Bracelet protein: a quaternary structure proposed for the giant extracellular hemoglobin of Lumbricus terrestris.

Abstract

The complete dissociation of the hexagonal bilayer structure ofLumbricus terrestris hemoglobin (3900 kDa) at neutral pH, in the presence of urea, guanidine hydrochloride, sodium perchlorate, potassium thiocyanate, sodium phosphotungstate, and sodium phosphomolybdate, followed by gel filtration at neutral pH on Sephacryl S-200 or Superose 6, produced two fragments, II (65 kDa) and m (17 kDa); NaDodSO4/polyacrylamide gel electrophoresis showed that peak II consisted of subunits D1 (31 kDa, chain V), D2 (37 kDa, chain VI), and T (50 kDa, disulfide-bonded trimer of chains H, HI, and IV) and that peak II consisted of subunit M (16 kDa, chain 1). When dissociation was incomplete, two additional peaks were present, peak Ia eluting at the same volume as the whole hemoglobin and peak lb (200 kDa). Scanning transmission electron micrographs of peak Ia showed it to consist of whole molecules and of incomplete hexagonal bilayer structures, missing an apparent 1/12th. Peak lb contained all four subunits but was usually deficient in subunits D1 and D2, was not always in equilibrium with the whole molecule, and could be dissociated further into II and HI. The patterns of dissociation observed at neutral pH were very similar to those observed previously at alkaline pH and at acid pH and appear to be incompatible with the generally accepted multimeric model of Lumrbncus hemoglobin subunit structure. A model is proposed in which it is postulated that the stoichiometries of some of the subunits need not be constant and that subunits D1 and D2 either form a "bracelet" decorated with complexes of T and M subunits or serve as "linkers" between the latter, to provide the appearance of a two-tiered hexagonal structure. Additional support for the proposed model comes from observations that the fragment II obtained subsequent to dissociation at pH 4, in sodium phosphotungstate, in sodium perchlorate, and in potassium thiocyanate was found to be in equilibrium with a hexagonal bilayer structure IaR(II), whose dimensions were =20% smaller than those of the native hemoglobin.

An electrospray ionization mass spectrometric study of the subunit structure of the giant hemoglobin from the leech Nephelopsis oscura

“…The direct observation of dodecamer globin subassemblies by ESI-TOF-MS in Nephelopsis Hb provides concrete evidence for their existence within these giant heteromultimeric complexes in solution. This result is in full accord with the "bracelet" model of the quaternary structure of HBL Hbs proposed earlier [20] and strongly supported by the recent low-resolution crystal structure of Lumbricus Hb [21]: An HBL complex of 12 dodecamer subassemblies tethered to 36 linker chains. Based on this model the expected mass of Nephelopsis Hb would be 208.33 ϫ 12 ϩ 24.95 ϫ 36 ϭ 3398 kDa , well within the observed range of masses of HBL Hbs [4].…”

The Multi-hemoglobin System of the Hydrothermal Vent Tube Worm Riftia pachyptila

“…As for other HBL Hbs analyzed with ESI-MS to date (13)(14)(15), our data for Riftia HBL Hb V1 fit well with the "bracelet model" proposed for L. terrestris (40). This model has recently been substantiated by three-dimensional reconstruction based on cryomicroscopy analysis (41).…”

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

“…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).…”