Observation of large, non-covalent globin subassemblies in the approximately 3600 kDa hexagonal bilayer hemoglobins by electrospray ionization time-of-flight mass spectrometry

A non-covalent globin subassembly comprising 12 globin chains (204 to 214 kDa) was observed directly by electrospray ionization time-of-flight mass spectrometry in the native hexagonal bilayer hemoglobins from the oligochaetes Lumbricus terrestris and Tubifex tubifex, the polychaetes Tylorrhynchus heterochaetus, Arenicola marina, Amphitrite ornata and Alvinella pompejana, the leeches Macrobdella decora, Haemopis grandis and Nephelopsis oscura and the chlorocruorin from the polychaete Myxicola infundibulum, over the pH range 3.5-7.0. The Hb from the deep-sea polychaete Alvinella exhibited in addition, peaks at approximately 107 kDa and at approximately 285 kDa, which were assigned to subassemblies of six globin chains and of 12 globin chains with three non-globin linker chains, respectively. The experimental masses decreased slightly with increased de-clustering potential (60 to 160 V) and were generally 0.1 to 0.2 % higher than the calculated masses, due probably to complexation with cations and water molecules.     

Immunological relatedness of annelid extracellular hemoglobins and chlorocruorins

1. The immunological relatedness of several annelid extracellular hemoglobins and chlorocruorins was investigated using ELISAs and Western blotting to determine the binding of purine polyclonal and monoclonal antibodies to Lumbricus terrestris hemoglobin with the hemoglobins of Tubifex tubifex, Tylorrhynchus heterochaetus, Arenicola marina and Macrobdella decora and the chlorocruoins of Myxicola infundibulum and Eudistylia vancouverii. 2. Polyclonal antibodies to Lumbricus terrestris hemoglobin bound to all the other hemoglobins and chlorocruorins. However, the titers were in all cases one to several orders of magnitude smaller than with Lumbricus terrestris hemoglobin. 3. Polyclonal antibodies to Eudistylia vancouverii chlorocruorin bound to the hemoglobins of Lumbricus terrestris, Tubifex tubifex, Arenicola marina, Tylorrhynchus heterochaetus and Macrobdella decora. 4. Of the nine monoclonal antibodies to Lumbricus terrestris hemoglobin isolated, two (No. 24 and No. 26) bound to the other hemoglobins and to Myxicola chlorocruorin, but the binding was again weaker than with Lumbricus hemoglobin. Antibody No. 26 also bound to Eudistylia chlorocruorin. Although antibody No. 24 appears to recognize a conformation-dependent epitope, antibody No. 26 recognizes a common epitope in each of the four subunits M, D1, D2, and T of unreduced Lumbricus hemoglobin. 4. An additional two monoclonal antibodies to Lumbricus hemoglobin (No. 21 and No. 25) bound also only to Tubifex hemoglobin. Antibody No. 21 recognizes subunits D1 and M of Lumbricus hemoglobin and No. 25 appears to recognize a conformation-dependent epitope.     

Revisiting the structure of Alvinella pompejana hemoglobin at 20A resolution by cryoelectron microscopy

The hemoglobin of the polychaete worm Alvinella pompejana was reconstructed at 20A resolution from frozen-hydrated samples observed by electron microscopy according to the random conical tilt series method. This three-dimensional reconstruction was mirror-inverted with respect to a previous volume published by de Haas et al. in 1996. In order to explain this handedness discrepancy, various 3D reconstructions using different reference volumes were carried out showing that the choice of the first volume was the keystone during the refinement process. The 3D reconstruction volume of A. pompejana Hb presented structural features characteristic of annelid Hbs with two hexagonal layers each comprising six hollow globular subassemblies and a complex of non-heme linker chains. Moreover, the eclipsed conformation of the two hexagonal layers and a HGS architecture similar to that described for Arenicola marina Hb led to the conclusion that A. pompejana Hb belonged to the architectural type II according to the definition of Jouan et al. (2001). A comparison between this cryo-EM volume and X-ray crystallography density maps of Lumbricus terrestris type-I Hb (Royer et al., 2000) showed that the triple stranded coiled coil structures of linker chains were different. Based on this observation, a model was proposed to explain the eclipsed conformation of the two hexagonal layers of type-II Hbs.     

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.     

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.     

