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.

Polypeptide chain composition diversity of hexagonal-bilayer haemoglobins within a single family of annelids, the alvinellidae.

Following previous analysis of the structure of Alvinella pompejana heaxagonal-bilayer haemoglobin (HBL Hb) [1], we report in this paper the structure of three other HBL Hbs belonging to Alvinella caudata, Paralvinella grasslei and Paralvinella palmiformis, members of the Alvinellidae, annelid family strictly endemic to deep-sea hydrothermal vents located on the ridge crests in the Pacific ocean. The multi-angle laser light scattering (MALLS) and fast protein liquid chromatography (FPLC) analysis revealed a broad range of molecular masses for the extracellular Hb molecules, 3517 +/- 14 kDa (A. caudata), 3822 +/- 28 kDa (P. grasslei) and 3750 +/- 150 kDa (P. palmiformis). Native and derivative Hbs (reduced, carbamidomethylated and deglycosylated) were analysed by electrospray ionization mass spectroscopy (ESI-MS) and the data was processed by the maximum entropy deconvolution system (MaxEnt). The most important difference between alvinellid HBL Hbs was the variation in their composition, from two to four monomeric globin chains, and from one to four linker chains. Therefore, despite the fact that all these species belong to a single family, notable differences in the polypeptide chain composition of their HBL Hbs were observed, probably accounting for their different functional properties as previously reported by this group Toulmond, A., El Idrissi Slitine, F., De Frescheville, J. & Jouin, C. (1990) Biol. Bull. 179, 366-373.

Gas transfer system in Alvinella pompejana (Annelida polychaeta, Terebellida): functional properties of intracellular and extracellular hemoglobins.

Alvinella pompejana is a tubicolous polychaete that dwells in the hottest part of the hydrothermal vent ecosystem in a highly variable mixture of vent (350 degrees C, anoxic, CO(2)- and sulfide-rich) and deep-sea (2 degrees C, mildly hypoxic) waters. This species has developed distinct-and specifically respiratory-adaptations to this challenging environment. An internal gas exchange system has recently been described, along with the report of an intracellular coelomic hemoglobin, in addition to the previously known extracellular vascular hemoglobin. This article reports the structure of coelomic hemoglobin and the functional properties of both hemoglobins in order to assess possible oxygen transfer. Coelomocytes contain a unique monomeric hemoglobin with a molecular weight of 14,810+/-1.5 Da, as determined by mass spectrometry. The functional properties of both hemoglobins are unexpectedly very similar under the same conditions of pH (6.1-8.2) and temperature (10 degrees -40 degrees C). The oxygen affinity of both proteins is relatively high (P50=0.66 Torr at 20 degrees C and pH 7), which facilitates oxygen uptake from the hypoxic environment. A strong Bohr effect (Phi ranging from -0.8 to -1.0) allows the release of oxygen to acidic tissues. Such similar properties imply a possible bidirectional transfer of oxygen between the two hemoglobins in the perioesophagal pouch, a mechanism that could moderate environmental variations of oxygen concentration and maintain brain oxygenation.

Electrospray ionization mass spectrometric determination of the molecular mass of the approximately 200-kDa globin dodecamer subassemblies in hexagonal bilayer hemoglobins.

