A dodecamer of globin chains is the principal functional subunit of the extracellular hemoglobin of Lumbricus terrestris

Repeated dissociation of the approximately 3600-kDa hexagonal bilayer extracellular hemoglobin of Lumbricus terrestris in 4 M urea followed by gel filtration at neutral pH produces a subunit that retains the oxygen affinity of the native molecule (approximately 12 torr), but only two-thirds of the cooperativity (nmax = 2.1 +/- 0.2 versus 3.3 +/- 0.3). The mass of this subunit was estimated to be 202 +/- 15 kDa by gel filtration and 202 +/- 26 kDa from mass measurements of unstained freeze-dried specimens by scanning transmission electron microscopy. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this subunit showed that it consists predominantly of the heme-containing subunits M (chain I, 17 kDa) and T (disulfide-bonded chains II-IV, 50 kDa). Mixing of subunits M and T isolated concurrently with the 200-kDa subunit resulted in partial association into particles that had a mass of 191 +/- 13 kDa determined by gel filtration and 200 +/- 38 kDa determined by scanning transmission electron microscopy and whose oxygen affinity and cooperativity were the same as those of the 200-kDa subunit. The results imply that the 200-kDa subunit is a dodecamer of globin chains, consisting of three copies each of subunits M and T (3 x chains (I + II + III + IV], in good agreement with the mass of 209 kDa calculated from the amino acid sequences of the four chains, and represents the largest functional subunit of Lumbricus hemoglobin. Twelve copies of this subunit would account for two-thirds of the total mass of the molecule, as suggested earlier (Vinogradov, S. N., Lugo, S. L., Mainwaring, M. G., Kapp, O. H., and Crewe, A. V. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 8034-8038). The retention of only partial cooperativity by the 200-kDa subunit implies that full cooperativity is dependent on the presence of a complete hexagonal bilayer structure, wherein 12 200-kDa subunits are linked together by approximately 30-kDa heme-deficient chains.     

The amino acid sequences of chains a, b, and c that form the trimer subunit of the extracellular hemoglobin from Lumbricus terrestris

The extracellular hemoglobin of Lumbricus terrestris comprises four major heme-containing chains, a, b, c, and d in equal proportions. We have determined the amino acid sequences of chains a, b, and c which form a disulfide-linked trimer. Chains a, b, and c have 151, 145, and 153 residues and calculated molecular weights of 17,525, 16,254, and 17,289, respectively. The sequence of chain b, reported previously (Garlick, R. L., and Riggs, A. F. (1982) J. Biol. Chem. 287, 9005-9015) has been completely redetermined and found to contain 12 fewer residues than originally reported. Chains a and c both contain unusual, highly polar NH2-terminal extensions of 7 residues before the A helix. These segments must be close together because they are joined by a disulfide bond. We suggest that this structure, with seven negatively charged groups, may be part of a functionally important Ca2+-binding site in the trimer. Comparison of the sequences of chains a, b, and c with those of chain d (Shishikura, F., Snow, J. W., Gotoh, T., Vinogradov, S. N., and Walz, D. A. (1987) J. Biol. Chem. 262, 3123-3131) and the four chains of the hemoglobin of Tylorrhynchus heterochaetus (Suzuki, T., and Gotoh, T. (1986) J. Biol. Chem. 261, 9257-9267) shows that the number and positions of the cysteinyl residues are all conserved. This suggests that the extracellular hemoglobins from both the Oligochaeta and Polychaeta have the same number and configuration of disulfide bonds within the molecule. Phylogenetic analysis suggests that gene duplication first generated an intracellular hemoglobin branch and an extracellular hemoglobin branch. DNA coding for a signal peptide would have been acquired by the extracellular globin gene after this event. At least two further gene duplications are required to account for the present four polypeptide chains.     

