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.     

The primary structure and functional properties of the hemoglobins of a ground squirrel (Spermophilus townsendii, Rodentia)

The hemoglobin of the ground squirrel Spermophilus townsendii consists of two components which are present in a ratio of ca. 2:1. The two hemoglobins have identical alpha-chains, but differ in their beta-chains. We present the primary structures of the alpha- and the two beta-globin chains. Following chain separation by chromatography on carboxymethyl-cellulose CM-52, the amino-acid sequences were established by automatic Edman degradation of the globin chains and the tryptic peptides, as well as of a peptide obtained by acid hydrolysis of the Asp-Pro bond of the beta-chains. The two beta-chains differ by only one amino-acid residue, Ala being present in the main and Asp in the minor component in position 58 (E2). The comparison with human hemoglobin showed only 14 exchanges in the alpha-chains but 33 in the beta-chains. Whereas no contact positions are affected in the alpha-chains, we found four such substitutions in the beta-chains, including one heme contact, two alpha 1/beta 1-contacts, and one alpha 1/beta 2-contact. It seems however, that the substitution found in the beta-chains has no effect on the oxygen affinity.     

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 primary structure of three hemoglobin chains from the indigo snake (Drymarchon corais erebennus,Serpentes): first evidence for alphaD chains and two beta chain types in snakes

The hemoglobin of the indigo snake (Drymarchon corais erebennus, Colubrinae) consists of two components, HbA and HbD, in the ratio of 1:1. They differ in both their alpha and beta chains. The amino acid sequences of both a chains (alphaA and alphaD) and one beta chain (betaI) were determined. The presence of an alphaD chain in a snake hemoglobin is described for the first time. A comparison of all snake beta chain sequences revealed the existence of two paralogous beta chain types in snakes as well, which are designated as betaI and betaII type. For the discussion of the physiological properties of Drymarchon hemoglobin, the sequences were compared with those of the human alpha and beta chains and those of the closely related water snake Liophis milians where functional data are available. Among the heme contacts, the substitution alphaD58(E7)His-->Gln is unusual but most likely without any effect. The residues responsible for the main part of the Bohr effect are the same as in mammalian hemoglobins. In each of the three globin chains only two residues at positions involved in the alpha1/beta2 interface contacts, most important for the stability and the properties of the hemoglobin molecule, are substituted with regard to human hemoglobin. On the contrary, nine, eleven, and six alpha1/beta1 contact residues are replaced in the alphaA, alphaD, betaI chains, respectively.     

Structure of the polypeptide chain of extracellular hemoglobin from the nematode Ascaris suum.

1. Ascaris suum extracellular hemoglobin is composed of eight identical single polypeptide chain subunits carrying two heme binding sites each. 2. Limited trypsinolysis followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis gave a major band corresponding to half the molecular mass of an intact subunit. 3. Peptide mapping of tryptic hydrolysates yielded 27 to 30 fluorescamine positive spots, about half the number of lysyl and arginyl residues in a polypeptide chain. 4. The findings indicate that a subunit of Ascaris hemoglobin consists of two structural units of roughly equal size, corresponding to two recurring sequences, connected together by the continuity of the polypeptide chain.     

Amino acid sequence of the monomer subunit of the giant multisubunit hemoglobin from the earthworm Pheretima sieboldi

The giant extracellular hemoglobin of the earthworm Pheretima sieboldi is mainly composed of two heme-containing subunits: a monomer; chain I and a disulfide-bonded trimer of chains II, III and IV. Both subunits can be separated easily by gel filtration under alkaline conditions. The amino acid sequence of chain I has been determined. It is composed of 141 residues, has two half-cystine residues forming a intrachain disulfide bridge, and has a molecular mass of 16911 Da including a heme group. Heterogeneity was found at position 37 (His or Ser). The amino acid sequence of Pheretima chain I showed 30-50% identity with those of eight heme-containing chains of Lumbricus and Tylorrhynchus hemoglobins. The sequences of nine chains of annelid giant hemoglobins were compared separately in the functionally essential central exonic region and structurally essential side exonic regions, and a phylogenetic tree was constructed. The amino acid substitution rate for the central exon was found to be about 1.5 times slower than that for the side exons.     