A dodecamer of globin chains is the principal functional subunit of the extracellular hemoglobin of Lumbricus terrestris

Repeated dissociation of the approximately 3600-kDa hexagonal bilayer extracellular hemoglobin of Lumbricus terrestris in 4 M urea followed by gel filtration at neutral pH produces a subunit that retains the oxygen affinity of the native molecule (approximately 12 torr), but only two-thirds of the cooperativity (nmax = 2.1 +/- 0.2 versus 3.3 +/- 0.3). The mass of this subunit was estimated to be 202 +/- 15 kDa by gel filtration and 202 +/- 26 kDa from mass measurements of unstained freeze-dried specimens by scanning transmission electron microscopy. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this subunit showed that it consists predominantly of the heme-containing subunits M (chain I, 17 kDa) and T (disulfide-bonded chains II-IV, 50 kDa). Mixing of subunits M and T isolated concurrently with the 200-kDa subunit resulted in partial association into particles that had a mass of 191 +/- 13 kDa determined by gel filtration and 200 +/- 38 kDa determined by scanning transmission electron microscopy and whose oxygen affinity and cooperativity were the same as those of the 200-kDa subunit. The results imply that the 200-kDa subunit is a dodecamer of globin chains, consisting of three copies each of subunits M and T (3 x chains (I + II + III + IV], in good agreement with the mass of 209 kDa calculated from the amino acid sequences of the four chains, and represents the largest functional subunit of Lumbricus hemoglobin. Twelve copies of this subunit would account for two-thirds of the total mass of the molecule, as suggested earlier (Vinogradov, S. N., Lugo, S. L., Mainwaring, M. G., Kapp, O. H., and Crewe, A. V. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 8034-8038). The retention of only partial cooperativity by the 200-kDa subunit implies that full cooperativity is dependent on the presence of a complete hexagonal bilayer structure, wherein 12 200-kDa subunits are linked together by approximately 30-kDa heme-deficient chains.     

Hemoglobins from deep-sea hydrothermal vent scaleworms of the genus Branchipolynoe: a new type of quaternary structure

Branchipolynoe symmytilida and B. seepensis are two scaleworms (Polychaeta; Polynoidae) living commensally in the mantle cavity of deep-sea hydrothermal vent and cold-seep mussels. In contrast with littoral members of this family, the two species exhibit a large amount of extracellular hemoglobin (Hb) in their coelomic fluid. Gel filtration revealed the existence of four different Hbs: one minor, high molecular mass (3x10(6) Da) Hb, V1-Hb, reminiscent of a vascular hexagonal bilayer annelid Hb; two major coelomic Hbs, C1-Hb, and C2-Hb, with unusual masses for extracellular annelid Hbs of 153 and 124 kDa respectively; and a minor probably coelomic Hb of 23 kDa (C3-Hb). Using electrospray ionization mass spectrometry, SDS-PAGE after subtilisin treatment, and tandem mass spectrometry, we showed that C1-Hb is a trimer of a 57,996 Da chain and C2-Hb is a dimer of a 57,648 Da chain, each chain being a four-domain/four-heme polypeptide. This multimeric, multidomain arrangement is unique among annelid Hbs and appears different from that of other known multidomain Hbs.     

Occurrence of two architectural types of hexagonal bilayer hemoglobin in annelids: comparison of 3D reconstruction volumes of Arenicola marina and Lumbricus terrestris hemoglobins

A 3D reconstruction at 25 A resolution of native hemoglobin of the polychaete worm Arenicola marina was carried out from frozen-hydrated specimens examined in the electron microscope. The reconstruction volume of this large extracellular multimeric respiratory pigment appears as a hexagonal bilayer structure with eclipsed vertices in its upper and lower hexagonal layers. Conversely, in hemoglobins of oligochaetes, achaetes, and vestimentiferans and in chlorocruorins of the Sabellidae (polychaete) family, the vertices of the upper layer are 16 degrees clockwise rotated with respect to those of the lower layer. The fact that two other polychaete hemoglobins (Alvinella pompejana and Tylorrhynchus heterochaetus) have the same architecture as Arenicola led us to define two types of hexagonal bilayer hemoglobins/chlorocruorins: (i) type-I present in oligochaete, achaete, and vestimentiferan hemoglobins and in Sabellidae chlorocruorins; and (ii) type-II present in polychaete hemoglobins. A comparative study of the hemoglobins of Lumbricus terrestris (type-I) and Arenicola marina (type-II) showed that only two small differences located in the c4 and c5 linking units are responsible of the important architectural difference present in oligomers. A likely scheme proposed to explain the phylogenic distribution of the two types suggests that Clitellata, Sabellida (polychaete), and vestimentiferan hemoglobins and chlorocruorins derive from a type-I ancestral molecule, while Terebellida (Alvinella), Phyllodocida (Tylorrhynchus), and Scolecida (Arenicola) and possibly other polychaetes derive from an ancestor molecule with type-II hemoglobin. The architectures of the hollow globular substructures are highly similar in Arenicola and Lumbricus hemoglobins, with 12 globin chains and three linking units (c3a, c3b, and c4). The central piece of Arenicola hemoglobin is an ellipsoid while that of Lumbricus is a toroid. No phylogenic correlation could be found between the structure of the central pieces and the architecture type.     