Amino acid sequence of the monomer subunit of the extracellular hemoglobin of Lumbricus terrestris

The giant extracellular hemoglobin (3,800 kDa) of the oligochaete Lumbricus terrestris consists of four subunits: a monomer (chain I), two subunits each of about 35 kDa (chains V and VI), and a disulfide-bonded trimer (50 kDa) of chains II, III, and IV. The complete amino acid sequence of chain I was determined: it consists of 142 amino acid residues and has a molecular weight of 16,750 including a heme group. Fifty-nine residues (42%) were found to be identical with those in the corresponding positions in Lumbricus chain II (Garlick, R. L., and Riggs, A. F. (1982) J. Biol. Chem. 257, 9005-9015); 45 (32%), 56 (40%), 44 (31%), and 45 (32%) residues were found to be in identical positions in the sequences of chains I, IIA, IIB, and IIC, respectively, of Tylorrhynchus heterochaetus hemoglobin (Suzuki, T., and Gotoh, T. (1986) J. Biol. Chem. 261, 9257-9267). When the sequences of all six annelid chains are compared, 18 invariant residues are found in the first 104 residues of the molecule; very little homology exists among the annelid chains in the carboxyl-terminal 38-residue region. Nine of the 18 invariant residues are also found in the human beta-globin chain.     

The extracellular hemoglobin of the earthworm, Lumbricus terrestris. Oxygenation properties of isolated chains, trimer, and a reassociated product

The extracellular hemoglobin of the earthworm Lumbricus terrestris has a two-tiered hexagonal structure that can be dissociated into 1/12 subunits. The Hb contains four major kinds of oxygen-binding chains, a, b, c, and d, of which a-c form a disulfide-linked trimer. Additional non-heme chains are necessary for the assembly of the intact 3800-kDa molecule of approximately 200 subunits. Oxygen equilibria have been measured for chains c and d, the abc trimer, the partially reassembled product of addition of chain d to the trimer, and the intact molecule. The results show that oxygenation of the trimer but not the isolated c or d subunits is modulated by both pH and Ca2+ ions. Cooperativity of oxygen binding by the trimer is low (Hill coefficient approximately 1.3). However, addition of chain d results in a substantial decrease in oxygen affinity and a large increase in cooperativity so that the oxygen equilibrium becomes indistinguishable from that of the intact native molecule at pH 6.8. Light-scattering data show that the smallest observed trimeric abc unit is the dimer (abc)2 at pH 6.8. Analysis of the major sedimentation velocity boundary of the product of the abc unit and chain d in the CO form in the absence of calcium surprisingly can be accounted for entirely in terms of a nondissociating dimer, (abc)2, and chain d. The data for the CO form in the presence of calcium are best fitted in terms of (abc)2.d. Although both subunits c and d also form dimers, oxygen binding by subunit c, but not d, is highly cooperative. These observations, taken together, suggest that the two dimers (abc)2 and d2 are likely to be the major participants in forming the primary functional unit, (abcd)2, which at pH 7.4 is partially dissociated when in the CO form. Subunit d is clearly necessary for the formation of a cooperative unit. The hypothesis that (abcd)2 is a primary functional unit is consistent with a stoichiometry of 2 (abcd)2 units per 1/12 subunit or 24 such units in each molecule of Hb which would contain, in all, 192 heme-containing chains.     

Heterogeneity of the subunits of Lumbricus terrestris extracellular hemoglobin dissociated at alkaline pH

1. The gel filtration profiles of dissociated extracellular hemoglobin of Lumbricus terrestris obtained in 0.1 M borate buffers at pH greater than 9, using columns of Sephadex G100, Sephacryl S200 and Ultragel AcA 44, consist of at least two peaks A and B. 2. SDS-PAGE of several fractions across the complete elution profile demonstrates that only the fractions under the right hand portion of peak B are homogenous and consist of the monomer (M) subunit (Mr = 17,000). 3. The fractions under the first peak contain the remaining subunits, a disulfide-bonded trimer (T) subunit (Mr = 50,000) and of two subunits (D1 and D2) of ca 30,000. 4. Densitometry of the SDS-PAGE patterns suggests that the proportions of these subunits vary across the two peaks, implying that peak A does not consist of a complex of fixed stoichiometry of the T and D1 and D2 subunits. 5. Furthermore, the D1 and D2 subunits overlap the M subunit in the trough between peaks A and B and are present in the left hand portion of peak B, probably because of the self-association of the M subunit. 6. In addition, SDS-PAGE experiments with a single fraction of peak A, where the load and the duration of staining were varied, suggests that the relative proportions of the subunits are independent of these two variables.     

