Assembly of the gigantic hemoglobin of the earthworm Lumbricus terrestris. Roles of subunit equilibria, non-globin linker chains, and valence of the heme iron.
The extracellular hemoglobin of the earthworm Lumbricus terrestris has four major kinds of O2-binding chains: a, b, and c (forming a disulfide-linked trimer), and chain d. Non-heme, non-globin structural chains, "linkers," are also present. Light-scattering techniques have been used to show that the ferrous CO-saturated abc trimer and chain d form an (abcd)4 complex of 285 kDa at neutral pH. Formation of the full-sized 4-MDa molecule requires the addition of linker chains in the proportion of two linkers per (abcd)4 and occurs much more rapidly in the presence of 10 mM calcium. This stoichiometry is supported not only by direct quantitative analysis of the intact hemoglobin but also by the fact that the addition of 50% of the proposed stoichiometric quantity of linkers results in the conversion of 50% of the (abcd)4 to full-sized molecules. Isolated CO-saturated abc trimers self-associate to (abc)2 and higher aggregates up to an apparent limit of (abc)10 approximately 550 kDa. The CO-saturated chain d forms dimers, (d)2, and tetramers, (d)4. Oxidation of the (abcd)4 complex with ferricyanide causes complete dissociation of chain d from the abc trimer, but addition of CN- maintains the (abcd)4 complex. Valence hybrids have also been studied. The ferrous CO-saturated abc trimer and met (ferric) chain d also associate to form (abcd)4, but the met abc trimer and ferrous CO-saturated chain d do not. Oxidation of the abc trimer and chain d to the ferric form causes the formation of a characteristic hemichrome spectrum with a maximum at 565 nm and a shoulder near 530 nm. These results show that interactions between the abc trimer and chain d are strongly dependent on the ligand and valence state of the heme iron. Light-scattering measurements reveal that oxidation of the intact Hb produces a significant drop in molecular mass from 4.1 to 3.6 MDa. Inclusion of CN- prevents this drop. These experiments indicate that oxidation causes the Hb to shed subunits. The observations provide an explanation for the wide variations in the molecular mass of L. terrestris Hb that have been observed previously.
Zhu
H
H
Department of Zoology, University of Texas, Austin, Texas 78712-1064, USA. Riggs@uts.cc.utexas.edu
Ownby
D W
DW
Riggs
C K
CK
Nolasco
N J
NJ
Stoops
J K
JK
Riggs
A F
AF
D000426:Alcohol Dehydrogenase / Q000737:chemistry; D000818:Animals; D001052:Apoferritins / Q000737:chemistry; D002256:Carbonic Anhydrases / Q000737:chemistry; D006418:Heme / Q000737:chemistry*; D006454:Hemoglobins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008027:Light; D008970:Molecular Weight; D009835:Oligochaeta / Q000737:chemistry*; D009955:Ornithine Decarboxylase / Q000737:chemistry; D010084:Oxidation-Reduction; D012542:Scattering, Radiation; D013057:Spectrum Analysis; D013954:Thyroglobulin / Q000737:chemistry
D006454:Hemoglobins; D006418:Heme; D013954:Thyroglobulin; D001052:Apoferritins; D000426:Alcohol Dehydrogenase; D009955:Ornithine Decarboxylase; D002256:Carbonic Anhydrases