The dissociation of the extracellular hemoglobin of Tubifex tubifex at extremes of pH and its reassociation upon return to neutrality

Polidori, Giampiero; Mainwaring, Mark G.; Kosinski, T.F.; Schwarz, Carolyn L.; Fingal, R A; Vinogradov, Serge N.

doi:10.1016/0003-9861(84)90509-5

Cited by 25 publications

(9 citation statements)

References 54 publications

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“…The peripheral location of subunit M is in harmony with the well-known fact that the dissociation of Lumbricus Hb at alkaline pH gives two fractions, one consisting of subunit M and the other of subunits D1, D2, and T (7). Such a pattern of dissociation at alkaline pH has been observed for many annelid hemoglobins, including those of the oligochaetes, Tubifex (19,20) and Eisenia (21); the polychaetes Abarenicola (22), Arenicola (23)(24)(25), and Ophelia (26); and the leeches Haemopis (27) and Dina (28).…”

Section: Methodsmentioning

confidence: 66%

Scanning transmission electron microscopic examination of the hexagonal bilayer structures formed by the reassociation of three of the four subunits of the extracellular hemoglobin of Lumbricus terrestris.

Kapp

Mainwaring²,

Vinogradov³

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

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A fraction obtained by gel filtration at neutral pH of the extracellular Hb of Lumbricus terrestris dissociated either at pH 9.8 or at pH 4.0, consisting of the three subunits D1 (31 kDa), D2 (37 kDa), and T (50 kDa), was found to produce two peaks when subjected to gel filtration on Superose 6 at pH 7. The first peak, which was eluted at a slightly greater volume than the native Hb, consisted of reassociated hexagonal bilayer structures when examined by scanning transmission electron microscopy. The dimensions of the two reassociated hexagonal bilayer structures were a vertex-to-vertex diameter of 25 nm and a height of 16 nm. The difference in size between the hexagonal bilayer structures and the native Hb is the contribution of subunit M, which consists of a single hemecontaining chain I (16.75 kDa). Although the reassociated hexagonal bilayer structures have overall dimensions smaller than the 30 nm x 20 nm dimensions of the native Hb, the diameters of the central cavities are not substantially altered. Subtraction of the three-dimensional reconstructions of the reassociated hexagonal bilayer structures from those of the native Hb showed that subunit M was primarily localized at the periphery of Lumbricus Hb. The formation of hexagonal bilayer structures in the complete absence of subunit M provides additional support for the "bracelet" model of the quaternary structure ofLumbricus Hb proposed recently by us in which subunits D1 and D2 were assumed to act as linkers for complexes of subunits M and T or to form a "bracelet" decorated with 12 complexes of subunits M and T.

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Section: Methodsmentioning

confidence: 66%

Scanning transmission electron microscopic examination of the hexagonal bilayer structures formed by the reassociation of three of the four subunits of the extracellular hemoglobin of Lumbricus terrestris.

Kapp

Mainwaring²,

Vinogradov³

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

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“…4C). Alkaline earth (Group IIA) cations are known to stabilize the HBL structure of annelid Hbs with respect to dissociation at alkaline pH (22,28,29), at acid pH (30,31), as well as thermal unfolding and autoxidation (32). In some cases, such as Amphitrite Hb (33) and Myxicola chlorocruorin (34), Ca 2ϩ is necessary for maintaining the HBL structure even at neutral pH.…”

