Abstract
The deep-sea tube worm Riftia pachyptila Jones possesses a complex of three extracellular Hbs: two in the vascular compartment, V1 (approximately 3500 kDa) and V2 (approximately 400 kDa), and one in the coelomic cavity, C1 (approximately 400 kDa). These native Hbs, their dissociation products and derivatives were subjected to electrospray ionization mass spectrometry (ESI-MS). The data were analyzed by the maximum entropy deconvolution system. We identified three groups of peaks for V1 Hb, at approximately 16, 23 27, and 30 kDa, corresponding to (i) two monomeric globin chains, b (Mr 16,133.5) and c (Mr 16,805.9); (ii) four linker subunits, L1 L4 (Mr 23,505.2, 23,851.4, 26,342.4, and 27,425.8, respectively); and (iii) one disulfide-bonded dimer D1 (Mr 31,720.7) composed of globin chains d (Mr 15,578.5) and e (Mr 16, 148.3). V2 and C1 Hbs had no linkers and contained a glycosylated monomeric globin chain, a (Mr 15,933.4) and a second dimer D2 (Mr 32,511.7) composed of chains e and f (Mr 16,368.1). The dimer D1 was absent from C1 Hb, clearly differentiating V2 and C1 Hbs. These Hbs were also subjected to SDS-PAGE analysis for comparative purposes. The following models are proposed ((cD1)(bD1)3) for the one-twelfth protomer of V1 Hb, ((cD)(bD)6(aD)) (D corresponding to either D1 or D2) for V2 and C1 Hbs. HBL V1 Hb would be composed of 180 polypeptide chains with 144 globin chains and 36 linker chains, each twelfth being in contact with three linker subunits, providing a total molecular mass = 3285 kDa. V2 and C1 would be composed of 24 globin chains providing a total molecular mass = 403 kDa and 406 kDa, respectively. These results are in excellent agreement with experimental Mr determined by STEM mass mapping and MALLS.
Highlights
The deep-sea tube worm Riftia pachyptila Jones possesses a complex of three extracellular Hbs: two in the vascular compartment, V1 (ϳ3500 kDa) and V2 (ϳ400 kDa), and one in the coelomic cavity, C1 (ϳ400 kDa)
HBL V1 Hb would be composed of 180 polypeptide chains with 144 globin chains and 36 linker chains, each twelfth being in contact with three linker subunits, providing a total molecular mass ؍3285 kDa
Polypeptide Chains Composition Determined by SDSPAGE—The nomenclature used to name the different subunits and polypeptide chains was that previously used for Lamellibrachia sp. [6]
Summary
The deep-sea tube worm Riftia pachyptila Jones possesses a complex of three extracellular Hbs: two in the vascular compartment, V1 (ϳ3500 kDa) and V2 (ϳ400 kDa), and one in the coelomic cavity, C1 (ϳ400 kDa). In a companion study [8] we have confirmed that Riftia pachyptila (Jones), a vestimentiferan living around deep-sea hydrothermal vents (9 –11), possesses three hemoglobins, two of them dissolved in the vascular blood (V1 and V2), and one in the coelomic fluid (C1) Their molecular weights have been determined by scanning transmission electron microscopy mass mapping (STEM) and by multi-angle laser light scattering (MALLS). A more accurate technique, ESI-MS, has been successfully applied to some HBL Hbs allowing the determination of the complete polypeptide chain compositions of these Hbs [13,14,15] They consist of 4 to 6 globin chains in the range 16,000 –18,000 Da and 3– 4 linker chains in the range 24,000 –27,000 Da. Previous studies by SDS-PAGE on Riftia vascular Hbs have shown that they consist of subunits of Mr ϳ15,000, 30,000, and 45,000 (16 –18). In this report we have determined the stoichiometry of the polypeptide chains and subunits constituting these Hbs by ESI-MS coupled with maximum entropy [19]
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