An electrospray ionization mass spectrometric study of the subunit structure of the giant hemoglobin from the leech Nephelopsis oscura

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Abstract
The subunit structure of the giant, extracellular hexagonal bilayer (HBL) hemoglobin (Hb) from the leech Nephelopsis oscura was investigated by electrospray ionization mass spectrometry (ESI-MS) employing a maximum entropy deconvolution of its complex, multiply charged ESI spectra. The denatured unreduced Hb consisted of three monomer globin chains (M), a1=16,535 Da, a2=17,171 Da and a3=17,315 Da, five nonglobin linker chains, L1=24,512 Da, L2=24,586 Da, L3=24,979 Da, L4=25,006 Da, and L5=25,566 Da and two subunits of 32,950 Da and 33,125 Da. ESI-MS of the denatured, reduced Hb showed that the latter were disulfide-bonded heterodimers (D) of globin chains b1=16,322 Da and b2=16,499 Da with chain c=16,632 Da. Time-of-flight ESI-MS of the Hb at pH 3.8, 4.5, 5.0, 5.8 and 7.0 revealed a distribution of charge states from 32+ to 37+ with masses decreasing from 211 to 208.5 kDa with increase in cone voltage from 60 to 160 V, indicating the presence of a subassembly comprising 12 globin chains. The subunit composition 6M + 3D + 12h, where M=16993 Da and D=33004 Da are the weighted masses and h=616.5 Da, provides a calculated mass, 208.37 kDa that is closest to 208.5 kDa. Our experimental findings are consistent with the bracelet model of HBL Hbs, verified by the recent low-resolution crystal structure of Lumbricus Hb, wherein an HBL arrangement of 12 globin dodecamer subassemblies is tethered to a central complex of 36 linker chains for a total mass of 208.37 × 12 + 24.94 × 36=3398 kDa.