An electrospray ionization mass spectrometric study of the giant, extracellular, hexagonal bilayer hemoglobin of the leech Haemopis grandis provides a complete enumeration of its subunits

Abstract The complex, multiply charged electrospray ionization mass spectra of the extracellular, ∼3500 kDa, hexagonal bilayer hemoglobin from the leech Haemopis grandis and its carbamidomethylated, reduced and reduced/carbamidomethylated forms were deconvoluted using a maximum entropy approach to provide the corresponding zero-charge spectra. Three groups of peaks were observed: monomeric globin chains a1–a4 at ∼17 kDa (16 634.1, 17 013.4, 17 592.9, and 17 573.3 Da with relative intensities of 16:4:12:1, respectively), linker chains L1–L3 at ∼24 kDa (24 004.2, 24 449.2 and 24 548.3 Da, with relative intensities of 5:1:2.5, respectively) and subunits D1 and D2 at 32 501.1 and 32 629.6 Da, respectively, with equal intensities. Reduction of the native hemoglobin with dithiothreitol resulted in a decrease in the mass of linker L2 by 115.1 Da, indicating cysteinylation, the disappearance of subunits D1 and D2 and the concomitant appearance of globin chains b (16 098.8 Da), c1 (16 403.9 Da), and c2 (16 532.5 Da), suggesting that subunits D1 and D2 are disulfide-bonded dimers b + c1 and b + c2, respectively. All the globin chains appear to have one intrachain disulfide bond, and globins b, c1, and c2 have an additional Cys which forms the interchain disulfide bond in D1 and D2. The linkers L1–L3 contain 10, 9, and 10 Cys residues, respectively, all forming intrachain disulfide bonds, except for the odd residue in L2 which is proposed to be the site of cysteinylation. The relative intensities of the three groups of peaks a1 + a2 + a3 + a4:L1 + L2 + L3:D1 + D2 are 1.65:1.7:0.8 in native hemoglobin. All the subunits in a second sample evinced substantial glycosylation, with up to five hexoses per subunit. A model of the quaternary structure of Haemopis hemoglobin is proposed, consisting of 72 monomeric globins and 36 globin dimers for a total of 144 globin chains and 42 linkers with a calculated total mass of 3506 kDa.