Calcium binding proteins: purification of hepatocalcin-55 from bovine liver

1. Bovine liver was homogenized and the proteins were fractionated by two DEAE-Sephadex steps and a gel filtration. 2. All 150 fractions collected from the DEAE-Sephadex column were electrophoresed and combined to give ten groups of proteins with different SDS-electrophoresis patterns. 3. Fractions G and H, showing fast migrating proteins, were transblotted to a polyvinylidenedifluoride membrane. By incubation with 45CaCl2 and subsequent autoradiography one protein revealed strong calcium binding. 4. This protein, named hepatocalcin-55, was obtained in a pure form from the gel filtration through Sephadex G-75. The molecular weight (55,000) was determined by gel filtration. Since SDS electrophoresis shows one protein band at Mr = 27,000 the native hepatocalcin must be a dimer. Its isoelectric point was found to be at 4.9. 5. Gamma-carboxyglutamate could not be detected in alkaline hydrolyzates of the protein under study. No carbohydrates were found in the hepatocalcin.     

Primary structure of human fibrinogen and fibrin. Isolation and partial characterization of chains of fragment D.

Fragment D has been isolated as an apparently single molecular weight species (molecular weight about 100,000) from plasmin digests of humman fibrinogen, using a combination of affinity chromatography on insolubilized "fibrin monomer" and gel filtration. This fragment consists of three chains with molecular weights of 15,000 (Dbeta), 42,500 (Dgamma1) or 39,500 (Dgamma2), and 14,000 (Dalpha) held together by disulfide bonds. The S-carboxymethyl derivatives of the chains have been separated by gel filtration and ion exchange chromatography, and their identity has been confirmed by peptide mapping and immunological analysis. The chain with a molecular weight of 45,000 is a fragment of the Bbeta chain of fibrinogen. The chain derived from the gamma chain of fibrinogen occurred in two molecular forms having molecular weight 42,500 and 39,500. The chain derivative with molecular weight 14,000 is most likely derived from the Aalpha chain of fibrinogen. The chains were characterized by NH2-terminal sequence analysis, amino acid composition, and carbohydrate staining. The two molecular analysis, amino acid composition, and carbohydrate staining. The two molecular forms of the gamma chain appeared to be identical except for an NH2-terminal peptide extension of 23 amino acid residues in the longer chain. The latter has sequences in common with the COOH-terminal part of the gamma chain of the NH2-terminal disulfide knot (BROMBACK, B., BRONDAHL, N. J., HESSEL, B., IWANAGA, S., and WALLEN, P. (1973) J. Biol. Chem. 248, 5806-5820); its NH2-terminal residue being Ala-63 of the gamma chain of fibrinogen.     

Aminoacyl-tRNA synthetases from Bacillus stearothermophilus. Asymmetry of substrate binding to tyrosyl-tRNA synthetase.

The interaction of L-tyrosine, L-tyrosyladenylate and tRNA-Tyr with tyrosyl-tRNA synthetase from Bacillus stearothermophilus was studied by equilibrium dialysis, gel filtration and fluorescence spectroscopy. The enzyme, which consists of two identical subunits (mol. wt 2 x 44000), binds only a single molecule of L-tyrosine per dimer with a K-d of 2 x 10-5 M at pH 7.8 and 23 degrees C. The tyrosyl-tRNA synthetase--tyrosyladenylate complex which was isolated by gel filtration also has one adenylate bound per dimeric enzyme molecule. In contrast, two tRNA-Tyr molecules bind per enzyme dimer, but the two binding sites are not equivalent having K-d values of 2 x 10-7 M and 1.3 x 10-6 M respectively at pH 6.5 and 25 degrees C. Since crystallographic analysis of the free enzyme [2] shows that the monomer is the asymmetric unit, the data indicate that substrate binding induces asymmetry in the enzyme.     

Trematode hemoglobins show exceptionally high oxygen affinity.

Ligand binding studies were made with hemoglobin (Hb) isolated from trematode species Gastrothylax crumenifer (Gc), Paramphistomum epiclitum (Pe), Explanatum explanatum (Ee), parasitic worms of water buffalo Bubalus bubalis, and Isoparorchis hypselobagri (Ih) parasitic in the catfish Wallago attu. The kinetics of oxygen and carbon monoxide binding show very fast association rates. Whereas oxygen can be displaced on a millisecond time scale from human Hb at 25 degrees C, the dissociation of oxygen from trematode Hb may require a few seconds to over 20 s (for Hb Pe). Carbon monoxide dissociation is faster, however, than for other monomeric hemoglobins or myoglobins. Trematode hemoglobins also show a reduced rate of autoxidation; the oxy form is not readily oxidized by potassium ferricyanide, indicating that only the deoxy form reacts rapidly with this oxidizing agent. Unlike most vertebrate Hbs, the trematodes have a tyrosine residue at position E7 instead of the usual distal histidine. As for Hb Ascaris, which also displays a high oxygen affinity, the trematodes have a tyrosine in position B10; two H-bonds to the oxygen molecule are thought to be responsible for the very high oxygen affinity. The trematode hemoglobins display a combination of high association rates and very low dissociation rates, resulting in some of the highest oxygen affinities ever observed.     

