form Dimers or Tetramers.

The nemertean Cerebratulus lacteus expresses hemogobin (Hb) in neural and body wall muscle tissues. Each tissue expresses a unique globin from separate genes encoding proteins with 109 amino acid residues, making them the smallest known Hbs. Relative to animal Hbs, both C. lacteus Hbs are truncated in regions corresponding to the A, B and H helices of a typical folded Hb. Both C. lacteus Hbs are known to have relatively high oxygen affinities and were shown in thin-layer oxygen binding experiments to have apparent high cooperativity. The Hill coefficients were about 2.8 and 2.0 for the neural and body wall Hbs, respectively. These data suggest the presence of subunit aggregation in the Hbs that would be required for cooperativity. The neural globin was recently cloned and expressed in E. coli in the laboratory of Luc Moens, Univ. of Antwerp. We determined the subunit aggregation state of both the body wall Hb and the recombinant neural Hb using laser light scattering and SDS-PAGE. We determined that the aggregation state of the C. lacteus recombinant neural Hb is a tetramer and the native body wall Hb is a mixture of dimers and monomers. These aggregated Hb subunits are not disulfide linked. Electrospray ionization mass spectrometry was used to determine the masses of the monomeric recombinant neural and native body wall globins (minus the hemes). The measured globin masses were 11,529 and 11,225 daltons, respectively. These values are within one a.m.u. of the values calculated from the amino acid sequences deduced from the gene sequences. These data support the globin sequences previously reported.