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.     

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.     

The effect of alkaline earth cations and of ionic strength on the dissociation of earthworm hemoglobin at alkaline pH

1. The effect of alkaline earth cations on the dissociation of the extracellular hemoglobin of Lumbricus terrestris and the effect of ionic strength on the dissociation of the hemoglobins of L. terrestris and Tubifex tubifex at concentrations of ca 2.5 mg/ml, over the pH range 9.0-10.5 was investigated using ultracentrifugation to separate the dissociated from the undissociated molecules. 2. Mg(II), Ca(II) and Sr(II) at concentrations of up to 0.2 M, decreased the dissociation of Lumbricus oxyhemoglobin from 70% at pH 9.0 and 100% at pH 9.5 and higher, to 20-30% at 0.05 M. The three cations were equally effective in decreasing the extent of dissociation of L. terrestris oxyhemoglobin over the pH range 9.0-10.5, with a K1/2 of ca 10 mM. 3. The dissociation of L. terrestris oxyhemoglobin over the pH range 9.0-10.5 was decreased only to 50-60% in the presence of up to 0.5 M NaCl or KCl; there was no further decrease in dissociation at concentrations of the two salts up to 1.5 M. 4. The dissociation of T. tubifex oxyhemoglobin over the pH range 9.0-10.0 was decreased from 100% to ca 40-50% in the presence of 0.5 M NaCl or KCl with little or no change at higher concentrations. At pH 10.5 and 11.0 the decrease in dissociation was more gradual, reaching ca 50% at 1.5 M NaCl.     

Amino acid sequence of the monomer subunit of the extracellular hemoglobin of Lumbricus terrestris

The giant extracellular hemoglobin (3,800 kDa) of the oligochaete Lumbricus terrestris consists of four subunits: a monomer (chain I), two subunits each of about 35 kDa (chains V and VI), and a disulfide-bonded trimer (50 kDa) of chains II, III, and IV. The complete amino acid sequence of chain I was determined: it consists of 142 amino acid residues and has a molecular weight of 16,750 including a heme group. Fifty-nine residues (42%) were found to be identical with those in the corresponding positions in Lumbricus chain II (Garlick, R. L., and Riggs, A. F. (1982) J. Biol. Chem. 257, 9005-9015); 45 (32%), 56 (40%), 44 (31%), and 45 (32%) residues were found to be in identical positions in the sequences of chains I, IIA, IIB, and IIC, respectively, of Tylorrhynchus heterochaetus hemoglobin (Suzuki, T., and Gotoh, T. (1986) J. Biol. Chem. 261, 9257-9267). When the sequences of all six annelid chains are compared, 18 invariant residues are found in the first 104 residues of the molecule; very little homology exists among the annelid chains in the carboxyl-terminal 38-residue region. Nine of the 18 invariant residues are also found in the human beta-globin chain.     

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.     

Purification and characterization of a Ca2+-binding protein in Lumbricus terrestris.

A Ca2+-binding protein which is capable of activating mammalian Ca2+-activatable cyclic nucleotide phosphodiesterase has been purified from Lumbricus terrestris and characterized. This protein and the Ca2+-dependent protein modulator from bovine tissues have many similar properties. Both proteins have molecular weights of approximately 18,000, isoelectric points of about pH 4, similar and characteristic ultraviolet spectra, and similar amino acid compositions. Both proteins bind calcium ions with high affinity. However, the protein from Lumbricus terrestris binds 2 mol of calcium ions with equal affinity, Kdiss = 6 X 10(-6) M, whereas the Ca2+-dependent protein modulator from bovine tissues binds 4 mol of calcium ions with differing affinities. Although the Ca2+-binding protein of Lumbricus terrestris activates the Ca2+-activatable cyclic nucleotide phosphodiesterase from mammalian tissues, we have failed to detect the existence of a Ca2+-activatable phosphodiesterase activity in Lumbricus terrestris. The activation of phosphodiesterase by the Ca2+-binding protein from Lumbricus terrestris is inhibited by the recently discovered bovine brain modulator binding protein (Wang, J. H., and Desai, R. (1977) J. Biol. Chem. 252, 4175-4184). Since the modulator binding protein has been shown to associate with the mammalian protein modulator to result in phosphodiesterase inhibition, it can be concluded that the Lumbricus terrestris Ca2+-binding protein also associates with the bovine brain modulator binding protein. Attempts to demonstrate the existence of a similar modulator binding protein in Lumbricus terrestris have been unsuccessful.     