Section: Reassembly Of Hbl Structurementioning

confidence: 99%

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

Sharma

Kuchumov

Chottard

et al. 1996

Journal of Biological Chemistry

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The dissociation of the ϳ3500-kDa hexagonal bilayer (HBL) hemoglobin (Hb) 27؊ (AsW) at neutral pH was followed by gel filtration, SDS-polyacrylamide gel electrophoresis, and scanning transmission electron microscopy. Elution curves were fitted to sums of exponentially modified gaussians to represent the peaks due to undissociated oxyHb, D (ϳ200 kDa), T؉L (ϳ50 kDa), and M (ϳ25 kDa) (T ‫؍‬ disulfide-bonded trimer of chains a-c, M ‫؍‬ chain d, and L ‫؍‬ linker chains). OxyHb dissociation decreased in the order Gdm⅐SCN > Gdm⅐Cl > urea > Gdm⅐OAc and AsW > SbW > SiW. Scanning transmission electron microscopy mass mapping of D showed ϳ10-nm particles with masses of ϳ200 kDa, suggesting them to be dodecamers (a؉b؉c) 3 d 3 . OxyHb dissociations in urea and Gdm⅐Cl and at alkaline pH could be fitted only as sums of 3 exponentials. The time course of D was bell-shaped, indicating it was an intermediate. Dissociations in SiW and upon conversion to metHb showed only two phases. The kinetic heterogeneity may be due to oxyHb structural heterogeneity. Formation of D was spontaneous during HBL reassembly, which was minimal ( 10%) without Group IIA cations. During reassembly, maximal (ϳ60%) at 10 mM cation, D occurs at constant levels (ϳ15%), implying the dodecamer to be an intermediate.

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“…Earlier work has demonstrated that: (a) the SDSjPAGE patterns of Tubifex and Lumbricus hemoglobins are very similar [16]; (b) their electron microscopic appearance and dimensions were identical [19]; and (c) their dissociation at extremes of pH were very similar [17]. The high extent of amino acid identity in the sequences of their monomeric globins provides an additional reason for proposing that the quarternary structures of the two extracellular hemoglobins are similar.…”

Section: Discussionmentioning

confidence: 99%

“…Both are hexagonal bilayer hemoglobins and exhibit very similar SDSjPAGE patterns [16]; each contains a disulfide-linked trimer and a monomer [17]. Here we have extended this comparison to include the amino acid sequence similarities between these two species, as well as a comparison with the monomeric globins of the oligochaete Pheretima [26] and of the marine polychaete, Tylorrhynchus [27].…”

Section: Discussionmentioning

confidence: 99%

“…The monomer subunit was isolated by gel filtration at alkaline pH as described by Polidori et al [17] and was further purified by Superose S-12 gel filtration and reversed-phase C8 chromatography [ll]. Reagents for protein modification were procured as follows: butanone (methylethylketone), trifluoroacetic acid and citraconic anhydride (Pierce Chemical Co.); dithiothreitol, iodoacetic acid, cyanogen bromide, trypsin and salts for buffers (Sigma Chemical Corp.).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Amino acid sequence of the monomer subunit of the giant extracellular hemoglobin of the aquatic oligochaete, Tubifex tubifex

Stern

Vinogradov

Sharma

et al. 1990

European Journal of Biochemistry

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The extracellular hemoglobin of the aquatic oligochaete Tubifex tubifex consists of four subunits: a monomer of 16.5 kDa, a disulfide-bonded trimer of about 50 kDa and at least two subunits of about 30 kDa. The complete amino acid sequence of the monomeric subunit was determined: it consists of 141 amino acid residues and has a molecular mass of 16286 Da including a heme group. 39 residues (28%) were found to be identical with those in the corresponding positions in the monomeric globin chains from Lumbricus terrestris, Pheretima sieboldi, and Tylorrhynchus heterochaetus. Tubifex and Lumbricus are most similar, with 1 5 amino acid identities (53%). There are eight invariant residues amongst these monomeric globins and the intracellular monomeric globin of Glycera and the human P-globin. The monomeric globin from Tubifex aligns best with those of group A, globins which have a Cys in their second position and an invariant Lys-Val-Lys at positions 9 -11 [Gotoh et al. (1987) Biochem. J. 241, 441 -4451. The two cysteine residues, at positions 2 and 131, appear to be disulfide-bonded.Hemoglobins occur episodically among the invertebrates, can be intra-or extra-cellular, and display a broad range of molecular size and subunit structure [l -61.

show abstract

The dissociation of the extracellular hemoglobin of Tubifex tubifex at extremes of pH and its reassociation upon return to neutrality

Cited by 25 publications

References 54 publications

Scanning transmission electron microscopic examination of the hexagonal bilayer structures formed by the reassociation of three of the four subunits of the extracellular hemoglobin of Lumbricus terrestris.

Scanning transmission electron microscopic examination of the hexagonal bilayer structures formed by the reassociation of three of the four subunits of the extracellular hemoglobin of Lumbricus terrestris.

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

Amino acid sequence of the monomer subunit of the giant extracellular hemoglobin of the aquatic oligochaete, Tubifex tubifex

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