Preparation and characterization of monoclonal antibodies to the extracellular hemoglobin of Lumbricus terrestris

Murine monoclonal antibodies to the extracellular hemoglobin of Lumbricus terrestris were prepared by a modification of the method of Kohler and Milstein. 224 hybridomas were found to produce antibodies which bound to the hemoglobin; they were tested for binding to the four subunits of the hemoglobin: M (chain I, 16 kDa), D1 (chain V, 31 kDa), D2 (chain VI, 37 kDa) and T (50 kDa), a disulfide-bonded trimer of chains II, III and IV, each of about 17 kDa. 150 hybridomas bound to all four subunits and 40 hybridomas bound to various combinations of subunits. The remaining 34 hybridomas combined only with the hemoglobin. The twelve hybridomas obtained after subculturing and cloning were tested for their binding to the two fractions II and III, consisting of subunits D1 + D2 + T and M, respectively, obtained by dissociation at pH 9.5 and at pH 4.0 and to the reassociated whole molecules, obtained subsequent to return to neutral pH. Eight hybridomas which combined only with the hemoglobin also combined with all the reassociated molecules but not with any of the fractions: these monoclonal antibodies probably recognize conformation-dependent antigenic sites that are present only in the hexagonal bilayer structure characteristic of the native and reassociated hemoglobin molecules. Of the remaining four hybridomas, two bound to subunit T and two combined with subunits T and D2; they also combined with the reassociated molecules and with the fractions II. In addition, the hybridomas did not bind to the hemoglobins of Tubifex, Limnodrilus, Arenicola, Tylorrhynchus and Macrobdella or to the chlorocruorins of Myxicola and Eudistylia.     

The effects of salts on the subunit structure and dissociation of Lumbricus terrestris hemoglobin.

The effects of the neutral salts of the Hofmeister series, NaCl, NaClO4, MgCl2, NaI, and also guanidine hydrochloride (Gdn-HCl)on the subunit organization and the state of association of Lumbricus terrestris hemoglobin were examined by light scattering molecular weight measurements. The subunit dissociation of the parent duodecameric structure of 3 x 10(6) molecular weight by various salts is similar in pattern to the sequential splitting of the associated protein to half-molecules of hexamers of 1.5 x 10(6) molecular weight, followed by further dissociation at higher reagent concentration to monomers of 250000 molecular weight. Duodecamer to hexamer dissociation is observed in 0.4 M MgCl2, 1-2 M NaCl, and 1 M Gdn-HCl, while hexamer to monomer dissociation is seen in the presence of 1 M MgCl2. All three species of duodecamers, hexamers, and monomers seem to be present in 1 M NaClO4. Further splitting of the monomers of A subunits to smaller B fragments of one-third to one-quarter molecular weight is observed in 1 M NaI solutions. Optical rotation in the peptide region and absorption measurements in the Soret region indicate the salt dissociation of Lumbricus terrestris hemoglobin is not accompanied by major changes in the folding of the subunits, except in the case of the strong protein denaturant, Gdn-HCl. Relative to the dissociation effects of the urea series of compounds reported in the preceding paper (Herskovits and Harrington, 1975), the neutral salts appear to be much more effective dissociating agents for L. terrestris hemoglobin. This suggests that polar and ionic interactions are relatively more important for the maintenance of the protein than hydrophobic interactions. This conclusion is also supported by calculations of the possible effects of binding of NaClO4, based on the Setschenow constants of the literature describing the interaction of salts with the peptide and hydrophobic alkyl group of the average amino acid found in proteins, on the standard free energy of dissociation of the duodecamer to hexamer.     