Hexagonal bilayer hemoglobins (Hbs) are approximately 3.6-MDa complexes of approximately 17-kDa globin chains and 24-32-kDa, nonglobin linker chains in a approximately 2:1 mass ratio found in annelids and related species. Studies of the dissociation and reassembly of Lumbricus terrestris Hb have provided ample evidence for the presence of a approximately 200-kDa linker-free subassembly consisting of monomer (M) and disulfide-bonded trimer (T) subunits. Electrospray ionization mass spectrometry (ESI-MS) of the subassemblies obtained by gel filtration of partially dissociated L. terrestris and Arenicola marina Hbs showed the presence of noncovalent complexes of M and T subunits with masses in the 213. 3-215.4 and 204.6-205.6 kDa ranges, respectively. The observed mass of the L. terrestris subassembly decreased linearly with an increase in de-clustering voltage from approximately 215,400 Da at 60 V to approximately 213,300 Da at 200 V. In contrast, the mass of the A. marina complex decreased linearly from 60 to 120 V and reached an asymptote at approximately 204,600 Da (180-200 V). The decrease in mass was probably due to the progressive removal of complexed water and alkali metal cations. ESI-MS at an acidic pH showed both subassemblies to consist of only M and T subunits, and the experimental masses demonstrated them to have the composition M(3)T(3). Because there are three isoforms of M and four isoforms of T in Lumbricus and two isoforms of M and 5 isoforms of T in Arenicola, the masses of the M(3)T(3) subassemblies are not unique. A random assembly model was used to calculate the mass distributions of the subassemblies, using the known ESI-MS masses and relative intensities of the M and T subunit isforms. The expected mass of randomly assembled subassemblies was 213,436 Da for Lumbricus Hb and 204,342 Da for Arenicola Hb, in good agreement with the experimental values.

Characterization and functional properties of the extracellular coelomic hemoglobins from the deep-sea, hydrothermal vent scaleworm Branchipolynoe symmytilida.

Polychaete species belonging to the genus Branchipolynoe are commensal with mussels from deep-sea hydrothermal vents and cold-seeps. Possessing hemoglobins (Hbs), the species B. symmytilida, which is found in the mussel Bathymodiolus thermophilus on the East Pacific Rise, is exceptional in a family normally devoid of respiratory pigments. In a previous paper we described two major coelomic extracellular hemoglobins with unique quaternary structures. Aiming to discern respiratory adaptations to the highly variable hydrothermal environment, this paper characterizes the functional properties of these Hbs and the coelomic fluid. The two major hemoglobins (C1 and C2) exhibit spectrophotometric characteristics of both intra- and extracellular hemoglobins. However, their amino acid content is very different from other known hemoglobins and is characterized by a high proportion of alanine and glycine (up to 40% cumulated in C1). C1 and C2 differ markedly by their cysteine content (0.8% and 13% respectively). The coelomic fluid exhibits a strong buffer capacity due to the high hemoglobin content (3 mM heme). In vitro, CO2 accumulation (up to 10-12 mM CO2 for PCO2 = 7.5 Torr) occurs with limited pH changes and is only partly accounted for by carbamino-Hb formation. The two hemoglobins exhibit high oxygen-affinities (P50 0.4 Torr for C1 and 0.9 Torr for C2, at 10 degrees C, pH 8) and a normal Bohr effect (phi values ranging from -0.54 and -0.37 at 10 degrees C, to -0.24 and -0.28 at 30 degrees C, for C1 and C2, respectively). Cooperativity values range from 0.8 to 1.9 for C1 and from 0.8 to 1.7 for C2. The temperature sensitivity of O2 affinity reflect deltaH values that decrease from -30 to -60 kJ x mol(-1) with increasing pH. C2 exhibits a slight specific effect of CO2 on oxygenation properties.

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.

S-Sulfohemoglobin and disulfide exchange: the mechanisms of sulfide binding by Riftia pachyptila hemoglobins.

The deep sea hydrothermal tube worm Riftia pachyptila possesses a multihemoglobin system with three different extracellular hemoglobins (Hbs; V1, V2, and C1): two dissolved in the vascular blood, V1 and V2, and one in the coelomic fluid, C1. V1 consists of four heme-containing chains and four linker chains. The globin chains making up V2 and C1 are, with one exception, common to V1. Remarkably these Hbs are able to bind oxygen and sulfide simultaneously and reversibly at two different sites. Two of the globin chains found in these three Riftia Hbs possess one free Cys residue and for at least one of the globins, the b-Cys75 is conserved among vestimentifera (Lamellibrachia sp.) and pogonophora (Oligobrachia mashikoi). By selectively blocking the free Cys with N-ethylmaleimide and using electrospray ionization mass spectrometry experiments, we show that these Cys residues are involved in sulfide binding by Riftia Hbs. Moreover, we also demonstrate that the larger V1 Hb can form persulfide groups on its linker chains, a mechanism that can account for the higher sulfide-binding potential of this Hb.

