The molecular dimensions of the extracellular hemoglobin of the leech Macrobdella decora, determined by scanning transmission electron microscopy were 29·8 nm × 19·5 nm (diameter × height) for negatively stained specimens. Measurements of molecular mass (Mm) of unstained specimens with the microscope gave Mm = 3560 ± 160 kDa. Small-angle X-ray scattering measurements gave a diameter of 28·0(±0·5) nm, radius of gyration 10·5(±0·2) nm and volume 7500(±300) nm3. The hemoglobin had no carbohydrate and its iron content was found to be 0·23(±0·02)% (w/w), corresponding to a minimum Mm of 24,000(±1300) kDa. SDS/polyacrylamide gel electrophoresis of the unreduced hemoglobin showed that it consisted of three subunits, which have apparent Mm values of 12 (1), 25 (2) and 29 kDa (3). The reduced hemoglobin consisted of four subunits, I (12 kDa), II(14 kDa), III (26 kDa) and IV (30 kDa). Subunit 1 corresponded to subunit I, subunit 2 to subunits III and IV and subunit 3 to subunit II. Partial N-terminal sequences were obtained for subunit 1, the two chains of subunit 2 and one of the two chains of subunit 3, suggesting that the hemoglobin consists of at least five different polypeptide chains. The percentage fraction of the three unreduced subunits was determined by densitometry of SDS/polyacrylamide gel patterns and quantitative determination of Coomassie R-250 dye bound to the individual bands in reduced and unreduced patterns to be, monomer (subunit I) : non-reducible subunit (subunit 2) : reducible dimer (subunit 3) = 0·35 : 0·29 : 0·35 (s.d. = ±0·05). This corresponded to a stoichiometry of 74 ± 11 : 37 ± 5 : 38 ± 6, assuming the molecular masses to be 17 kDa, 30 kDa and 34 kDa, taking into account the anomalously high mobility of annelid globins in SDS-containing gels. The stoichiometry calculated from the amino acid compositions of the hemoglobin and the three subunits was 82 ± 12 : 29 ± 4 : 40 ± 8. Gel filtration of the hemoglobin at pH 9·8, at neutral pH subsequent to dissociation at pH 4 and at neutral pH in the presence of urea and Gu · HCl provided no evidence for the existence of a putative 1/12 of the whole molecule (Mm approx. 300 kDa). Furthermore, the largest subunits obtained had Mm of 60 to 100 kDa and had a much decreased content of subunit 2, suggesting that the hemoglobin was not a simple multimeric protein. Three-dimensional reconstruction from microscope images provided a model of Macrobdella hemoglobin that is very similar to the reconstruction of Lumbricus hemoglobin: the radial mass distribution curves are virtually superimposable. The quaternary structure of Macrobdella is thus likely to be very similar to that of Lumbricus hemoglobin and can be described by the "bracelet" model, in which a scaffolding consisting of approximately 36 copies of subunit 2, the "structural", non-heme-containing subunit, is decorated with 12 identical complexes (approx. 200 kDa) of "functional", heme-containing chains, each consisting of six copies of subunit 1 and three copies of subunit 3, to give the appearance of a hexagonal bilayer structure. This model is consistent with the Mm values derived by microscopy and explains the low iron content, typical of annelid hemoglobins. © 1990 Academic Press Limited.