Carnivora: the primary structure of the Pacific Walrus (Odobenus rosmarus divergens, Pinnipedia) hemoglobin

The primary structure of the alpha- and beta-chains of the hemoglobin from the Pacific Walrus (Odobenus rosmarus divergens, Pinnipedia) is presented. Sequence analysis revealed only one hemoglobin component whereas two bands were found in polyacrylamide gel electrophoresis. The globin chains were separated by high-performance liquid chromatography and the sequences determined by automatic liquid- and gas-phase sequencing of the chains and their tryptic peptides. The alpha-chains show 20 and the beta-chains 12 exchanges compared to the corresponding human chains. In the alpha-chains one heme- and two alpha 1/beta 1-contacts were exchanged whereas in the beta-chains one alpha 1/beta 1-, one alpha 1/beta 2-and one heme-contact are substituted. Compared to Harbour Seal (Phoca vitulina) the Walrus hemoglobin shows 9 amino-acid replacements in the alpha-chains and 5 in the beta-chains. The relation between Pinnipedia and Arctoidea is discussed.     

The complete amino acid sequence of giant multisubunit hemoglobin from the polychaete Tylorrhynchus heterochaetus

The extracellular hemoglobin from the polychaete Tylorrhynchus heterochaetus is a "giant," multisubunit protein with an apparent molecular weight of 3.37 X 10(6), and consists of two types of subunits: a "monomeric" chain (chain I) and a disulfide-bonded "trimer" of chains IIA, IIB, and IIC. We reported the amino acid sequences of chains I, IIB, and IIC previously (Suzuki, T., Yasunaga, H., Furukohri, T., Nakamura, K., and Gotoh, T. (1985) J. Biol. Chem. 260, 11481-11487). The sequence of chain IIA has now been determined. Chain IIA consists of 146 amino acid residues with a heme group and has a molecular weight of 17,236. All of the constituent chains of Tylorrhynchus hemoglobin appear to be homologous with those of vertebrate hemoglobins and contain heme. Distal (E7) His, distal (E11) Val, and proximal (F8) His are all conserved in the four chains. Phylogenetically, chain IIA appears more closely related to the monomeric chain I than to either of the other "trimeric" chains IIB and IIC. This is the first giant extracellular hemoglobin to be sequenced completely.     

Preparation and chemical characterization of the three chains of the major hemoglobin of the sea snake, Pelamis platurus.

One of the 2 main hemoglobins of the sea snake Pelamis platurus, (the Yellow-bellied sea snake) comprising about 70% of the total hemoglobin, was separated by DEAE-Sephadex column chromatography. From results of gel filtration and iron content determination, both intact sea snake hemoglobin, and the isolated major hemoglobin, were concluded to be composed of 4 subunits with a molecular weight of 66,000-67,000 daltons. Separation of the chains of globin of the major hemoglobin by CM 52 column chromatography gave 3 peaks, named, chains a, b, and c. The approximate molecular weights of chains a, b, and c were deduced by SDS gel electrophoresis to be 14,000, 16,000, and 20,000 daltons, respectively. The peptide maps and amino acid compositions of the three chains were distinctly different. N-Terminal and C-terminal amino acid sequence studies reveal that chains a, b, and c represented the alpha-chain, beta-chain, and beta'-chain differing from the normal beta-chain in having a C-terminal sequence of -Arg-Leu-His-Tyr. From the peak areas of the 3 chains obtained by CM 52 column chromatography, and the peak sizes of the 3 bands separated by SDS gel electrophoresis, it was concluded that the sea snake hemoglobin is composed of a hybrid tetramer, alpha2betabeta'.     

