Subunit distribution of calcium-binding sites in Lumbricus terrestris hemoglobin.

The giant, approximately 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 approximately 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 approximately 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 approximately 2.2, had 1 Ca per mole and very little Cu and Zn. Electrospray ionization mass spectrometry of linker L3 at pH approximately 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) approximately 30 sites with affinities higher than EDTA occurring within the cysteine-rich domains of linker L3 and dodecamer subassembly, and (iii) approximately 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.

The role of linkers in the reassembly of the 3.6 MDa hexagonal bilayer hemoglobin from Lumbricus terrestris.

The extent and kinetics of reassembly of the four groups of linkers L1-L4 with 213 kDa subassemblies of twelve globin chains D, (bac)3(d)3, isolated from the approximately 3.6 MDa hexagonal bilayer (HBL) hemoglobin (Hb) of Lumbricus terrestris, was investigated using gel filtration. The reassembled HBL's were characterized by scanning transmission electron microscopic (STEM) mass mapping and their subunit content determined by reversed-phase chromatography. In reassembly by method (A), the linkers isolated by RP-HPLC at pH approximately 2.2 were added to D at neutral pH; in method (B), the linkers were renatured at neutral pH and then added to D. With method (A) the percentage of HBL reassembly varied from >/=13% in the absence of Ca(II) to /=75%), with ternary and binary linker combinations (40-50%) and with individual linkers producing yields increasing in the following order: L1=1-3%, L2 approximately L3=10-20% and L4=35-55%. The yield was two- to eightfold lower with method (B), except in the case of linkers L1-L3. Although the reassembly kinetics were always biphasic, with t1/2=0.3-3.3 hours and 10-480 hours, the ratio of the amplitudes fast:slow was 1:0.6 with method (A) and 1:2.5 with method (B). These results are consistent with a scheme in which the slow HBL reassembly is dependent on a slow conversion of linker conformation at neutral pH from a reassembly incompetent to a reassembly competent conformation. Although all the linkers self-associate extensively at neutral pH, forming complexes ranging from dimers to >18-mers, the size of the complex does not affect the extent or rate of reassembly. The oxygen binding affinity of reassembled HBLs was similar to that of the native Hb, but their cooperativity was lower. A model of HBL reassembly was proposed which postulates that binding of linker dimers to two of the three T subunits of D causes conformational alterations resulting in the formation of complementary binding sites which permit lateral self-association of D subassemblies, and thus dictate the formation of a hexagonal structure due to the 3-fold symmetry of D.

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 Oncley's 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.

Analysis of a 3.6-MDa hexagonal bilayer hemoglobin from Lumbricus terrestris using a gas-phase electrophoretic mobility molecular analyzer.

The recent successful use of electrospray gas-phase electrophoretic mobility molecular analysis (GEMMA) to separate globular proteins (mass 6 to 670 kDa) and the excellent correlation found between the electrophoretic mobility diameter (EMD), or Millikan diameter, and the protein mass (S. L. Kaufman et al., 1996, Anal. Chem. 68, 1895-1904; 1996, Anal. Chem. 68, 3703), prompted the examination of a large protein complex, the 3.6-MDa, heteromultimeric, hexagonal bilayer hemoglobin (Hb) and its subunits from the earthworm Lumbricus terrestris. The native Hb had an EMD of 25.7 nm and the products of its dissociation at pH >8 and <5 were resolved into peaks with EMDs of 10.5, 6.3, 5.0, and 4.2 nm, identified as a dodecamer of globin chains ([a+b+c]3d3, 213 kDa), the disulfide-bonded trimer of globin chains ([a+b+c], 52.7 kDa), all the linker chains (L1, 27.5 kDa; L2, 32.1 kDa; L3, 24.9 kDa; L4, 24. 1 kDa), and the monomer subunit (chain d, 17 kDa), respectively. Reassembly of the Hb complex was observed on restoring the pH from >8 to 7. The EMDs and the masses of the Hb and its subunits are in excellent agreement with the correlation found earlier, under the assumption of nearly spherical shape with an effective density around 0.7 g/cm3. GEMMA also provided a profile of the Hb completely dissociated in 0.1% SDS; its deconvolution permitted a quantitative determination of the subunit stoichiometry, providing a globin to linker ratio of 3 to 1.

A hierarchy of disulfide-bonded subunits: the quaternary structure of Eudistylia chlorocruorin.

