Askar R. Kuchumov

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

The dissociation of the 3500-kDa hexagonal bilayer (HBL) hemoglobin (Hb) of Lumbricus terrestris upon exposure to Gdm salts, urea and the heteropolytungstates [SiWO] (SiW), [NaSbWO] (SbW) and [BaAsWO] (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 (200 kDa), T+L (50 kDa), and M (25 kDa) (T = disulfide-bonded trimer of chains a-c, M = chain d, and L = linker chains). OxyHb dissociation decreased in the order Gdm•SCN > Gdm•Cl > urea > Gdm•OAc and AsW > SbW > SiW. Scanning transmission electron microscopy mass mapping of D showed 10-nm particles with masses of 200 kDa, suggesting them to be dodecamers (a+b+c)d. OxyHb dissociations in urea and Gdm•Cl 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 (60%) at 10 mM cation, D occurs at constant levels (15%), implying the dodecamer to be an intermediate.

Molecular Shape, Dissociation, and Oxygen Binding of the Dodecamer Subunit of Lumbricus terrestris Hemoglobin

Small angle x-ray scattering of the 213-kDa dodecamer of Lumbricus terrestris Hb yielded radius of gyration = 3.74 ± 0.01 nm, maximum diameter = 10.59 ± 0.01 nm, and volume = 255 ± 10 nm3, with no difference between the oxy and deoxy states. Sedimentation velocity studies indicate the dodecamer to have a spherical shape and concentration- and Ca2+-dependent equilibria with its constituent subunits, the disulfide-bonded trimer of chains a-c and chain d. Equilibrium sedimentation data were fitted best with a trimer-dodecamer model, ln K4 = 7 (association K in liters3/g3) at 1°C and 4 at 25°C, providing ΔH = −20 kcal/mol and ΔS = 4.4 eu/mol. Oxydodecamer dissociation at pH 8.0, in urea, GdmCl, heteropolytungstate K8[SiW11O39] and of metdodecamer at pH 7, was followed by gel filtration. Elution profiles were fitted with exponentially modified gaussians to represent the three peaks. Two exponentials were necessary to fit all the dissociations except in [SiW11O39]−8. Equilibrium oxygen binding measurements at pH 6.5-8.5, provided P50 = 8.5, 11.5-11.9 and 11.9-13.5 torr, and n50 = 5.2-9.5, 3.2-4.9, and 1.8-2.7 for blood, Hb, and dodecamer, respectively, at pH 7.5, 25°C. P50 was decreased 3- and 2-fold in ~100 mM Ca2+ and Mg2+, respectively, with concomitant but smaller increases in cooperativity.

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.

An electrospray ionization mass spectrometric study of the extracellular hemoglobins from Chironomus thummi thummi.

The aquatic larvae of the dipteran, Chironomus thummi thummi contain extracellular hemoglobins which exhibit stage-specific expression. We have used maximum entropy-based deconvolution of the complex, multiply charged electrospray ionization mass spectra, to demonstrate the presence of more than 20 components, ranging in mass from 14,417.3 Da to 17,356.5 Da in the 4th instar larvae. Of the 15 major peaks with intensities > 10 relative to 100 for the 14,417.3 Da-component (CTT-IV), only the 15,528.2-Da peak does not correspond to a known amino acid sequence. Since the number of C. thummi thummi globin genes now stands at 27, including one cDNA and not counting three that must encode known globins, our results suggest that only a limited number of the globin genes are expressed in the 4th instar larvae.

Electrospray ionization mass spectrometric study of the multiple intracellular monomeric and polymeric hemoglobins of Glycera dibranchiata

The intracellular hemoglobin (Hb) of the marine polychaete Glycera dibranchiata is comprised of two groups of globins differing in their primary structures and state of aggregation. About six electrophoretically and chromatographically distinct monomeric Hbs which have Leu as the distal residue, and an equal number of polymeric Hbs which have the usual distal His, have been identified to date. Deconvolution of the electrospray ionization mass spectra (ESI-MS) of the Hbs and of their carbamidomethylated, reduced, and reduced/carbamidomethylated forms, using a maximum entropy-based approach (MaxEnt), showed the presence of at least 18 peaks attributable to monomer Hbs (14,500–15,200 Da) and an approximately equal number of polymer Hb peaks (15,500–16,400 Da). Although the ratio of the monomer to polymer components in pooled Hb preparations remained constant at 60:40, Hb from individuals had generally less than 6 monomer and 6 polymer components; 2 of the 19 individuals appeared to be deficient in polymer Hbs. Taking into account possible fragmentations of the known monomeric and polymeric globin sequences, we estimate conservatively that there are 10 monomeric and an equal number of polymeric Hbs, the majority comprising a single free Cys. Surprisingly, the calculated mass of the sequence deduced from the high-resolution monomer Hb crystal structures does not correspond to any of the observed masses. ESI-MS of the monomer Hb crystal revealed 11 components, of which 5, accounting for 67% of total, were related to the three major sequences GMG2–4. These findings underline the need for routine mass spectrometric characterization of all protein preparations. The complete resolution of the Glycera Hb ESI-MS using MaxEnt processing illustrates the power of this method to resolve complex protein mixtures.

