Purification, characterization and sequence analyses of the extracellular giant hemoglobin from Oligobrachia mashikoi.
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 P50 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 P50 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.
Nakagawa
Taro
T
Department of Life Science, Graduate School of Natural Science and Technology, Kanazawa University, Japan.
Onoda
Seiko
S
Kanemori
Masaaki
M
Sasayama
Yuichi
Y
Fukumori
Yoshihiro
Y
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D006454:Hemoglobins / Q000235:genetics* / Q000378:metabolism; D007448:Invertebrates / Q000235:genetics*; D007564:Japan; D016013:Likelihood Functions; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009841:Oligonucleotides; D010100:Oxygen / Q000378:metabolism; D010802:Phylogeny; D011487:Protein Conformation; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D013053:Spectrophotometry
D018076:DNA, Complementary; D006454:Hemoglobins; D009841:Oligonucleotides; D010100:Oxygen