Primary structure of the common polypeptide chain b from the multi-hemoglobin system of the hydrothermal vent tube worm Riftia pachyptila: an insight on the sulfide binding-site.

The deep-sea tube worm Riftia pachyptila Jones possesses a multi-hemoglobin system with three different extracellular Hbs: two dissolved in the vascular blood, V1 (ca. 3,500 kDa) and V2 (ca. 400 kDa), and one in the coelomic fluid, C1 (ca. 400 kDa). V1 Hb consists of four heme-containing, globin chains (b-e) and four linker chains (L1-L4). V2 and C1 Hbs are exclusively built from globin chains, six for V2 (a-f) and five for C1 (a-e). The complete amino acid sequence of the isolated monomeric globin chain b, common to all Riftia Hbs, has been determined by automated Edman degradation sequencing of the peptides derived by digestion with trypsin, chymotrypsin, thermolysin, and CNBr. This polypeptide chain is composed of 144 amino acid residues, providing a M(r) of 16, 135.0 Da. Moreover, the primary sequence of chain b revealed 3 Cys residues at position 4, 75, and 134. Cys-4 and Cys-134 are located at positions where an intra-chain disulfide bridge is formed in all annelid, vestimentiferan, or pogonophoran chains, but Cys-75 is located at a unique position only found in three globin chains belonging to Lamellibrachia and Oligobrachia, a vestimentiferan and a pogonophoran. In both groups, Hbs can bind sulfide reversibly to fuel the chemosynthetic process of the symbiotic bacteria they harbor. Sulfide-binding experiments performed on purified Hb fractions (i.e., V1, V2, and C1 Hbs) suggest that free Cys residues on globin chains, and the numerous Cys found in linker chains, as determined previously by ESI-MS, may be the sulfide binding-sites. Blocking the free Cys by N-ethylmaleimide, we confirmed that free cysteines were involved in sulfide-binding but did not account for the whole sulfide-binding capacity of V1 Hb. Furthermore, a phylogenetic tree was constructed from 18 globin-like chains of annelid, vestimentiferan, and pogonophoran extracellular Hbs to clarify the systematic position of tubeworms. Riftia chain b clearly belongs to the "strain A" family with 30 to 80% identity with the other sequences analyzed. Its position in the tree confirmed a close relationship between vestimentiferan, pogonophoran, and annelid Hbs.

Investigation by electrospray ionization mass spectrometry of the extracellular hemoglobin from the polychaete annelid Alvinella pompejana: an unusual hexagonal bilayer hemoglobin.

Alvinella pompejana inhabits deep-sea hydrothermal vent sites along the East-Pacific Rise, where it colonizes the walls of actively venting high-temperature chimneys. This worm is the most thermophilic metazoan known to date. In Alvinella, as in other alvinellids, oxygen transport is mainly achieved by an extracellular Hb dissolved in the vascular blood. This Hb has a molecular mass of 3833 +/- 14 kDa as revealed by multiangle laser light scattering (MALLS). Native and derivative Hb (reduced, carbamidomethylated, and deglycosylated) were analyzed by electrospray ionization mass spectrometry (ESI-MS). The data were processed by the maximum entropy deconvolution system (MaxEnt). We identified three groups of peaks for Alvinella Hb, at ca. 16, 23-26, and 50 kDa corresponding to (i) four monomeric globin chains, a1 (16 633.4), a2(16 532.4), a3 (16 419.6), and a4(16 348.9); (ii) four linker chains, L1-L4 (22 887. 1, 24 230.5, 26 233.6, and 25 974.4); and (iii) one disulfide-bonded trimer T (51 431.9) composed of globin chains b (16 477.5), c (16 916.1), and d (18 048.8). These Hbs were also subjected to SDS-PAGE analysis for comparative purposes. In addition, using the ESI-MS data we propose two alternative models for the quaternary structure of Alvinella's Hb.