An electrospray ionization mass spectrometric study of the giant, extracellular, hexagonal bilayer hemoglobin of the leech Haemopis grandis provides a complete enumeration of its subunits

Abstract The complex, multiply charged electrospray ionization mass spectra of the extracellular, ∼3500 kDa, hexagonal bilayer hemoglobin from the leech Haemopis grandis and its carbamidomethylated, reduced and reduced/carbamidomethylated forms were deconvoluted using a maximum entropy approach to provide the corresponding zero-charge spectra. Three groups of peaks were observed: monomeric globin chains a1–a4 at ∼17 kDa (16 634.1, 17 013.4, 17 592.9, and 17 573.3 Da with relative intensities of 16:4:12:1, respectively), linker chains L1–L3 at ∼24 kDa (24 004.2, 24 449.2 and 24 548.3 Da, with relative intensities of 5:1:2.5, respectively) and subunits D1 and D2 at 32 501.1 and 32 629.6 Da, respectively, with equal intensities. Reduction of the native hemoglobin with dithiothreitol resulted in a decrease in the mass of linker L2 by 115.1 Da, indicating cysteinylation, the disappearance of subunits D1 and D2 and the concomitant appearance of globin chains b (16 098.8 Da), c1 (16 403.9 Da), and c2 (16 532.5 Da), suggesting that subunits D1 and D2 are disulfide-bonded dimers b + c1 and b + c2, respectively. All the globin chains appear to have one intrachain disulfide bond, and globins b, c1, and c2 have an additional Cys which forms the interchain disulfide bond in D1 and D2. The linkers L1–L3 contain 10, 9, and 10 Cys residues, respectively, all forming intrachain disulfide bonds, except for the odd residue in L2 which is proposed to be the site of cysteinylation. The relative intensities of the three groups of peaks a1 + a2 + a3 + a4:L1 + L2 + L3:D1 + D2 are 1.65:1.7:0.8 in native hemoglobin. All the subunits in a second sample evinced substantial glycosylation, with up to five hexoses per subunit. A model of the quaternary structure of Haemopis hemoglobin is proposed, consisting of 72 monomeric globins and 36 globin dimers for a total of 144 globin chains and 42 linkers with a calculated total mass of 3506 kDa.

A mass spectrometric study of the heterogeneity of the monomer subunit of Lumbricus terrestris hemoglobin

The subunits of the hemoglobin of Lumbricus terrestris consist of heme-binding globin chains, designated a, b, c, and d, and linker chains. The sequence of chain d, which is also referred to as a monomer subunit, has been reported by Shishikura et al. (Biol. Chem.1987, 262, 3123–3131). This subunit has been found to be heterogeneous and in this study three fractions were separated by C18 reverse-phase high-performance liquid chromatography. The major chain d1 with molecular weight 15,993. 5 ± 2. 1 u possesses S7 instead of G7, the partial sequence RDIIDD (33–38) instead of KGILRE, E instead of Q in positions 23 and 58, and T84 instead of A84. The C-terminus is K140 instead of D141I142. Chain d3 with molecular weight 15,963. 2 ± 1. 6 u showed high sequence homology with chain d1, differing apparently only in residue 84 where A84 replaces T84. Analysis of the third chromatographie fraction revealed two additional chains with molecular weights of 15,996. 1 ± 1. 4 and 15,937. 6 ± 1. 2 u. These are designated chains d2 and d4, respectively, but their sequence assignments are not yet certain.

The apparently symmetrical hexagonal bilayer hemoglobin from Lumbricus terrestris has a large dipole moment.

The giant approximately 3.6 MDa hexagonal bilayer hemoglobin (HBL Hb) from Lumbricus terrestris consists of 12 213-kDa dodecamers of four globin chains ([b + a + c]3[d]3) tethered to a central scaffold of approximately 36 non-globin, linker subunits L1-L4 (24-32 kDa). Three-dimensional reconstructions obtained by electron cryomicroscopy showed it to have a D6 point-group symmetry, with the two layers rotated approximately 16 degrees relative to each other. Measurement of the dielectric constants of the Hb and the dodecamer over the frequency range 5-100 kHz indicated relaxation frequencies occurring at 20-40 and 300 kHz, respectively, substantially lower than the 700-800 kHz in HbA. The dipole moments calculated using Oncleys equation were 17,300 +/- 2300 D and 1400 D for the Hb and dodecamer, respectively. The approximately threefold higher dipole moment of the dodecamer relative to HbA is consistent with an asymmetric shape in solution suggested by small-angle X-ray scattering. Although a two-term Debye equation and a prolate ellipsoid of revolution model provided a good fit to the experimental dielectric dispersion of the dodecamer, a three-term Debye equation based on an oblate ellipsoid of revolution model was required to fit the asymmetric dielectric dispersion curve of the Hb: the required additional term may represent either an induced dipole moment or a substructure which rotates independently of the main permanent dipole component of the Hb. The D6 point-group symmetry implies that the dipole moments of the dodecamers cancel out. Thus, in addition to a possible contribution from fluctuations of the proton distribution, the large dipole moment of the Hb may be due to an asymmetric distribution of the heterogeneous linker subunits.