Towards a resolution of the long-standing controversy regarding the molecular mass of extracellular erythrocruorin of the earthworm Lumbricus terrestris

The published molecular mass of erythrocruorin of Lumbricus terrestris and related earthworm species covers a bewildering range of 3.23-4.5 MDa. A critical reexamination reveals that some mass determinations were underestimated and the results do cluster, not at one, but at two values of the molecular mass. One cluster corresponds to approximately 3.6 MDa, as predicted for a stoichiometry of 144 globin and 36 linker chains-the Vinogradov model for the hexagonal bilayer (HBL) assembly of Lumbricus erythrocruorin-and as estimated from the crystal structure of HBL at 5.5 A resolution [Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 7107]. The other cluster corresponds to approximately 4.4 MDa. In addition, a molecular mass of 4.1 MDa, determined by multiangle laser light scattering (MALLS), stands apart of the two clusters, separated from the masses obtained by other methods of molecular mass determination.We propose a stoichiometry of 192 globin and 36 linker chains for the 4.4-MDa molecule. The 36 linkers and 144 out of 192 globin chains are identified with the HBL and the remaining 48 globins are allotted equally to the two halves of the axial cavity above and below the central torus of the structure. The proposed model is supported by the occurrence in some annelid species of erythrocruorin with centrally placed subunits [Biochim. Biophys. Acta 359 (1974) 210], and by the oxidation-dependent shedding of subunits in Lumbricus erythrocruorin. We propose further that the 4.1 MDa determination represents the weight average molecular mass of a population of molecules resulting from a partial dissociation of 4.4-MDa erythrocruorin. This interpretation seems reasonable on the background of the very low protein concentrations ( approximately 100 microg/ml and lower) prevailing at the MALLS experiment.     

Mass distributions of a macromolecular assembly based on electrospray ionization mass spectrometric masses of the constituent subunits

Macromolecular assemblies containing multiple protein subunits and having masses in the megadalton (MDa) range are involved in most of the functions of a living cell. Because of variation in the number and masses of subunits, macromolecular assemblies do not have a unique mass, but rather a mass distribution. The giant extracelular erythrocruorins (Ers), ∼ 3.5 MDa, comprized of at least 180 polypeptide chains, are one of the best characterized assemblies. Three-dimensional reconstructions from cryoelectron microscopic images show them to be hexagonal bilayer complexes of 12 subassemblies, each comprised of 12 globin chains, anchored to a subassembly of 36 nonglobin linker chains. We have calculated the most probable mass distributions forLumbricus andRiftia assemblies and their globin and linker subassemblies, based on theLumbricus Er stoichiometry and using accurate subunit masses obtained by electrospray ionization mass spectrometry. The expected masses ofLumbricus andRiftia Ers are 3.517 MDa and 3.284 MDa, respectively, with a possible variation of ∼ 9% due to the breadth of the mass distributions. TheLumbricus Er mass is in astonishingly good agreement with the mean of 23 known masses, 3.524 ± 0.481 MDa.     

