Purification, Characterization and Sequence Analyses of the Extracellular Giant Hemoglobin from Oligobrachia mashikoi

Abstract

-We purified an extracellular hemoglobin with the molecular mass of ca. 440 kDa from the whole homogenates of Oligobrachia mashikoi (phylum Pogonophora) by a one-step gel-filtration. The preparation was pure to be crystallized. The P 50 values of the hemoglobin and the fresh blood prepared from O. mashikoi were about 0.82 Torr and 0.9 Torr, respectively, which were much lower than the P 50 value of human hemoglobin. However, the n values of the hemoglobin and the blood were about 1.2 and 1.1, respectively.Using the improved tricine SDS-PAGE, we could separate O. mashikoi hemoglobin into four kinds of the globin chains, A1, A2, B1 and B2, and succeeded for the first time in cloning and sequencing of the complete cDNA encoding B1 globin gene, in addition to A1, A2 and B2 globin genes in full length. We found that all globin genes have the extracellular signal sequences in each molecule and the distal His of the B1 globin chain is replaced to Gln. Finally, we constructed phylogenetic trees of the hemoglobins from Pogonophora, Vestimentifera and Annelida.

Alpha-Glucosidase-like Activity Detected in a Siboglinid Polychaete, Oligobrachia mashikoi

“…In the digestive tract of mammals, there are digestive enzyme complexes such as the sucrase-isomaltase complex. Consequently, a giant type of α-glucosidase combined with other molecules may be possible, as seen in the giant hemoglobin of this species (Nakagawa et al, 2005).…”

Direct Evidence That Extracellular Giant Hemoglobin is Produced in Chloragogen Tissues in a Beard Worm, Oligobrachia mashikoi (Frenulata, Siboglinidae, Annelida)

“…A fragment from nucleotide positions 199 to 339 of the A2-globin subunit was amplified by PCR by using a plasmid carrying the A2-globin cDNA (Nakagawa et al, 2005) as a template and a set of primers: an A2-forward primer (5'-AGATAAGCTTGGTAA TGACATCGATTCTTC-3') containing a Hind III site (underlined), and an A2-reverse primer (5'-TATAGAGCTCACGAGAGTCATG CTGAGA-3') containing a Sac I site (underlined). The nucleotide sequence of the PCR-amplified region codes for the hem pocket, including the proximal and distal histidine residues of the A2-globin, and the sequence similarity to A1-, B1-, and B2-globin cDNA sequences is 52.5%, 51.1%, and 54.6%, respectively.…”

“…PCR conditions were 95°C for 2 min, followed by 32 cycles of 95°C for 30 sec, 60°C for 30 sec, and 74°C for 1 min. To detect the cDNA of each globin subunit, the following primers were designed on the basis of globin cDNA sequences (Nakagawa et al, 2005): for the A1-globin cDNA, forward A1 primer 5'-GTAT-GTAACCGACTTGAGCAGATCCTGG-3' and reverse A1-KpnI primer 5'-CTGGTACCTTAGCCGGAAATACCGCTAGCA-3'; for the A-2 globin cDNA, forward A2 primer 5'-GATTGCACTTCCCT-CAATCGCCTGTTTGG-3' and reverse A2-KpnI primer 5'-CTGG-TACCTTAACCAGAAATGCCGCTGACA-3'; for the B1-globin cDNA, forward B1 primer 5'-GAATGCTGCAGTAGAGGTGATGC-CGAGG-3' and reverse B1-KpnI primer 5'-CTGGTACCTTA-CAAGCCTGCACCAATACCA-3'; and for the B2-globin cDNA, forward B2 primer 5'-TCGAGCTGTTGTTCCTCCGAGGCAGG-3' and reverse B2-KpnI primer 5'-CTGGTACCTTACAAGCCTGCT-GAGATGCCG-3'.…”

“…They possess a closed blood-vessel system and have a large quantity of extracellular hemoglobin in the blood (Southward, 1993). In Oligoblachi mashikoi, the binding affinity of hemoglobin to oxygen has been reported to be extremely high (Nakagawa et al, 2005;Aki et al, 2007). This result seems to reflect the fact that these polychaetes inhabit a weakly reductive environment in which a small quantity of toxic gas is produced (Tervilliger et al, 1987).…”

“…400 kDa) of O. mashikoi is composed of four kinds of globin subunits (A1, A2, B1, and B2) (Nakagawa et al, 2005). Furthermore, Numoto et al (2005 a, b) succeeded in analyzing the three-dimensional structure of this hemoglobin by X-ray crystallography, and this analysis suggested the positions of unpaired free cysteine residues to which the sulfur atom in hydrogen sulfide should bind.…”