Subunit distribution of calcium-binding sites in Lumbricus terrestris hemoglobin

The giant, 3.6-MDa hexagonal bilayer hemoglobin (Hb) of Lumbricus terrestris consist of twelve 213-kDa globin subassemblies, each comprised of three disulfide-bonded trimers and three monomer globin chains, tethered to a central scaffolding of 36–42 linkers L1–L4 (24–32 kDa). It is known to contain 50–80 Ca and 2–4 Cu and Zn; the latter are thought to be responsible for the superoxide dismutase activity of the Hb. Total reflection X-ray fluorescence spectrometry was used to determine the Ca, Cu, and Zn contents of the Hb dissociated at pH 2.2, the globin dodecamer subassembly, and linker subunits L2 and L4. Although the dissociated Hb retained 20 Ca²⁺ and all the Cu and Zn, the globin subassembly had 0.4 to 3 Ca²⁺, depending on the method of isolation, and only traces of Cu and Zn. The linkers L2 and L4, isolated by reversed-phase high-pressure liquid chromatography at pH 2.2, had 1 Ca per mole and very little Cu and Zn. Electrospray ionization mass spectrometry of linker L3 at pH 2.2 and at neutral pH demonstrated avid binding of 1 Ca²⁺ and additional weaker binding of 7 Ca²⁺ in the presence of added Ca²⁺. Based on these and previous results which document the heterogeneous nature of the Ca²⁺-binding sites in Lumbricus Hb, we propose three classes of Ca²⁺-binding sites with affinities increasing in the following order: (i) a large number of sites (>100) with affinities lower than EDTA associated with linker L3 and dodecamer subassembly, (ii) 30 sites with affinities higher than EDTA occurring within the cysteine-rich domains of linker L3 and dodecamer subassembly, and (iii) 25 very high affinity sites associated with the linker subunits L1, L2, and L4. It is likely that the low-affinity type (i) sites are the ones involved in the effects of 1–100 mM Group IIA cations on Lumbricus Hb structure and function, namely increased stability of its quaternary structure and increased affinity and cooperativity of its oxygen binding.     

Three-dimensional reconstruction of Lumbricus terrestris hemoglobin at 22 A resolution: intramolecular localization of the globin and linker chains.

A 3D reconstruction of the hemoglobin (Hb) of the earthworm Lumbricus terrestris was carried out by the 3D projection alignment method from electron microscopy images of a frozen-hydrated specimen at 22 A resolution. The results were analyzed by a new approach taking into account the evolution of the 210 densities forming the 3D volume as a function of the threshold of surface representation. The whole oligomer with D6point-group symmetry is comprised of 12 hollow globular substructures (HGS) with local 3-fold symmetry tethered to a complex network of linking subunits (linker complex). The 12 globin subunits of each HGS are distributed around local 3-fold axis in four layers of three subunits. The first layer, the most external, contains monomeric globin chains 2A, 3A, and 5A. The three trimers corresponding to the nine remaining subunits have one subunit in each of the second (2B, 3B, 5B), third (1A, 4A, 6A), and fourth (1B, 4B, 6B) layer. The distances between the centers of the globin chains forming the trimers are in the ranges 20-32 A and 45-52 A. The linker complex is made up of two types of linking units. The first type forms three loops connecting globin chains of the second, third and fourth layers. The average molecular mass (Mm) of these subunits was 25 kDa. The second type forms the central structure, termed hexagonal toroid, and its 12 connections to the HGS. This structure corresponds to a hexamer of a single linking unit with a Mm (31.2 kDa), size and a shape different from those of the HGS loops. A careful study of 3D volume architecture shows that each toroid linking unit is bound to the three loops of a HGS pair located in the upper and lower hexagonal layers, respectively. As shown in a model of architecture, hexagonal bilayered (HBL) Hbs can be built very simply from 144 globin chains and 42 linker chains belonging to two different types. We also propose a simple assembly sequence for the construction of HBL Hbs based on the architecture model.     

Lumbricus terrestris hemoglobin: A comparison of small-angle X-ray scattering and cryoelectron microscopy data

The quaternary structure of Lumbricus terrestris hemoglobin was investigated by small-angle x-ray scattering (SAXS). Based on the SAXS data from several independent experiments, a three-dimensional (3D) consensus model was established to simulate the solution structure of this complex protein at low resolution (about 3 nm) and to yield the particle dimensions. The model is built up from a large number of small spheres of different weights, a result of the two-step procedure used to calculate the SAXS model. It accounts for the arrangement of 12 subunits in a hexagonal bilayer structure and for an additional central unit of cylinder-like shape. This model provides an excellent fit of the experimental scattering curve of the protein up to h = 1 nm⁻¹ and a nearly perfect fit of the experimental distance distribution function p(r) in the whole range. Scattering curves and p(r) functions were also calculated for low-resolution models based on 3D reconstructions obtained by cryoelectron microscopy (EM). The calculated functions of these models also provide a very good fit of the experimental scattering curve (even at h > 1 nm⁻¹) and p(r) function, if hydration is taken into account and the original model coordinates are slightly rescaled. The comparison of models reveals that both the SAXS-based and the EM-based model lead to a similar simulation of the protein structure and to similar particle dimensions. The essential differences between the models concern the hexagonal bilayer arrangement (eclipsed in the SAXS model, one layer slightly rotated in the EM model), and the mass distribution, mainly on the surface and in the central part of the protein complex. © John Wiley & Sons, Inc. Biopoly 45: 289–298, 1998     

Kinetic light scattering studies on the dissociation of hemoglobin from Lumbricus terrestris.