Novel dissociation mechanism of a polychaetous annelid extracellular haemoglobin

The extracellular haemoglobin of the marine polychaete, Arenicola marina, is a hexagonal bilayer haemoglobin of approximately 3600 kDa, formed by the covalent and noncovalent association of many copies of both globin subunits (monomer and trimer) and nonglobin or 'linker' subunits. In order to analyse the interactions between globin and linker subunits, dissociation and reassociation experiments were carried out under whereby Arenicola hexagonal bilayer haemoglobin was exposed to urea and alkaline pH and the effect was followed by gel filtration, SDS/PAGE, UV-visible spectrophotometry, electrospray-ionization MS, multiangle laser light scattering and transmission electron microscopy. The analysis of Arenicola haemoglobin dissociation indicates a novel and complex mechanism of dissociation compared with other annelid extracellular haemoglobins studied to date. Even though the chemically induced dissociation triggers partial degradation of some subunits, spontaneous reassociation was observed, to some extent. Parallel dissociation of Lumbricus haemoglobin under similar conditions shows striking differences that allow us to propose a hypothesis on the nature of the intersubunit contacts that are essential to form and to hold such a complex quaternary structure.     

Preparation and characterization of monoclonal antibodies to the extracellular hemoglobin of Lumbricus terrestris

Murine monoclonal antibodies to the extracellular hemoglobin of Lumbricus terrestris were prepared by a modification of the method of Kohler and Milstein. 224 hybridomas were found to produce antibodies which bound to the hemoglobin; they were tested for binding to the four subunits of the hemoglobin: M (chain I, 16 kDa), D1 (chain V, 31 kDa), D2 (chain VI, 37 kDa) and T (50 kDa), a disulfide-bonded trimer of chains II, III and IV, each of about 17 kDa. 150 hybridomas bound to all four subunits and 40 hybridomas bound to various combinations of subunits. The remaining 34 hybridomas combined only with the hemoglobin. The twelve hybridomas obtained after subculturing and cloning were tested for their binding to the two fractions II and III, consisting of subunits D1 + D2 + T and M, respectively, obtained by dissociation at pH 9.5 and at pH 4.0 and to the reassociated whole molecules, obtained subsequent to return to neutral pH. Eight hybridomas which combined only with the hemoglobin also combined with all the reassociated molecules but not with any of the fractions: these monoclonal antibodies probably recognize conformation-dependent antigenic sites that are present only in the hexagonal bilayer structure characteristic of the native and reassociated hemoglobin molecules. Of the remaining four hybridomas, two bound to subunit T and two combined with subunits T and D2; they also combined with the reassociated molecules and with the fractions II. In addition, the hybridomas did not bind to the hemoglobins of Tubifex, Limnodrilus, Arenicola, Tylorrhynchus and Macrobdella or to the chlorocruorins of Myxicola and Eudistylia.     

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.     

A re-evaluation of the molecular mass of earthworm extracellular hemoglobin from meniscus depletion sedimentation equilibrium. Nature of the 10 S dissociation species.

Previous calculations from meniscus depletion sedimentation equilibrium earthworm hemoglobin from Lumbricus terrestris (E.J. Wood et al., Biochem. J. 153 (1976) 589-96) and from the related species Lumbricus sp. (L. sp.) (M.M. David and E. D Mol. Biol. 87 (1974) 89--101) were made on the assumption that the solutions behaved ideally. Re-examination of their results reveals, however, a dependence of the apparent molecular mass on concentration. Taking this effect into consideration, we have nowrecalculated from their data molecular masses of 4.4--4.5 MDa for the hemoglobin of both L. terrestris and L. sp. On the basis of the new determinations, we propose for the polypeptide chain composition of L. terrestris hemoglobin a model [(abcd )4L1L2L3]12 where a,b,c,d are the four globin and L1,L2,L3 are the three major linker chain constituents of the protein. The model is consistent with the D6 symmetry of the molecule. A 10 S intermediate product in the alkaline dissociation Lumbricus hemoglobin is viewed as a binary mixture of products resulting from a disproportionation reaction involving the structural unit. The present interpretation is shown to be consistent with observed relations between molecular masses and SDS gel electrophoretic band patterns of 10 S species and intact hemoglobin.     