Quaternary structure of the extracellular haemoglobin of the lugworm Arenicola marina: a multi-angle-laser-light-scattering and electrospray-ionisation-mass-spectrometry analysis.

To elucidate the quaternary structure of the extracellular haemoglobin (Hb) of the marine polychaete Arenicola marina (lugworm) it was subjected to multi-angle laser-light scattering (MALLS) and to electrospray-ionisation mass spectrometry (ESI-MS). It was also subjected to SDS/PAGE analysis for comparative purposes. MALLS analysis gave a molecular mass of 3648 +/- 24 kDa and a gyration radius of 11.3 +/- 1.7 nm. Maximum entropy analysis of the multiply charged electrospray spectra of the native, dehaemed, reduced and carbamidomethylated Hb forms, provided its complete polypeptide chain and subunit composition. We found, in the reduced condition, eight globin chains of molecular masses 15952.5 Da (a1), 15974.8 Da (a2), 15920.9 Da (b1), 16020.1 Da (b2), 16036.2 Da (b3), 16664.8 Da (c), 16983.2 Da (d1), 17033.1 Da (d2) and two linker chains L1, 25174.1 Da, and L2, 26829.7 Da. In the native Hb, chains b, c, d occur as five disulphide-bonded trimer subunits T with masses of 49560.4 Da (T1), 49613.9 Da (T2), 49658.6 Da (T3), 49706.8 Da (T4), 49724.5 Da (T5). Linker chains L1 and L2 occur as one disulphide-bonded homodimer 2L1 (D1) of 50323.1 Da and one disulphide-bonded heterodimer L1-L2 (D2) of 51 981.5 Da. Polypeptide chains a and d possess one free cysteine residue and chains d possess an unusual total of five cysteine residues. Semi-quantitative analysis of ESI-MS data allowed us to propose the following model for the one-twelfth protomer: [(3a1)(3a2)2T] (T corresponding to either T3, T4 or T5). From electron micrograph data T1 and T2 are probably located at the centre of the molecule as mentioned in previous studies. The Hb would thus be composed of 198 polypeptide chains with 156 globin chains and 42 linker chains, each twelfth being in contact with 3.5 linker subunits, providing a total mass of 3682 kDa including haems in agreement with the experimental molecular mass determined by MALLS. From ESI-MS relative intensities and the model proposed above, the globin/linker ratio gave 0.71:0.29 and 0.73:0.27, respectively. The estimation of haem content by pyridine haemochromogen and by cyanmethaemoglobin (HiCN) methods also support the globin chain number provided by ESI-MS.

Three-dimensional reconstruction of the hexagonal bilayer hemoglobin of the hydrothermal vent tube worm Riftia pachyptila by cryoelectron microscopy.

A frozen-hydrated specimen of the V1 hemoglobin of the hydrothermal vent tube worm Riftia pachyptila was observed in the electron microscope and subjected to three-dimensional reconstruction by the method of random conical tilt series. The 3D volume possesses a D6 point-group symmetry. When viewed along its 6-fold axis the vertices of its upper hexagonal layer are 16 degrees clockwise rotated compared to those of the lower layer. A central linker complex is decorated by 12 hollow globular substructures. The linker complex comprises (i) a central hexagonal toroid, (ii) two internal bracelets onto which the hollow globular substructures are built, and (iii) six structures connecting the two hexagonal layers. The hollow globular substructures, related to the dodecamers of globin chains resulting from the dissociation of the hexagonal bilayer hemoglobin, have a local pseudo 3-fold symmetry and are composed each of three elongated structures visible when the volume is displayed at high threshold. At a resolution of 36 A, the 3D volumes of the hexagonal bilayer hemoglobins of Riftia pachyptyla and of the leech Macrobdella decora look almost perfectly identical.