The kinetics of the pH-induced dissociation of the 3 X 10(6) mol wt hemoglobin from Lumbricus terrestris (the earthworm) have been studied in a light-scattering stopped-flow apparatus. The ligand dependent dissociation data were fit well by a simple sequential model. The data for CO and oxyhemoglobin are consistent with Hb12 leads to 2Hb6 leads to 12Hb. Methemoglobin at pH 7 appears to be hexameric and the dissociation is consistent with the model: Hb6 leads to 6Hb. In a sequential decay scheme for which light-scattering changes are monitored, the relative amounts of rapid and slow phase are determined by the rate constants as well as the molecular weights of intermediate species. Assignment of the hexameric intermediate is supported by an investigation of the sensitivity of the theoretical kinetic curves to the molecular weights of the intermediates. This assignment is further supported by the following: (1) the same model will fit the data for oxy- and CO-hemoglobin at all three temperatures (a 24-29-fold variation in rate constants), (2) evidence from electron microscopy shows hexameric forms, and (3) methemoglobin is apparently stable as a hexamer at pH 7. When CO replaces O2 as the ligand, the dissociation rate increases by a factor of four. The met is about 20 times faster than the initial oxyhemoglobin dissociation rate, but perhaps more relevant for comparing dissociation of the hexamer, the met rate was respectively 100 times and 500 times faster than that for the assumed hexameric forms of CO- and oxy-hemoglobin. The activation energies for the dodecamer to hexamer dissociation and for the dissociation of the hexamer to smaller forms were about 30 kcal/mol for oxy-, CO-, and methemoglobin.     

Solution studies on heme proteins: subunit structure, dissociation, and unfolding of Lumbricus terrestris hemoglobin by the ureas.

The subunit structure, dissociation, and unfolding of the hemoglobin of the earthworm, Lumbricus terrestris, were investigated by light scattering molecular weight methods and changes in optical rotatory dispersion (at 233 nm) and absorption in the Soret region. Urea and the alkylureas, methyl-, ethyl-, propyl-, and butylurea, were employed as the reagents to cause both dissociation and unfolding of the protein. Analysis of the light scattering data suggests that the dissociation patterns as a function of hemoglobin concentration in the various dissociating solvents can be described in quantitative terms, either as an equilibrium mixture consisting of parent duodecamers and hexamers of 3 x 10(6) and 1.5 x 10(6) molecular weight (in 1-3 M urea, 1-2 M methyl- and ethylurea, and 1 M propylurea), as a mixture of hexamers and monomers, the latter with a molecular weight of 250000 (i.e., in 4 M urea), or as a mixture of all three species of duodecamers, hexamers, and monomers, seen in 2 M propylurea. Parallel studies by optical rotation and absorption measurements indicate that there is little or no unfolding of the subunits at urea and alkylurea concentrations where complete dissociation to hexamers and extensive dissociation to monomers can be achieved. Further splitting of the monomers (A subunits) to smaller fragments of one-third to one-quarter of the molecular weight of the monomers (B subunits) is seen in the presence of 7 and 8 M urea (pH 7) and in alkaline urea to propylurea solutions. Analysis of the dissociation data of duodecamers to monomers, based on equations used in studies of the urea and amide dissociation of human hemoglobin A from our laboratory, suggests few urea and alkylurea binding sites at the areas of hexamer contacts in the associated duodecameric form of L. terrestris hemoglobin. This suggests that hydrophobic interactions are not the dominant forces that govern the state of association of L. terrestris hemoglobin relative to polar and ionic interactions. The unfolding effects of the ureas, at concentrations above the dissociation transitions, are closely similar to their effects on other globular proteins, suggesting that hydrophobic interactions play an important role in the maintenance of the folded conformation of the subunits. Use of the Peller-Flory equation, with binding constants based on free energy transfer data of hydrophobic amino acid side chains and denaturation data used in previous denaturation studies, gave a relatively good acount of the observed denaturation midpoints obtained with the various ureas supporting these conclusions.     