Interaction of giant extracellular Glossoscolex paulistus hemoglobin (HbGp) with ionic surfactants: a MALDI-TOF-MS study

The giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) is constituted by approximately 144 subunits containing heme groups with molecular masses in the range of 16-19kDa forming a monomer (d) and a trimer (abc), and around 36 non-heme structures, named linkers (L). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF-MS) analysis was performed recently, to obtain directly information on the molecular masses of the different subunits from HbGp in the oxy-form. This technique demonstrated structural similarity between HbGp and the widely studied hemoglobin of Lumbricus terrestris (HbLt). Indeed, two major isoforms (d(1) and d(2)) of identical proportions with masses of 16,355+/-25 and 16,428+/-24Da, respectively, and two minor isoforms (d(3) and d(4)) with masses around 16.6kDa were detected for monomer d of HbGp. In the present work, the effects of anionic sodium dodecyl sulfate (SDS) and cationic cethyltrimethylammonium chloride (CTAC) on the oligomeric structure of HbGp have been studied by MALDI-TOF-MS in order to evaluate the interaction between ionic surfactants and HbGp. The data obtained with this technique show an effective interaction of cationic surfactant CTAC with the two isoforms of monomer d, d(1) and d(2), both in the whole protein as well as in the pure isolated monomer. The results show that up to 10 molecules of CTAC are bound to each isoform of the monomer. Differently, the mass spectra obtained for SDS-HbGp system showed that the addition of the anionic surfactant SDS does not originate any mass increment of the monomeric subunits, indicating that SDS-HbGp interaction is, probably, significantly less effective as compared to CTAC-HbGp one. The acid pI of the protein around 5.5 is, probably, responsible for this behavior. The results of this work suggest also some interaction of both surfactants with linker chains as well as with trimers, as judged from observed mass increments. Our data are consistent with a recent spectroscopic study showing a strong interaction between CTAC and HbGp at physiological pH [P.S.Santiago, et al, Biochim. Biophys. Acta 1770 (2007) 506-517.].     

The dissociation of the extracellular hemoglobin of Lumbricus terrestris at acid pH and its reassociation at neutral pH. A new model of its quaternary structure

Lumbricus terrestris HbO2 and HbCO dissociated below pH 5.0; a time-dependent alteration to the met form occurred at pH less than 5 and pH less than 4.5, respectively. The extent of dissociation was unaffected by alkaline earth cations but was decreased by an increase in ionic strength. HbO2 and HbCO exposed to pH 4.0-4.8 were centrifuged to obtain the undissociated pellet (P1) and dissociated supernatant (S1) fractions. S1 was reassociated at pH 7.0 by dialysis against various buffers and then centrifuged to obtain the reassociated pellet (P2) and unreassociated supernatant (S2) fractions. Reassociation was possible only if S1 was dialyzed against water prior to return to neutral pH; otherwise precipitation occurred starting at about pH 5.3. The extent of reassociation varied from about 40 to 80%, was usually higher for HbCO than HbO2, and was unaffected by an increase in ionic strength or by Ca(II). Gel filtration of P2 on Sephacryl S-300 at neutral pH gave one peak IaR, eluting at a slightly greater volume than the native Hb; S1 and S2 gave in addition, three peaks, Ib (200 kDa), II (65 kDa), and III (18 kDa). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that P2 was slightly deficient in subunit M relative to the Hb, that Ib was deficient in subunits D1 and D2 and that II and III consisted of subunits D1 + D2 + T and subunit M, respectively. Scanning transmission electron microscopy of P2 showed that it was smaller than the native hemoglobin: 25 nm in diameter and 16 nm in height, instead of 30 X 20 nm. Comparison of the results of the dissociations of Lumbricus Hb at alkaline pH (Kapp, O. H., Polidori, G., Mainwaring, M., Crewe, A. V., Vinogradov, S. N. (1984) J. Biol. Chem. 259, 628-639) with those obtained in this study suggested that the Hb quaternary structure was not multimeric and that an alternative model had to be considered. In the proposed model it is assumed that subunits D1 and D2 form a scaffolding or "bracelet," decorated with 12 complexes of M and T subunits.     

Effects of magnesium and calcium on the oxygenation reaction of erythrocruorin from the marine polychaete Arenicola marina and the terrestrial oligochaete Lumbricus terrestris

Oxygenation function of annelid erythrocruorin (Er) is affected by Mg and Ca concentration in the blood. Four classes of responses may be encountered in different species: 1) Mg=Ca (equal effects), 2) Mg>Ca, 3) Mg相似文献   

Native and subunit molecular mass and quarternary structure of the hemoglobin from the primitive branchiopod crustacean Triops cancriformis

