The multi-hemoglobin system of the hydrothermal vent tube worm Riftia pachyptila. II. Complete polypeptide chain composition investigated by maximum entropy analysis of mass spectra.
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.
Zal
F
F
Equipe Ecophysiologie, UPM-CNRS-INSU, Station Biologique, BP 74, 29682 Roscoff Cedex, France.
Lallier
F H
FH
Green
B N
BN
Vinogradov
S N
SN
Toulmond
A
A
D000818:Animals; D003545:Cysteine / Q000737:chemistry; D006454:Hemoglobins / Q000737:chemistry*; D046911:Macromolecular Substances; D013058:Mass Spectrometry; D008970:Molecular Weight; D011077:Polychaeta / Q000737:chemistry*; D011134:Polysaccharides / Q000737:chemistry; D011487:Protein Conformation; D012623:Seawater; D013816:Thermodynamics
D006454:Hemoglobins; D046911:Macromolecular Substances; D011134:Polysaccharides; D003545:Cysteine