ELECTROSPRAY-IONIZATION MASS-SPECTROMETRIC COMPOSITION OF THE 400 KDAHEMOGLOBIN FROM THE POGONOPHORAN OLIGOBRACHIA-MASHIKOI AND THE PRIMARY STRUCTURES OF 3 MAJOR GLOBIN CHAINS

Maximum entropy analysis of the electrospray ionization mass spectra o f the native, carbamidomethylated, reduced and reduced and carbamidome thylated forms of the extracellular ca. 400 kDa hemoglobin of the pogo nophoran Oligobrachia mashikoi has shown it to consist of eight globin chains: (a1-a5), 14861.1, 14937.1, 15040.7, 15070.6 and 15310.6 Da an d b-d1, 15173.2, 15605.1 and 14775.4 Da, respectively. Although chains a1-a5 are monomeric, chains b+c form a disulfide-bonded dimer of 3077 6.8 Da and chains b+c+d1 form a disulfide-bonded trimer of 45551.9 Da. The major chains a5, b and c were separated by reverse-phase chromato graphy, and their cDNA's amplified by PCR using redundant oligomers ba sed on their N-terminal amino-acid sequences. The complete amino-acid sequences of chains a5 (142 residues), b (140 residues) and c (147 res idues) were derived from protein and cDNA sequencing and represent the first pogonophoran globin sequences. They have a high percent identit y (35-52%) with the globin chains of the approximate to 3500 kDa hexag onal bilayer hemoglobins from the annelids Lumbricus and Tylorrhynchus and the vestimentiferan Lamellibrachia, suggesting a very close relat ionship among the phyla Annelida, Pogonophora and Vestimentifera. Two free cysteine residues (Cys-73 and Cys-83), which we proposed to be th e most probable candidates for the sulfide-binding sites in the Lamell ibrachia chains (Suzuki, T., Takagi, T. and Ohta, S. (1990) Biochem. J . 266, 221-225), are also conserved in the three chains (Cys-73 for ch ains b and c, and Cys-83 for chain a5) of Oligobrachia hemoglobin, in agreement with the probable role of the hemoglobin in the binding and transport of sulfide to the symbiotic bacteria which provide the metab olic fuel in the two phyla.