Many branchiopod crustaceans are endowed with extracellular, high-molecular-weight hemoglobins whose exact structural characteristics have remained a matter of conjecture. By using a broad spectrum of techniques, we provide precise and coherent information on the hemoglobin of one of the phylogenetically 'oldest' extant branchiopods, the tadpole shrimp Triops cancriformis. The hemoglobin dissociated under reducing conditions into two subunits, designated TcHbA and TcHbB, with masses of 35,775+/-4 and 36,055+/-4 Da, respectively, determined by ESI-MS. Nonreducing conditions showed only two disulfide-bridged dimers, a homodimer of TcHbA, designated D1 (71,548+/-5 Da), and the heterodimer D2 (71,828+/-5 Da). Carbamidomethylation of free SH groups revealed the presence of three cysteines per subunit and indicated one intrasubunit and one intersubunit disulfide bridge. Ultracentrifugation and light-scattering experiments under nondenaturating conditions yielded mass estimates that suggested an uneven number of 17 subunits forming the native hemoglobin. This unrealistic number resulted from the presence of two size classes (16-mer and 18-mer), which were recognized by native PAGE and Ferguson plot analysis. ESI-MS revealed three hemoglobin isoforms with masses of 588.1 kDa, 662.0 kDa, and 665.0 kDa. The 16-mer and the smaller 18-mer species are supposed to be composed of TcHbA only, given the dominance of this subunit type in SDS/PAGE. Transmission electron microscopy of negatively stained specimens showed a population of compact molecules with geometrical extensions of 14, 16 and 9 nm. The proposed stoichiometric model of quarternary structure provides the missing link to achieve a mechanistic understanding of the structure-function relationships among the multimeric arthropodan hemoglobins.     

Low resolution crystal structure of Arenicola erythrocruorin: influence of coiled coils on the architecture of a megadalton respiratory protein

Annelid erythrocruorins are extracellular respiratory complexes assembled from 180 subunits into hexagonal bilayers. Cryo-electron microscopic experiments have identified two different architectural classes. In one, designated type I, the vertices of the two hexagonal layers are partially staggered, with one hexagonal layer rotated by about 16 degrees relative to the other layer, whereas in the other class, termed type II, the vertices are essentially eclipsed. We report here the first crystal structure of a type II erythrocruorin, that from Arenicola marina, at 6.2 A resolution. The structure reveals the presence of long continuous triple-stranded coiled-coil "spokes" projecting towards the molecular center from each one-twelfth unit; interdigitation of these spokes provides the only contacts between the two hexagonal layers of the complex. This arrangement contrasts with that of a type I erythrocruorin from Lumbricus terrestris in which the spokes are broken into two triple-stranded coiled coils with a disjointed connection. The disjointed connection allows formation of a more compact structure in the type I architecture, with the two hexagonal layers closer together and additional extensive contacts between the layers. Comparison of sequences of the coiled-coil regions of various linker subunits shows that the linker subunits from type II erythrocruorins possess continuous heptad repeats, whereas a sequence gap places these repeats out of register in the type I linker subunits, consistent with a disjointed coiled-coil arrangement.     

Ligand binding in a hierarchy of globin complexes. The hexagonal bilayer hemoglobin of Lumbricus terrestris and its heme-containing subunits

The kinetics of CO and NO recombination with the giant approximately 3600-kDa hexagonal bilayer hemoglobin of Lumbricus terrestris and its subunits, the approximately 200-kDa dodecamer of globin chains (3 x chains (I + II + III + IV] (see preceding paper (Vinogradov S.N., Sharma, P.K., Qabor, A.N., Wall, J.S., Westrick, J.A., Simmons, J.H., and Gill, S.J. (1991) J. Biol. Chem. 266, 13091-13096], the 50-kDa disulfide-bonded trimer (chains II-IV), the monomer (chain I), and the approximately 30-kDa linker (chains VA, VB, and VI), were measured following photolysis over time scales ranging from picosecond to millisecond. CO recombination at 436 nm subsequent to excitation (9 ns) at 532 nm showed three phases covering a 100-fold range for the Hb, dodecamer, trimer, and linker protein. The proportion of the fast phase was 0.1-0.2 for the trimer, dodecamer, and Hb. The relative rates and amplitudes of the phases were not affected by changes in CO concentration or excitation intensity. The monomer showed a single phase with a rate of 2 x 10(6) M-1 s-1. The second-order reaction with NO showed two rates. The faster rate was 90 x 10(6) M-1 s-1 and accounted for approximately 0.7 of the reaction for all species except the monomer, where it accounted for the full reaction. The slower rate was 15 x 10(6) M-1 s-1 for all species except the monomer.