Stoichiometry of Subunits and Heme Content of Hemoglobin from the Earthworm Lumbricus terrestris

Zhu, Hao; Hargrove, Mark S.; Xie, Qiang; Nozaki, Yoshiyuki; Linse, Klaus D.; Smith, Sandra Susan; Olson, John S.; Riggs, Austen

doi:10.1074/jbc.271.47.29999

Cited by 32 publications

(37 citation statements)

References 35 publications

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“…2 The weight proportions of linkers were found to be approximately 16.4% by two independent procedures: the integrated absorption of the HPLC peaks and amino acid analysis of these peaks. These results led to the conclusion that the overall stoichiometry is (abcd) 2 L. This stoichiometry is further supported by the results of SDS-capillary gel electrophoresis monitored at 214 nm (see our companion study (16)). …”

supporting

confidence: 63%

“…(ii) In the second set of experiments, HbO 2 (freshly prepared, unfrozen) with and without calcium was prepared with Ca 2ϩ , 25 mM bis-tris propane, 100 mM NaCl, 10 mM CaCl 2 , pH 7.0, or without Ca 2ϩ , in 5 mM sodium phosphate, 100 mM NaCl, 1 mM EDTA, pH 7.0. The latter preparation without calcium is the same solution used for the dry weight determinations (16). Aliquots of each of these solutions were oxidized in the dark by adding K 3 Fe(CN) 6 as 5, 10, 25, 50, 75, 90, or 100% of the molar quantity of heme-iron.…”

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confidence: 99%

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Assembly of the Gigantic Hemoglobin of the Earthworm Lumbricus terrestris

Zhu

Ownby

Riggs

et al. 1996

Journal of Biological Chemistry

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The extracellular hemoglobin of the earthworm Lumbricus terrestris has four major kinds of O 2 -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 COsaturated abc trimers self-associate to (abc) 2 and higher aggregates up to an apparent limit of (abc) 10 ϳ550 kDa. The extracellular hemoglobins of annelids were first shown by Svedberg and Eriksson (1) to be gigantic molecules of at least 3 MDa in molecular mass. Although several subsequent studies gave molecular masses close to 4 MDa or higher for the Hb of Lumbricus terrestris and related species (2-4), recent studies by scanning transmission electron microscopy (5) have suggested masses of 3.5-3.6 MDa, and early light-scattering measurements suggested even lower values (6, 7). A possible reason for this great variation in reported molecular masses is oxidation which has been shown to cause extensive dissociation of many annelid Hbs (8 -10). Goss et al. (9) used light scattering to show that oxidation of L. terrestris Hb at neutral pH caused a large drop in molecular weight. Similarly, Ascoli et al. (10) found that oxidation of the similar Hb from Octolasium complanatum 1 caused hemichrome formation and dissociation outside of a narrow range near pH 7.The stoichiometry has also been uncertain. The Hb of L. terrestris has four major kinds of O 2 -binding globin chains: a, b, and c (forming the disulfide-linked abc trimer) and chain d, together with non-heme structural chains, "linkers," designated L. Kapp et al. (11) and Vinogradov et al. (12) have reported that linkers comprise 33-36% of the total mass, but this conclusion was based largely on the staining of SDS-gels with Coomassie Blue. This dye, however, binds to different proteins to quite different extents (13,14). Ownby et al. (15) redetermined the stoichiometry by reverse-phase high performance liquid chromatography (HPLC).2 The weight proportions of linkers were found to be approximately 16.4% by two independent procedures: the integrated absorption of the HPLC peaks and amino acid analysis of these peaks. These results led to the conclusion that the overall stoichiometry is (abcd) 2 L. This stoichiometry is further supported by the results of SDS-capillary gel electrophoresis monitored at 214 nm (see our companion study (16)).The goals of the present experiments are to redetermine the molecu...

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supporting

confidence: 63%

mentioning

confidence: 99%

Assembly of the Gigantic Hemoglobin of the Earthworm Lumbricus terrestris

Zhu

Ownby

Riggs

et al. 1996

Journal of Biological Chemistry

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“…In 1991, Fushitani and Riggs reassembled at a neutral pH the trimer and monomer subunits isolated by dissociation of the Hb at pH Ͼ 9, observed a 5.8 S species using sedimentation velocity, and suggested it to be an octamer globin complex M 2 T 2 . In subsequent experiments, the CO forms of the trimer and monomer subunits isolated by dissociation at an alkaline pH were mixed at a neutral pH to obtain a subassembly with a mass of ϳ280 kDa as determined by multiple angle laser light scattering, in agreement with a mass of 286 kDa calculated for a hexadecamer [M] 4 [T] 4 (22)(23)(24) (22)(23)(24). Apart from the fact that the proposed globin to linker stoichiometry corresponds to iron and heme contents that are higher than those generally observed for over 30 HBL Hbs by many different investigators (4,32), the most telling shortcoming of this model is that the proposed hexadecamer subassembly cannot have a 3-fold symmetry and is thus unable to account for the 12 local 3-fold axes observed in the threedimensional reconstructions based on cryoelectron microscopy images in frozen, hydrated samples of Lumbricus Hb obtained by the groups of Van Heel and colleagues (16) and Lamy and colleagues (21).…”