Determination of the formal reduction potential of Lumbricus terrestris hemoglobin using thin layer spectroelectrochemistry

The formal reduction potential (Eo') of Lumbricus terrestris hemoglobin was determined using thin layer spectroelectrochemistry as 0.073 (+/-0.005) V vs Ag/AgCl (0.281 V vs SHE, standard hydrogen electrode). Nernst plots of Lumbricus terrestris hemoglobin with tris-bipyridinecobalt(II) as a mediator titrant have similar linear slopes as Nernst plots of horse heart myoglobin with hexaamineruthenium(II) as a mediator titrant.     

Lumbricus terrestris hemoglobin--the architecture of linker chains and structural variation of the central toroid

The extracellular giant hemoglobin from the earthworm Lumbricus terrestris was reconstructed at 14.9-A resolution from cryo-electron microscope images, using a new procedure for estimating parameters of the contrast transfer (CTF) function. In this approach, two important CTF parameters, defocus and amplitude contrast ratio, can be refined iteratively within the framework of 3D projection alignment procedure, using minimization of sign disagreement between theoretical CTF and cross-resolution curves. The 3D cryo-EM map is in overall good agreement with the recent X-ray crystallography map of Royer et al. (2000, Proc. Natl. Acad. Sci. USA 97, 7107-7111), and it reveals the local threefold arrangement of the three linker chains present within each 1/12 of the complex. The 144 globin chains and 36 linker chains within the complex are clearly visible, and the interdigitation of the 12 coiled-coil helical spokes forming the central toroidal piece is confirmed. Based on these findings, two mechanisms of the dodecameric unit assembly are proposed and termed "zigzag" and "pairwise" polymerizations. However, the detection by cryo-EM of 12 additional rod-like bodies within the toroid raises the possibility that the architecture of the toroid is more complex than previously thought or that yet unknown ligands or allosteric effectors for this oxygen carrier are present.     

The molecular weight of the extracellular haemoglobin of Lumbricus terrestris determined by electron microscopy

1. The mol. wt of the extracellular haemoglobin of the oligochaete Lumbricus terrestris was determined by counting in negatively stained electron micrographs. 2. The value obtained using apoferritin as a mol. wt standard is (3.8 +/- 0.3) x 10(6), in agreement with recent determinations employing different physical methods. 3. We conclude that all annelid extracellular haemoglobins and chlorocruorins which have the same dimensions as Lumbricus haemoglobin probably have the same mol. wt.     

The dissociation of the extracellular hemoglobin of Lumbricus terrestris at acid pH and its reassociation at neutral pH. A new model of its quaternary structure

Lumbricus terrestris HbO2 and HbCO dissociated below pH 5.0; a time-dependent alteration to the met form occurred at pH less than 5 and pH less than 4.5, respectively. The extent of dissociation was unaffected by alkaline earth cations but was decreased by an increase in ionic strength. HbO2 and HbCO exposed to pH 4.0-4.8 were centrifuged to obtain the undissociated pellet (P1) and dissociated supernatant (S1) fractions. S1 was reassociated at pH 7.0 by dialysis against various buffers and then centrifuged to obtain the reassociated pellet (P2) and unreassociated supernatant (S2) fractions. Reassociation was possible only if S1 was dialyzed against water prior to return to neutral pH; otherwise precipitation occurred starting at about pH 5.3. The extent of reassociation varied from about 40 to 80%, was usually higher for HbCO than HbO2, and was unaffected by an increase in ionic strength or by Ca(II). Gel filtration of P2 on Sephacryl S-300 at neutral pH gave one peak IaR, eluting at a slightly greater volume than the native Hb; S1 and S2 gave in addition, three peaks, Ib (200 kDa), II (65 kDa), and III (18 kDa). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that P2 was slightly deficient in subunit M relative to the Hb, that Ib was deficient in subunits D1 and D2 and that II and III consisted of subunits D1 + D2 + T and subunit M, respectively. Scanning transmission electron microscopy of P2 showed that it was smaller than the native hemoglobin: 25 nm in diameter and 16 nm in height, instead of 30 X 20 nm. Comparison of the results of the dissociations of Lumbricus Hb at alkaline pH (Kapp, O. H., Polidori, G., Mainwaring, M., Crewe, A. V., Vinogradov, S. N. (1984) J. Biol. Chem. 259, 628-639) with those obtained in this study suggested that the Hb quaternary structure was not multimeric and that an alternative model had to be considered. In the proposed model it is assumed that subunits D1 and D2 form a scaffolding or "bracelet," decorated with 12 complexes of M and T subunits.