The quaternary structure of the cysteine-rich, approximately 3500-kDa chlorocruorin (Chl) from the marine polychaete Eudistylia vancouverii was investigated using maximum entropy deconvolution of the electrospray ionization mass spectra (ESIMS). The native Chl provided two groups of peaks, at approximately 25 and approximately 33 kDa, and one peak at approximately 66 kDa. ESIMS of the reduced and reduced and carbamidomethylated Chl and of its subunits obtained by HPLC provided the complete subunit composition of the Chl. Two groups of nonglobin linker chains were observed: L1a-f (25 000.4, 25 017.9, 25 039.6, 25 057.0, 25 074.4 and 25 096.8 Da) and L2a-d (25 402.7, 25 446.0, 25 461.6 and 25 478.3Da) (+/-2.5 Da), with relative intensities L1:L2 = 5:2. Six globin chains were found, a1, a2, and b1-4, with reduced masses of 16 051.5, 16 172.4, 16 853.5, 17 088.9, 17 161.2 and 17 103.6 (+/-1.0 Da) and relative intensities of 8:4:1:4:2:1, respectively. Disulfide-bonded dimers and a tetramer of globin chains were identified: D1 = a1 + b3 at 33 207.1; D2 at 33 374.1, which had a cysteinylated Cys (a2 + b2 + Cys); and D3 = a1 + b4 at 33 149.4 Da (+/-3.0 Da), with relative intensities D1:D2:D3 = 5:4:1 and T = a1 + a2 + b1 + b2 at 66 154.8 +/- 4.0 Da. A 206-kDa dodecamer subunit obtained by dissociation of the Chl in 4 M urea [Qabar, A. N., et al. (1991) J. Mol. Biol. 222, 1109-1129], was found to consist only of tetramers T. A model was proposed for the Chl, based on a dimer:tetramer ratio of 2:1: four 206-kDa dodecamers (trimer of tetramers) and 48 dimers tethered to a framework of 30 L1 and 12 L2 linker chains. The 144 globin chains (2480 kDa) and 42 linker chains (1059 kDa) provide a total mass of 3539 kDa, in good agreement with the 3480 +/- 225 kDa determined previously by STEM mass mapping. The hierarchy of disulfide-bonded globin subunits observed for Eudistylia Chl provides a built-in heterogeneity of hexagonal bilayer structures.

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.

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 degrees C and 4 at 25 degrees C, providing DeltaH = -20 kcal/mol and DeltaS = 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 degrees C. P50 was decreased 3- and 2-fold in approximately 100 mM Ca2+ and Mg2+, respectively, with concomitant but smaller increases in cooperativity.

Superoxide dismutase activity in the giant hemoglobin of the earthworm, Lumbricus terrestris.

The giant hemoglobin, which occurs in free solution in the blood of earthworms, contains copper and zinc and exhibits superoxide dismutase activity as assessed by competition assays using cytochrome c or nitroblue tetrazolium. On a molar basis, the activity of this hemoglobin was approximately 10% that of the mammalian copper, zinc superoxide dismutase. The dodecamer, which contains the globin chains but lacks the linker subunits, had very little activity when compared to the complete native molecule. This suggests that the superoxide dismutase activity resides in one of the linker subunits. This is the first report of a superoxide dismutase bound to a respiratory pigment.

The role of the dodecamer subunit in the dissociation and reassembly of the hexagonal bilayer structure of Lumbricus terrestris hemoglobin.

The dissociation of the approximately 3500-kDa hexagonal bilayer (HBL) hemoglobin (Hb) of Lumbricus terrestris upon exposure to Gdm salts, urea and the heteropolytungstates [SiW11O39]8- (SiW), [NaSb9W21O86]18- (SbW) and [BaAs4W40O140]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 (approximately 200 kDa), T+L (approximately 50 kDa), and M (approximately 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 approximately 10-nm particles with masses of approximately 200 kDa, suggesting them to be dodecamers (a+b+c)3d3. 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 (

Mass spectrometric composition and molecular mass of Lumbricus terrestris hemoglobin: a refined model of its quaternary structure.