Three-dimensional reconstruction by cryoelectron microscopy of the giant hemoglobin of the polychaete worm Alvinella pompejana.

A frozen-hydrated specimen of the hexagonal bilayer hemoglobin (HBL Hb) from the deep-sea hydrothermal vent polychaete worm Alvinella pompejana, the most thermophilic metazoan known to date, was observed in the electron microscope and subjected to three-dimensional (3D) reconstruction by the method of random conical tilt series. At a resolution of 34.6 A by the differential phase residual method and 27.7 A by the Fourier shell correlation method, the 3D volume possesses a D6 point-group symmetry. While in previous 3D reconstructions of annelid and vestimentiferan Hbs the vertices of the upper layer were 16 degrees rotated compared with those of the lower layer, in Alvinella Hb the vertices of the two hexagonal layers are almost perfectly eclipsed when viewed along the 6-fold axis. As in the HBL Hbs of Riftia pachyptila and Macrobdella decora, a central linker complex is decorated by 12 hollow globular substructures (HGS). The linker complex comprises (1) a central hexagonal toroid, (2) two internal bracelets onto which the HGSs are built, and (3) six connections between the two hexagonal layers. Each HGS is composed of six masses, which are separated when the volume is displayed at high threshold, plus one additional mass involved in the bracelet connecting the six HGSs in both hexagonal layers. The HGSs have a local pseudo 3-fold symmetry and a disposition of the high-density masses different from those of Riftia V1 Hb.

The multi-hemoglobin system of the hydrothermal vent tube worm Riftia pachyptila. I. Reexamination of the number and masses of its constituents.

The deep-sea tube worm Riftia pachyptila Jones possesses a well developed circulatory system and a large coelomic compartment, both containing extracellular hemoglobins. Fresh vascular blood is heterogeneous and contains two different hemoglobins (V1 and V2), whereas the coelomic fluid is homogeneous and comprises only one hemoglobin (C1). Their molecular weights have been determined by scanning transmission electron microscopy mass mapping (STEM) and by multi-angle laser light scattering (MALLS). Both methods yielded approximately the same molecular weights with masses significantly higher than the literature data for V1. V1, V2, and C1 had Mr of 3396 +/- 540 x 10(3), 393 +/- 71 x 10(3), and 410 +/- 51 x 10(3) by STEM, and 3503 +/- 13 x 10(3), 433 +/- 8 x 10(3), and 380 +/- 4 x 10(3) by MALLS, respectively. Transmission electron micrographs of V1 are typical of an hexagonal bilayer hemoglobin (HBL Hb). When submitted to dilution or osmotic shock, V1 dissociates into halves and one-twelfth subunits like annelid HBL Hbs. V1 is resistant to urea treatment, indicating that hydrophobic interactions play a small role in its quaternary structure. Conversely, V1 Hb is rather unstable in solution without denaturant, a property which seems to be characteristic of vestimentiferan HBL Hbs and could be explained by an important number of hydrogen bonds.

The multi-hemoglobin system of the hydrothermal vent tube worm Riftia pachyptila. II. Complete polypeptide chain composition investigated by maximum entropy analysis of mass spectra.

