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

The complex, multiply charged electrospray ionization mass spectra of the e xtracellular, similar to 3500 kDa, hexagonal bilayer hemoglobin from the le ech Haemopis grandis and its carbamidomethylated, reduced and reduced/carba midomethylated forms were deconvoluted using a maximum entropy approach to provide the corresponding zero-charge spectra. Three groups of peaks were o bserved: monomeric globin chains a1-a4 at similar to 17 kDa (16 634.1, 17 0 13.4, 17 592.9, and 17 573.3 Da with relative intensities of 16:4:12:1, res pectively), linker chains L1-L3 at similar to 24 kDa (24 004.2, 24 449.2 an d 24 548.3 Da, with relative intensities of 5:1:2.5, respectively) and subu nits D1 and D2 at 32 501.1 and 32 629.6 Da, respectively, with equal intens ities. 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 g lobin chains b (16 098.8 Da), c1 (16 403.9 Da), and c2 (16 532.5 Da), sugge sting that subunits D1 and D2 are disulfide-bonded dimers b + c1 and b + c2 , respectively. All the globin chains appear to have one intrachain disulfi de bond, and globins b, c1, and c2 have an additional Cys which forms the i nterchain disulfide bond in D1 and D2. The Linkers L1-L3 contain 10, 9, and 10 Cys residues, respectively, all forming intrachain disulfide bonds, exc ept for the odd residue in L2 which is proposed to be the site of cysteinyl ation. The relative intensities of the three groups of peaks al + a2 + a3 a4:L1 + L2 + L3:D1 + D2 are 1.65:1.7:0.8 in native hemoglobin. All the sub units in a second sample evinced substantial glycosylation, with up to five hexoses per subunit. A model of the quaternary structure of Haemopis hemog lobin 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 mas s of 3506 kDa. (Int J Mass Spectrom 188 (1999) 105-112) (C) 1999 Elsevier S cience B.V.