The Role of the Dodecamer Subunit in the Dissociation and Reassembly of the Hexagonal Bilayer Structure of Lumbricus terrestris Hemoglobin

Sharma, Pawan K.; Kuchumov, Askar R.; Chottard, Geneviève; Martin, Philip D.; Wall, Joseph S.; Vinogradov, Serge N.

doi:10.1074/jbc.271.15.8754

Cited by 39 publications

(38 citation statements)

References 35 publications

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“…Furthermore, the data of Herskovits and Harrington (7) on the effects of 4 M urea on L. terrestris Hb suggest that 5-9% of the Hb may become denatured by this treatment. Because some of the "dodecamer" is oxidized and therefore dissociated, the preparation cannot be completely homogeneous and must contain both free abc trimer and chain d. This conclusion is consistent with the rather broad distribution of molecular masses observed for the dodecamer by scanning transmission electron microscopy (66,76).…”

Section: Model Of Assemblysupporting

confidence: 69%

“…Sixth, Sharma et al (76) have assumed that high concentrations of guanidinium salts, urea, or heteropolytungstates are required to dissociate the Hb at neutral pH to produce the presumed dodecamer and other products. The dissociation and subsequent reassociation upon removal of the agent were followed by HPLC chromatography with the fitting of the resulting patterns to a series of modified Gaussian distributions.…”

Section: Model Of Assemblymentioning

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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Section: Model Of Assemblysupporting

confidence: 69%

Section: Model Of Assemblymentioning

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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“…It is now well known that the stability of HBL Hb is strongly dependent on the presence of group IIA cations (i.e. Ca2+, Mg" and Srz') [3,45,461. At present, there are several techniques available for the determination of the molecular masses of huge molecules like the HBL Hh, i.e.…”

Section: Discussionmentioning

confidence: 99%

Quaternary Structure of the Extracellular Haemoglobin of the Lugworm Arenicola marina

Zal

Green²,

Lallier

et al. 1997

European Journal of Biochemistry

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To elucidate the quaternary structure of the extracellular haemoglobin (Hb) of the marine polychaete Arenicola marina (lugworm) it was subjected to multi-angle laser-light scattering (MALLS) and to electrospray-ionisation mass spectrometry (ESI-MS). It was also subjected to SDS/PAGE analysis for comparative purposes. MALLS analysis gave a molecular mass of 3648 % 24 kDa and a gyration radius of 11.3 rt 1.7 nm. Maximum entropy analysis of the multiply charged electrospray spectra of the native, dehaemed, reduced and carbamidomethylated Hb forms, provided its complete polypeptide chain and subunit composition. We found, in the reduced condition, eight globin chains of molecular masses 15952. Keywords: Arenicola ; haemoglobin ; quaternary structure ; multi-angle laser-light scattering; electrospray mass spectrometry.The giant extracellular haemoglobins (Hb) and chlorocruorins found in annelids [1] and vestimentiferans [2, 31 are characterised by an acidic isoelectric point and by an hexagonal symmetry in electron micrographs consisting of two superimposed hexagonal arrays of twelve spherical subunits, the hexagonal bilayer (HBL). These HBL Hb also contain a low haem and iron contents (about 67% of those observed in other Hbs) and consist of two types of chains, globin chains (~1 6 -1 8 kDa), accounting for approximately 70% of the total mass, and haem-deficient linker chains (~2 4 -2 8 kDa) necessary for the assemblage into the HBL structure [1,. The intertidal marine polychaete Arenicola marina (L.) was one of the first 60s (~3 6 0 0 kDa)

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“…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] 3 [bac] 3, consisting of three copies each of the monomer M (chain d) and the disulfidebonded trimer T (chains b ϩ a ϩ c) (6); in addition, this subassembly was found to be an obligate intermediate in the dissociation and reassembly of the HBL structure (7). A complete ESI-MS determination of the masses of the constituent globin and linker chains and the disulfide-bonded trimer subunit of Lumbricus Hb provided a calculated mass of 213.434 kDa for the subassembly [M] 3 [T] 3 (8).…”

mentioning

confidence: 99%

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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The Role of the Dodecamer Subunit in the Dissociation and Reassembly of the Hexagonal Bilayer Structure of Lumbricus terrestris Hemoglobin

Cited by 39 publications

References 35 publications

Assembly of the Gigantic Hemoglobin of the Earthworm Lumbricus terrestris

Assembly of the Gigantic Hemoglobin of the Earthworm Lumbricus terrestris

Quaternary Structure of the Extracellular Haemoglobin of the Lugworm Arenicola marina

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

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