The deep-sea tube worm Riftia pachyptila Jones possesses a complex of three extracellular Hbs: two in the vascular compartment, V1 (approximately 3500 kDa) and V2 (approximately 400 kDa), and one in the coelomic cavity, C1 (approximately 400 kDa). These native Hbs, their dissociation products and derivatives were subjected to electrospray ionization mass spectrometry (ESI-MS). The data were analyzed by the maximum entropy deconvolution system. We identified three groups of peaks for V1 Hb, at approximately 16, 23 27, and 30 kDa, corresponding to (i) two monomeric globin chains, b (Mr 16,133.5) and c (Mr 16,805.9); (ii) four linker subunits, L1 L4 (Mr 23,505.2, 23,851.4, 26,342.4, and 27,425.8, respectively); and (iii) one disulfide-bonded dimer D1 (Mr 31,720.7) composed of globin chains d (Mr 15,578.5) and e (Mr 16, 148.3). V2 and C1 Hbs had no linkers and contained a glycosylated monomeric globin chain, a (Mr 15,933.4) and a second dimer D2 (Mr 32,511.7) composed of chains e and f (Mr 16,368.1). The dimer D1 was absent from C1 Hb, clearly differentiating V2 and C1 Hbs. These Hbs were also subjected to SDS-PAGE analysis for comparative purposes. The following models are proposed ((cD1)(bD1)3) for the one-twelfth protomer of V1 Hb, ((cD)(bD)6(aD)) (D corresponding to either D1 or D2) for V2 and C1 Hbs. HBL V1 Hb would be composed of 180 polypeptide chains with 144 globin chains and 36 linker chains, each twelfth being in contact with three linker subunits, providing a total molecular mass = 3285 kDa. V2 and C1 would be composed of 24 globin chains providing a total molecular mass = 403 kDa and 406 kDa, respectively. These results are in excellent agreement with experimental Mr determined by STEM mass mapping and MALLS.

Energetics of the Ventilatory Piston Pump of the Lugworm, a Deposit-feeding Polychaete Living in a Burrow.

The aim of this study was to tentatively estimate the energy cost of breathing in the lugworm, Arenicola marina (L.), a gallery-dwelling, piston-pump breather that moves water in a tail-to-head direction. Each tested lugworm was placed in a horizontal glass tube. The caudal end of the tube was connected to a well-aerated seawater reservoir at 20{deg}C, and the cephalic end attached to a drop meter through a tube resistance. At the exit of the cephalic chamber the O2 tension was recorded via an in situ O2 electrode, and the hydrostatic pressure of the exhaled water was also recorded. Water flow rate, total O2 uptake rate {Mdot}TOTO2, O2 extraction coefficient, and the mechanical power necessary to pump water through the resistive anterior exit of the apparatus ({Wdot}MEC), were computed. The basal metabolic rate of each animal ({Mdot}CONFO2) was separately estimated by the confinement method. {Mdot}CONFO2 subtracted from {Mdot}TOTO2 approximates {Mdot}CBO2, the O2 uptake rate necessary to activate the piston-pump breathing mechanism and to ensure the corresponding mechanical work rate, {Wdot}MEC. The results show that the energy cost of breathing, {Mdot}CBO2, of the piston-pump-breathing Arenicola is very high, with mean values approximating 47% of the {Mdot}TOTO2 value; that the mechanical power we measured, {Wdot}MEC, is very low; and that the mechanical-to-metabolic efficiency, the ratio {Wdot}MEC/{Mdot}CBO2, does not exceed 1%. These observations are compared to those obtained in other piston-pump breathers, such as Chaetopterus variopedatus and Urechis caupo, and in ciliary filter feeders including polychaetes, bivalves, and ascidians.

Seawater salinity and blood acid-base balance in the lugworm, Arenicola marina (L.).