A disulfide-bonded trimer of myoglobin-like chains is the principal subunit of the extracellular hemoglobin of Lumbricus terrestris

The extracellular hemoglobin of Lumbricus terrestris (3900 kDa) consists of at least six different polypeptide chains: I through IV (16-19 kDa), V (31 kDa) and IV (37 kDa) (Vinogradov, S.N., Shlom, J.M., Hall, B.C., Kapp, O.H. and Mizukami, H. (1977) Biochim. Biophys. Acta 492, 136-155). SDS-polyacrylamide gel electrophoresis of the unreduced hemoglobin shows that chains II, III and IV form a disulfide-bonded 50 kDa subunit. This subunit was isolated by gel filtration of the hemoglobin on Sephacryl S-200 (a) at neutral pH in 0.1% SDS and (b) in 0.1 M sodium acetate buffer (pH 4.0); in the latter case it retains heme. The 50 kDa subunit obtained by method (b) was reduced and subjected to chromatofocusing on PBE 94 column: the elution pattern obtained with Polybuffer 74 (pH 4.5) and monitored at 280 nm, consisted of three peaks A, B and C; peaks A and B but not C, had absorbance at 410 nm. SDS-polyacrylamide gel electrophoresis showed that peaks A, B and C corresponded to chains II, IV and III, respectively. Amino acid analyses and N-terminal sequence determinations identified chain II as the whose primary structure had been determined (Garlick, R. and Riggs, A. (1982) J. Biol. Chem. 257, 9005-9015). Carbohydrate analysis of the native hemoglobin shows it to contain 2.0 +/- 0.5% carbohydrate consisting of mannose and N-acetylglucosamine in a mole ratio of about 9:1. The carbohydrate content of the 50 kDa subunit is 1.8 +/- 0.5%; it consists of mannose and N-acetylglucosamine in the same ratio and it appears to be associated with chain IV. Rabbit polyclonal antisera to 50 kDa subunit, prepared by method (a), and to the native hemoglobin were shown to cross-react with the hemoglobin and the 50 kDa subunit, respectively, by immunodiffusion. One of eight mouse monoclonal antibodies directed against the native hemoglobin reacted strongly with the 50 kDa subunit prepared by methods (a) and (b) in an enzyme-linked immunosorbent assay (ELISA). Another monoclonal antibody reacted strongly with the 50 kDa subunit obtained by method (b). Neither of the two hybridomas exhibited a strong reaction with any of the three constituent chains of the 50 kDa subunit. These results suggest that the unusual disulfide-bonded 50 kDa subunit, consisting of three myoglobin-like polypeptide chains of which only two have heme, is an integral part of the native Lumbricus hemoglobin molecule.     

Chloride cotransport in the membrane of earthworm body wall muscles

The resting membrane potential (V(m)) of isolated somatic longitudinal muscles of the earthworm Lumbricus terrestris was studied by glass microelectrodes. The inhibition of chloride permeability by low pH did not affect V(m) of the muscle fibers in isolated somatic longitudinal muscles of the earthworm Lumbricus terrestris which was -48.7 mV (inside negative) at pH 7.3 and -49.1 at pH 5.6. On the other hand, bathing the muscles in Cl(-) and Na(+)-free solutions, or application of the chloride transporter inhibitor furosemide and Na(+)-K(+)-ATPase inhibitor ouabain depolarized the V(m) by 3-5 mV. The effects of a Cl(-) -free solution and ouabain were not additive. This demonstrates relatively small contribution of equilibrium potential for Cl(-) to the resting membrane potential and electrogenic effect of Na(+)K(+)-ATPase which is dependent on the supply of Na(+)(i) ions by furosemide-sensitive and Cl(-)(e)- and Na(+)(e)-dependent electroneutral transport (most probably Na(+)K(+)Cl(-) cotransport).     

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.     

The effects of salts on the subunit structure and dissociation of Lumbricus terrestris hemoglobin.

