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

• Published in: Journal of the American Society for Mass Spectrometry 2004, Vol.15 (1), p.22-27
• Main Authors: Green, Brian N, Vinogradov, Serge N
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Chains; Charge distribution; Crystal structure; Electrospraying; Fundamental and applied biological sciences. Psychology; Hemoglobin; Hemoglobins - chemistry; Hemoproteins; Ionization; Ions; Leeches - chemistry; Mass spectrometry; Maximum entropy; Metalloproteins; Molecular Weight; Protein Denaturation; Proteins; Spectrometry, Mass, Electrospray Ionization - methods; Subassemblies
• ISSN: 1044-0305; 1879-1123
• DOI: 10.1016/j.jasms.2003.08.013

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.