The kinetics of variations in the blood acid base balance (ABB) were investigated in a moderately euryhaline osmoconformer, the lugworm Arenicola marina (L.), exposed to natural and experimental hypo- or hyperosmotic shocks. In natural as well as in experimental conditions, a hyposmotic shock induced a transient and essentially metabolic acidosis, probably linked to the ionic readjustments following the shock, which was rapidly overridden by a metabolic alkalosis. In field conditions, a new ABB equilibrium was then attained, the metabolic alkalosis being neutralized by the respiratory and metabolic acidosis occurring normally in the lugworm during low tide. Conversely, in the normoxic conditions of our laboratory experiments, a new ABB equilibrium was never reached. Under experimental conditions, a hyperosmotic shock always induced a respiratory and metabolic acidosis. In the field, this phenomenon must occur at the beginning of high tide and must help to restore normal blood ABB in lugworms submitted to a moderate hyposmotic shock during low tide. All the observed blood ABB variations reveal the complex intracellular processes through which the lugworm submitted to moderate osmotic shocks tentatively regulates, sometimes without any real success, its osmoticity and volume. Obviously, complementary physical, physiological and behavioral mechanisms allow the lugworm to live in sediments washed by almost fresh water during a 7-8 h 'low tide'.

Two-dimensional electrophoresis of Arenicola marina extracellular hemoglobin: separation of chains with identical molecular mass but different isoelectric point.

1. On the basis of their molecular masses, four types of polypeptides (A, B, C, D) were obtained by SDS-PAGE of the extracellular hemoglobin of the polychaete annelid Arenicola marina. 2. On 2-dimensional polyacrylamide gel electrophoresis, the erythrocruorin dissociated into six different types of polypeptide chains; A1, A2, B1, B2, C and D. 3. A1 and B1 migrate in 2-dimensional electrophoresis at the same position as alpha and beta chains of human hemoglobin.

Extracellular Hemoglobins of Hydrothermal Vent Annelids: Structural and Functional Characteristics in Three Alvinellid Species.

The polychaete annelids Alvinella pompejana, Alvinella caudata, and Paralvinella grasslei are strictly associated with deep sea hydrothermal vents. Each species possesses an extracellular hemoglobin, Hb, which has been studied and compared to that of a common intertidal polychaete, the lugworm Arenicola marina. The four Hbs exhibit very similar quaternary structures and spectral properties, and only small differences appeared in the gross polypeptide compositions after reduction and sodium dodecyl sulfate denaturation of the native molecules. Conversely, by a comparison of the effects of pH (6.6-7.6) and temperature (10-40{deg}C) on their intrinsic O affinities, Bohr factors, cooperativities, and apparent heats of oxygenation, lugworm Hb can be differentiated from that of the alvinellids, and the Hb of A. pompejana from that of A. caudata. The known biology of the lugworm and a further analysis of the data suggest several hypotheses concerning the in vivo O2 transport function of the alvinellid Hbs, the in vivo blood pH value in the two alvinellid species, their respective range of optimal temperature, and their ability to create a differentiated and stable external microenvironment.

Ventilation and respiratory gas exchanges of the lugworm Arenicola marina (L.) as functions of ambient PO2 (20-700 torr).

Ventilatory regulation of intact, unrestrained lugworms Arenicola marina living in glass-tube artificial burrows was examined for values of inspired seawater PO2, PIO2, from 20 to 700 torr, at constant ambient pH and PCO2 values. The water ventilation rate and the respiratory characteristics of the ventilated seawater were measured. The water convection requirement and the corresponding specific rates of O2 uptake and CO2 production were calculated. The mean ventilatory water flow was a complex function of PIO2: decrease in hyperoxia, increase in hypoxia, decrease in extreme hypoxia. Compared to the normoxic responses, hyperoxia led to a hypercapnia (and acidosis) and moderate hypoxia to a hypocapnia (and alkalosis) in the expired water, variations which presumably reflect blood acid-base balance changes. Thus, as in other water breathers, the regulation of the organism's oxygenation may override the regulation of its acid-base balance. The lugworm's oxygen exchanger is highly efficient. However, below a critical partial pressure, PIO2 ca 120 torr, values of O2 consumption and ventilation decreased. A second critical O2 partial pressure appeared at PIO2 values between 80 and 40 torr; a 'switch-on' of anaerobic metabolism. These phenomena may be viewed as features of an adaptative respiratory strategy selected for in relation with the lugworm's particular peristaltic ventilatory mechanism and its intertidal mode of life.