The effects of the neutral salts of the Hofmeister series, NaCl, NaClO4, MgCl2, NaI, and also guanidine hydrochloride (Gdn-HCl)on the subunit organization and the state of association of Lumbricus terrestris hemoglobin were examined by light scattering molecular weight measurements. The subunit dissociation of the parent duodecameric structure of 3 x 10(6) molecular weight by various salts is similar in pattern to the sequential splitting of the associated protein to half-molecules of hexamers of 1.5 x 10(6) molecular weight, followed by further dissociation at higher reagent concentration to monomers of 250000 molecular weight. Duodecamer to hexamer dissociation is observed in 0.4 M MgCl2, 1-2 M NaCl, and 1 M Gdn-HCl, while hexamer to monomer dissociation is seen in the presence of 1 M MgCl2. All three species of duodecamers, hexamers, and monomers seem to be present in 1 M NaClO4. Further splitting of the monomers of A subunits to smaller B fragments of one-third to one-quarter molecular weight is observed in 1 M NaI solutions. Optical rotation in the peptide region and absorption measurements in the Soret region indicate the salt dissociation of Lumbricus terrestris hemoglobin is not accompanied by major changes in the folding of the subunits, except in the case of the strong protein denaturant, Gdn-HCl. Relative to the dissociation effects of the urea series of compounds reported in the preceding paper (Herskovits and Harrington, 1975), the neutral salts appear to be much more effective dissociating agents for L. terrestris hemoglobin. This suggests that polar and ionic interactions are relatively more important for the maintenance of the protein than hydrophobic interactions. This conclusion is also supported by calculations of the possible effects of binding of NaClO4, based on the Setschenow constants of the literature describing the interaction of salts with the peptide and hydrophobic alkyl group of the average amino acid found in proteins, on the standard free energy of dissociation of the duodecamer to hexamer.     

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.     

Lumbricus erythrocruorin at 3.5 A resolution: architecture of a megadalton respiratory complex

Annelid erythrocruorins are highly cooperative extracellular respiratory proteins with molecular masses on the order of 3.6 million Daltons. We report here the 3.5 A crystal structure of erythrocruorin from the earthworm Lumbricus terrestris. This structure reveals details of symmetrical and quasi-symmetrical interactions that dictate the self-limited assembly of 144 hemoglobin and 36 linker subunits. The linker subunits assemble into a core complex with D(6) symmetry onto which 12 hemoglobin dodecamers bind to form the entire complex. Although the three unique linker subunits share structural similarity, their interactions with each other and the hemoglobin subunits display striking diversity. The observed diversity includes design features that have been incorporated into the linker subunits and may be critical for efficient assembly of large quantities of this complex respiratory protein.     

Molecular shape of Lumbricus terrestris erythrocruorin studied by electron microscopy and image analysis

The molecular structure of erythrocruorin (hemoglobin) from Lumbricus terrestris has been studied by electron microscopy of negatively stained particles. Over 1000 molecular projections were selected from a number of electron micrographs and were then classified by multivariate statistical image-processing techniques. The two main groups of top and side views were each subdivided into smaller classes with significantly different features. About half of the top-view projections exhibit perfect hexagonal symmetry at the current resolution of about 2.0 nm, while the other top views lack this symmetry, probably as a result of tilting of the molecules relative to the carbon support film. The side views were separated into two 'families', each associated with the two different stable side-view positions the molecules can take. From these narrow stable side-views, the two families of projections are, again, generated by tilting. The symmetry properties of the three non-tilted projections show that Lumbricus erythrocruorin has a pointgroup D6 (622) symmetry rather than D3 (32).     

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.     

The molecular weight of the extracellular haemoglobin of Lumbricus terrestris determined by electron microscopy

1. The mol. wt of the extracellular haemoglobin of the oligochaete Lumbricus terrestris was determined by counting in negatively stained electron micrographs. 2. The value obtained using apoferritin as a mol. wt standard is (3.8 +/- 0.3) x 10(6), in agreement with recent determinations employing different physical methods. 3. We conclude that all annelid extracellular haemoglobins and chlorocruorins which have the same dimensions as Lumbricus haemoglobin probably have the same mol. wt.     

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.     

The interaction of Staphylococcal protein A with free coelomocytes of annelids.

