Electrospray ionization mass spectrometric composition of the 400 kDa hemoglobin from the pogonophoran Oligobrachia mashikoi and the primary structures of three major globin chains

Abstract

Maximum entropy analysis of the electrospray ionization mass spectra of the native, carbamidomethylated, reduced and reduced and carbamidomethylated forms of the extracellular ca. 400 kDa hemoglobin of the pogonophoran 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 and b− dl, 15173.2, 15605.1 and 14775.4 Da, respectively. Although chains a1– a5 are monomeric, chains b + c form a disulfide-bonded dimer of 30776.8 Da and chains b + c + dl form a disulfide, bonded trimer of 45551.9 Da. The major chains a5, b and c were separated by reverse-phase chromatography, and their cDNA's amplified by PCR using redundant oligomers based on their N-terminal amino-acid sequences. The complete amino-acid sequences of chains a5 (142 residues), b (140 residues) and c (147 residues) were derived from protein and cDNA sequencing and represent the first pogonophoran globin sequences. They have a high percent identity (35–52%) with the globin chains of the ∼ 3500 kDa hexagonal bilayer hemoglobins from the annelids Lumbricus and Tylorrhynchus and the vestimentiferan Lamellibrachia, suggesting a very close relationship among the phyla Annelida, Pogonophora and Vestimentifera. Two free cysteine residues (Cys-73 and Cys-83), which we proposed to be the most probable candidates for the sulfide-binding sites in the Lamellibrachia chains (Suzuki, T., Takagi, T. and Obta, S. (1990) Biochem. J. 266, 221–225), are also conserved in the three chains (Cys-73 for chains 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 metabolic fuel in the two phyla.