Primary structure of a linker subunit of the tube worm 3000-kDa hemoglobin

The deep-sea tube worm Lamellibrachia contains two giant extracellular hemoglobins, a 3000-kDa hemoglobin and a 440-kDa hemoglobin. The former consists of four heme-containing chains (AI-AIV) and two linker chains (AV and AVI) for the assembly of the heme-containing chains. The 440-kDa hemoglobin consists of only four heme-containing chains (Suzuki, T., Takagi, T., and Ohta, S. (1988) Biochem. J. 255, 541-545). The complete amino acid sequence of a linker subunit (chain AV) has been determined by automated Edman sequencing of the peptides derived by digestions with lysyl endopeptidase and endoproteinase Asp-N. The chain is composed of 224 amino acid residues, and the molecular mass for the protein moiety was calculated to be 24,894 Da. An Asn-X-Thr sequence which is possible as a glycosylation site was suggested at positions 108-110. A computer-assisted homology search showed that the sequence shows no notable homology with any other globins and proteins. However a careful alignment of the linker sequence with a heme-containing chain sequence suggested that there is a slight, but significant homology between the two sequences. The alignment also suggested that the linker resulted from gene duplication of a heme-containing chain with a three exon-two intron structure, and that the first exon of domain 1 and the last exon of domain 2 had been lost during evolution. In our alignment, domain 1 has the heme-binding proximal histidine, but domain 2 does not. This is the first linker subunit to be sequenced completely.     

Chemical properties and amino acid composition of beta1-bungarotoxin from the venom of Bungarus multicinctus (Formosan banded krait)

beta-Bungarotoxin purified from the venom of Bungarus multicinctus (Formosan banded krait) contained no carbohydrate and behaved as a homogeneous protein on polyacrylamide gel electrophoresis at pH 4.1 and SDS-polyacrylamide gel electrophoresis without 2-mercaptoethanol treatment. Its molecular weight and isoelectric point were estimated to be about 21,000 by gel filtration and about 9.5 by isoelectric focusing, respectively. The toxin treated with the reducing agent was split into two polypeptide chains as revealed by SDS-polyacrylamide gel electrophoresis and their molecular weights were calculated to be about 13,000 and 7,000. The two polypeptide chains (the large one named the A chain and the small one the B chain) were isolated by gel filtration after reduction of disulfide bonds in the toxin followed by alkylation. The A chain contained 120 amino acid residues including 13 half-cystines and the B chain 60 residues including 7 half-cystines. The two chains were supposed to link by disulfide bond(s) in the intact toxin which contained no free sulfhydryl groups. The N-terminal residues of the A and B chains were asparagine and arginine and the C-terminal ones were glutamine and proline, respectively, in accordance with the results of the terminal analyses of the intact toxin.     

Amino-acid sequences of the alpha and beta chains of adult hemoglobins of the Grand Galago, Galago crassicaudatus

The adult Grand Galago (Galago crassicaudatus) was found to have two hemoglobin components (Hb I and Hb II) which were separated by carboxymethyl cellulose column chromatography. The alpha and beta chains of each component were isolated. The tryptic peptides of the alpha and beta chains were each isolated and sequenced by the conventional method. The alignment of these peptides in each chain was deduced from the homology of their sequences with that of human adult hemoglobin. The alpha chains from Hb I and Hb II were considered to be identical. On the other hand, there was only one amino-acid difference between the two beta chains at the 125th residue from the N-terminus.     

Human Immunoglobulin Light Chains λ Form Amyloid Fibrils and Granular Aggregates in Solution

Myeloma nephropathy is a disorder characterized by deposition of monoclonal immunoglobulin light chains in the kidneys. The chains deposited form either amyloid fibrils or granular (amorphous) aggregates. Distinct molecular mechanisms leading to the formation of different aggregate types in kidney of patients with multiple myeloma are poorly understood. Here we describe the self-association kinetics of human monoclonal immunoglobulin light chains lambda (GRY) isolated from urine of a patient with multiple myeloma. Under physiological conditions, the isolated light chain exists predominantly in a form of covalent dimer with apparent molecular mass of 50.1 kD. Spectral probe binding, analytical gel filtration, Western blot analysis, and electron microscopy indicate that GRY dimer aggregation occurs via two different pathways producing either amyloid fibrils or amorphous aggregates depending on microenvironment. Incubation of GRY (25 microM) for 4-14 days at 37 degrees C in phosphate buffered saline (PBS), pH 7.0, or in PBS containing urea (0.8 M), pH 6.5, leads to amyloid fibril formation. Under electron microscopy, the fibrils show unbranched thread-like structures, approximately 60-80 x 1000 A in size, which can bind thioflavin T and Congo Red. GRY maintained in acetate buffer, pH 3.5, forms granular aggregates. The structure of GRY oligomers formed during the early stage of amyloid fibril formation (1-4 days) has been examined by means of protein cross-linking with homobifunctional reagents. These oligomers are predominantly trimers and tetramers.     