Section: Discussionmentioning

confidence: 94%

“…Based on the finding of a ϳ200-kDa globin subassembly upon mild, partial dissociation of the Hb at neutral pH, a "bracelet" model of its quaternary structure was proposed to consist of twelve ϳ200-kDa globin subassemblies attached to a central scaffolding of 36 -42 linker chains (24 -32 kDa) (5). Scanning transmission electron microscopy mass mapping of the isolated globin subassembly showed it to have a mass of 202 Ϯ 26 kDa, consonant with it being a dodecamer of globin chains (ϳ17 ϫ 12 ϭ 204 kDa), [d] [22][23][24]. Based on these results, a Hb model was proposed consisting of 12 hexadecamer subassemblies and 24 linker chains.…”

mentioning

confidence: 99%

“…Furthermore, three-dimensional reconstructions of several HBL Hbs using cryoelectron microscopy have demonstrated unequivocally the overall correctness of the bracelet model (16 -21). Concurrent studies of Lumbricus Hb and its reassembly subsequent to dissociation at an alkaline pH by A. F. Riggs and his group have yielded quite different results (22)(23)(24). Multiple angle laser light scattering was used to determine the mass of the Hb, 4.1 Ϯ 0.1 MDa, and reassembly of the isolated M and T subunits was interpreted to indicate a hexadecamer subassembly [M] 4 [T] 4 , ϳ285 kDa (22)(23)(24).…”

mentioning

confidence: 99%

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Electrospray Ionization Mass Spectrometric Determination of the Molecular Mass of the ∼200-kDa Globin Dodecamer Subassemblies in Hexagonal Bilayer Hemoglobins

Green¹,

Bordoli²,

Hanin³

et al. 1999

Journal of Biological Chemistry

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The giant extracellular HBL 1 Hbs found in most terrestrial, aquatic, and marine annelids and in deep sea annelids and vestimentiferans are ϳ3.6-MDa complexes of globin subunits and nonglobin, linker chains and represent a summit of complexity for oxygen-binding heme proteins (1-4). The most extensively studied complex is the Hb from the common earthworm Lumbricus terrestris. Based on the finding of a ϳ200-kDa globin subassembly upon mild, partial dissociation of the Hb at neutral pH, a "bracelet" model of its quaternary structure was proposed to consist of twelve ϳ200-kDa globin subassemblies attached to a central scaffolding of 36 -42 linker chains (24 -32 kDa) (5). Scanning transmission electron microscopy mass mapping of the isolated globin subassembly showed it to have a mass of 202 Ϯ 26 kDa, consonant with it being a dodecamer of globin chains (ϳ17 ϫ 12 ϭ 204 kDa), [d] [22][23][24]. Based on these results, a Hb model was proposed consisting of 12 hexadecamer subassemblies and 24 linker chains. A key point in resolving the differences is a reliable mass for the globin subassembly. We present the results of an ESI-MS study that provides accurate masses for the globin subassemblies isolated from the HBL Hbs of L. terrestris and the marine polychaete Arenicola marina.

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Comparison of three different fluorescent visualization strategies for detectingEscherichia coli ATP synthase subunits after sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Berggren¹,

Chernokalskaya

Lopez³

et al. 2001

Proteomics

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The correlation between protein molecular weight and the number of lysine or basic amino acid residues was found to be high for broad range molecular weight standards, subunits of Escherichia coli F1F0-ATP synthase and the translated open reading frame of E. coli. A relatively poor correlation between protein molecular weight and the number of cysteine residues was observed in all cases. The ability of amine-reactive, thiol-reactive and basic amino acid-binding fluorophores to detect the eight subunits of F1F0-ATP synthase complex was assessed using 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF), monobromobimane (MBB) and SYPRO Ruby protein gel stain, respectively. Though experimentally none of the fluorophores provided accurate estimates of the subunit stoichiometry of this complex, MDPF and SYPRO Ruby protein gel stain were capable of semiquantitative detection of every subunit. MBB, however, failed to detect subunits a, b and c of the hydrophobic F0 complex, as well as subunit epsilon of the F1 complex. All three fluorescent detection procedures permitted subsequent identification of representative subunits by peptide mass profiling using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The use of thiol-reactive fluorophores for the global analysis of protein expression profiles does not appear to be advisable as a significant number of proteins have few or no cysteine residues, thus escaping detection.

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Stoichiometry of Subunits and Heme Content of Hemoglobin from the Earthworm Lumbricus terrestris

Cited by 32 publications

References 35 publications

Assembly of the Gigantic Hemoglobin of the Earthworm Lumbricus terrestris

Assembly of the Gigantic Hemoglobin of the Earthworm Lumbricus terrestris

Electrospray Ionization Mass Spectrometric Determination of the Molecular Mass of the ∼200-kDa Globin Dodecamer Subassemblies in Hexagonal Bilayer Hemoglobins

Comparison of three different fluorescent visualization strategies for detectingEscherichia coli ATP synthase subunits after sodium dodecyl sulfate-polyacrylamide gel electrophoresis

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