The Staphylococcal protein A (SpA) binding protein was detected on the surface of annelid coelomocytes. The flow cytometric analysis revealed that 50% coelomocytes of Lumbricus terrestris react with SpA, a figure six times higher than the number of positive coelomocytes found in Eisenia foetida.     

Solution studies on heme proteins: subunit structure, dissociation, and unfolding of Lumbricus terrestris hemoglobin by the ureas.

The subunit structure, dissociation, and unfolding of the hemoglobin of the earthworm, Lumbricus terrestris, were investigated by light scattering molecular weight methods and changes in optical rotatory dispersion (at 233 nm) and absorption in the Soret region. Urea and the alkylureas, methyl-, ethyl-, propyl-, and butylurea, were employed as the reagents to cause both dissociation and unfolding of the protein. Analysis of the light scattering data suggests that the dissociation patterns as a function of hemoglobin concentration in the various dissociating solvents can be described in quantitative terms, either as an equilibrium mixture consisting of parent duodecamers and hexamers of 3 x 10(6) and 1.5 x 10(6) molecular weight (in 1-3 M urea, 1-2 M methyl- and ethylurea, and 1 M propylurea), as a mixture of hexamers and monomers, the latter with a molecular weight of 250000 (i.e., in 4 M urea), or as a mixture of all three species of duodecamers, hexamers, and monomers, seen in 2 M propylurea. Parallel studies by optical rotation and absorption measurements indicate that there is little or no unfolding of the subunits at urea and alkylurea concentrations where complete dissociation to hexamers and extensive dissociation to monomers can be achieved. Further splitting of the monomers (A subunits) to smaller fragments of one-third to one-quarter of the molecular weight of the monomers (B subunits) is seen in the presence of 7 and 8 M urea (pH 7) and in alkaline urea to propylurea solutions. Analysis of the dissociation data of duodecamers to monomers, based on equations used in studies of the urea and amide dissociation of human hemoglobin A from our laboratory, suggests few urea and alkylurea binding sites at the areas of hexamer contacts in the associated duodecameric form of L. terrestris hemoglobin. This suggests that hydrophobic interactions are not the dominant forces that govern the state of association of L. terrestris hemoglobin relative to polar and ionic interactions. The unfolding effects of the ureas, at concentrations above the dissociation transitions, are closely similar to their effects on other globular proteins, suggesting that hydrophobic interactions play an important role in the maintenance of the folded conformation of the subunits. Use of the Peller-Flory equation, with binding constants based on free energy transfer data of hydrophobic amino acid side chains and denaturation data used in previous denaturation studies, gave a relatively good acount of the observed denaturation midpoints obtained with the various ureas supporting these conclusions.     

Analysis of the acid and alkaline dissociation of earthworm hemoglobin, Lumbricus terrestris, by front-face fluorescence spectroscopy

The steady-state fluorescence properties of the multisubunit hemoglobin isolated from the earthworm, Lumbricus terrestris, were studied by front-face fluorometry. Acid and alkaline dissociation of this high-molecular-weight hemoglobin were examined over the pH range 3.7-12.5 using different liganded states (oxy, CO, met). The relative intensity of the emission maximum at 320 nm (exc. 280 nm) is ligand-dependent increasing as follows: oxy less than deoxy less than CO less than met at pH 7.0. The intensity of the emission maximum of oxyhemoglobin at the alkaline acid end point, pH 10.5 (333 nm), is significantly greater than that observed at the acid end point, pH 4.18 (320 nm), suggesting different subunit dissociation. The spectra of oxyhemoglobin at pH 4.18 and the spectrum of carbonmonoxy hemoglobin at pH 7.0 in the presence of 1 M magnesium chloride were almost identical, indicating similar subunit dissociation. Difference spectrum (pH 9.0-7.2) of fluorescence emission (exc. 305) resulted in a maximum at 341 nm, indicative of tyrosinate formation. This suggests that tyrosine(s) may also be located at the subunit interface(s) of this hemoglobin. These studies indicate that several aromatic amino acid residues are associated with the critical sites of subunit interactions within this molecule. Analysis of the fluorescence spectra also suggests that the formation of different subunit species resulting from acid and alkaline dissociation cannot be ruled out.