Sedimentation equilibrium measurements and scanning transmission electron microscopy (STEM) mass mapping of the extracellular, hexagonal bilayer hemoglobin (HBL Hb) of the earthworm Lumbricus terrestris provided masses of 3.41 to 3.66 MDa and 3.56 (+/- 0.13) MDa, respectively. The Hb also contains 57.2 (+/- 6.0) moles of tightly bound Ca per mole of protein. The Hb and its subunits obtained by dissociation, in native, dehemed and reduced carbamidomethylated forms, were subjected to electrospray ionization mass spectroscopy (ESI-MS). Maximum entropy deconvolution identified three groups of peaks, at approximately 16 kDa, 24 to 32 kDa and approximately 53 kDa corresponding to the monomer subunit M (globin chain d), four linker subunits and the disulfide-bonded trimer T (globin chains a + b + c). Subunit M consisted of three components, d1 (15, 992.4), d2 (15, 978.0) and d3 (15, 962.1) (+/- 1.0 Da), with relative intensities 1.0:5:0.3, respectively. Subunit T consisted of four major components, T1 (52, 922.6), T2 (52, 760.0), T3 (52, 598.5) and T4 (52, 435.4) (+/- 4.0 Da), with relative intensities 0.6:1.0:0.2:0.7, respectively. ESI-MS of carbamidomethylated T, demonstrated that, unlike chains b (16, 254.4) and c (17, 289.2), chain a exists as a series of four, hexose-connected, glycosylated isoforms, a1 to a4 (19, 389.9, 19, 227.4, 19, 065.3 and 18, 902.9) (+/- 1.0 Da). The mass differences between the deglycosylated chain a (17, 524.0) and a1 to a4 correspond to glycan side-chains (GlcNAc)2 (Man)n (n = 6 to 9). Four groups of peaks were observed in the 24 to 32 kDa region. Linkers L1a (27, 540.8) and L1b (27, 702.4) (+/- 2.0 Da) are isoforms of L1 (25, 837.5 in N-deglycosylated Hb) with glycan side-chains (GlcNAc)2 (Man)n (n = 8,9). Linkers L2 (32, 104.3 (+/- 5.0) Da) and L3 (24, 912.9 (+/- 2.0) Da) occur as single species. Linkers L4a to L4c (24, 019.0, 24, 102.3 and 24, 169.9) (+/- 2.0 Da) with relative intensities 1.0:0.8:0.8, have not been identified previously. From ESI-MS relative intensities, L1:L2:L3:L4 = 0.6:0.4:1.0:0.5 and globin linker = 0.78:0.22. HPLC of Lumbricus Hb provided a globin linker = 0.73:0.27 (+/- 0.02) and a heme content of 2.52 (+/- 0.14) wt%. A model is proposed for the HBL structure, wherein 12 213.4 kDa dodecamers (144 globin chains, 2561 kDa) decorate a hexagonal framework of 36 linker chains (12L1 + 6L2 + 12L3 + 6L4) to provide a total mass of 3.531 MDa, each dodecamer being in contact with three linker subunits.

Molecular symmetry of the dodecamer subunit of Lumbricus terrestris hemoglobin.

The principal functional subunit of the approximately 3500 kDa extracellular Lumbricus terrestris hemoglobin is a 213 kDa dodecamer of four chemically distinct globin chains, consisting of a non-covalent complex of three trimer submits (disulfide-bonded chains a, b and c) and three monomer subunits (chain d). X-ray diffraction of crystals of the dodecamer grown at neutral pH, were found to be monoclinic, with the unit cell dimensions: a = 112.3 A, b = 190.0 A, c = 69.6 A, beta = 102.0 degrees with h + k + l = 2n + 1 absent, characteristic of space group I121. In addition, these crystals exhibit a pseudo trigonal P321 symmetry with unit cell dimensions a = 190.5 A, b = 190.5 A, c = 69.5 A, gamma = 120.0 degrees. Assuming that the assymetric unit contains an entire dodecamer, a model of the latter was constructed that satisfies the symmetry of the trigonal pseudo cell and is consistent with the symmetry of the I121 crystallographic cell. The resulting model has strong implications concerning the hexagonal bilayer structure of the native hemoglobin.

Electrospray ionization mass spectrometric determination of the complete polypeptide chain composition of Tylorrhynchus heterochaetus hemoglobin.

Electrospray ionization mass spectrometry (ESI-MS) of the native, reduced, and carbamidomethylated forms of the extracellular, 3.38-MDa hemoglobin from the marine polychaete Tylorrhynchus heterochaetus, when combined with a maximum entropy (MaxEnt) analysis, provided a complete description of the polypeptide chain composition. This hemoglobin, a hetero-multimeric complex of approximately 180 polypeptide chains, consisting of globin and linker subunits in an approximately 3:1 mass ratio, is among the largest protein complexes investigated by ESI-MS. The globin subunits consist of a monomer subunit (chain I, 15575.4 Da) and a disulfide-bonded trimer subunit, 50068.4 Da, consisting of globin chains IIA (16601.9 Da), IIB (16680.4 Da), and IIC (16,794.0 Da). Linker subunits L1-L5, 23233.8, 24835.4, 25326.9, 28202.2, and 26317.2 Da, respectively, were found together with a disulfide-bonded dimer of L2, 52609.4 Da. Using the exact masses of the subunits, a plausible model of the hemoglobin consisting of 144 globin chains (36 monomers and 36 trimers) and 36 linker chains provides a calculated mass of 3.42 MDa.