Purification,characterization and molecular cloning of a monocot mannose-binding lectin from <Emphasis Type="Italic">Remusatia vivipara</Emphasis> with nematicidal activity

A mannose-binding lectin (RVL) was purified from the tubers of Remusatia vivipara, a monocot plant by single-step affinity chromatography on asialofetuin-Sepharose 4B. RVL agglutinated only rabbit erythrocytes and was inhibited by mucin, asialomucin, asialofetuin and thyroglobulin. Lectin activity was stable up to 80°C and under wide range of pH (2.0–9.3). SDS-PAGE and gel filtration results showed the lectin is a homotetramer of Mr 49.5 kDa, but MALDI analysis showed two distinct peaks corresponding to subunit mass of 12 kDa and 12.7 kDa. Also the N-terminal sequencing gave two different sequences indicating presence of two polypeptide chains. Cloning of RVL gene indicated posttranslational cleavage of RVL precursor into two mature polypeptides of 116 and 117 amino-acid residues. Dynamic light scattering (DLS) and gel filtration studies together confirmed the homogeneity of the purified lectin and supported RVL as a dimer with Mr 49.5 kDa derived from single polypeptide precursor of 233 amino acids. Purified RVL exerts potent nematicidal activity on Meloidogyne incognita, a root knot nematode. Fluorescent confocal microscopic studies demonstrated the binding of RVL to specific regions of the alimentary-tract and exhibited a potent toxic effect on M. incognita. RVL-mucin complex failed to interact with the gut confirming the receptor mediated lectin interaction. Very high mortality (88%) rate was observed at lectin concentration as low as 30 μg/ml, suggesting its potential application in the development of nematode resistant transgenic-crops.     

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.     

The structure of invertebrate extracellular hemoglobins (erythrocruorins and chlorocruorins)

The knowledge accumulated over the last 30 years concerning the subunit structures of the invertebrate extracellular hemoglobins permits us to classify them into four distinct groups. Single-domain, single-subunit hemoglobins consisting of single, heme-binding polypeptide chains which have a molecular mass of ca. 16 KDa. These molecules are found in multicellular parasitic organisms such as the trematodes Dicrocoelium and Fasciolopsis and in a few insects, namely in the adult Anisops and in the larvae of Chironomus and of Buenoa. Two-domain, multi-subunit hemoglobins consisting of 30-37 KDa polypeptide chains each containing two, linearly connected heme-binding domains, which form polymeric aggregates with molecular masses ranging from 250 to 800 KDa. These hemoglobins are found extensively among the carapaced branchiopod crustaceans: Caenestheria, Daphnia and Lepidurus hemoglobins have been found to consist of 10, 16 and 24 two-domain chains, respectively. Judging from their electron microscopic appearances, some of the hemoglobins may possess different molecular symmetries. Multi-domain, multi-subunit hemoglobins consisting of two or more polypeptide chains, each comprising many heme-binding domains of ca. 15-20 KDa each. Examples of this class are found among the carapaceless branchiopod crustaceans, the planorbid snails and the clams from the families Astartidae and Carditidae. Artemia hemoglobin consists of two chains of ca. 125 KDa, each containing 8 heme-binding domains. Planorbis and Helisoma hemoglobins possess a molecular mass of ca. 1700 KDa and consist of 10 chains of 170-200 KDa. Astarte and Cardita hemoglobins appear in electron micrographs as rod-like polymers of variable dimensions, 20-30 nm in diameter and 20-100 nm in length and consist of polypeptide chains of ca. 300 KDa. The crustacean and gastropod hemoglobins vary in their electron microscopic appearance and may possess different molecular symmetries. Single-domain, multi-subunit hemoglobins consisting of aggregates of several small subunits, some of which are disulfide-bonded and not all of which contain heme. These molecules are widely distributed among the annelids and possibly also among the pogonophores. They are characterized by a two-tiered, hexagonal electron microscopic appearance, with a vertex-to-vertex diameter of 30 nm and a height of 20 nm, an acidic isoelectric point, a sedimentation coefficient of 50-60 S and a low iron content of 0.24 +/- 0.03%.(ABSTRACT TRUNCATED AT 400 WORDS)