ANALYSIS OF A 3.6-MDA HEXAGONAL BILAYER HEMOGLOBIN FROM LUMBRICUS-TERRESTRIS USING A GAS-PHASE ELECTROPHORETIC MOBILITY MOLECULAR ANALYZER

The recent successful use of electrospray gas-phase electrophoretic mo bility molecular analysis (GEMMA) to separate globular proteins (mass 6 to 670 kDa) and the excellent correlation found between the electrop horetic mobility diameter (ER ID), or Millikan diameter, and the prote in mass (S. L. Kaufman ct at, 1996, Anal, Chem. 68, 1895-1904; 1996, A nal. Chem. 68, 3703), prompted the examination of a large protein comp lex, the 3.6-MDa, heteromultimeric, hexagonal bilayer hemoglobin (Hb) and its subunits from the earthworm Lumbricus terrestris. The native H b had an EMD of 25.7 nm and the products of its dissociation at pH >8 and <5 were resolved into peaks with EMDs of 10.5, 6.3, 5.0, and 4.2 n m, identified as a dodecamer of globin chains ([a+b+c](3)d(3), 213 kDa ), the disulfide-bonded trimer of globin chains ([a+b+c], 52.7 kDa), a ll the linker chains (L1, 27.5 kDa; L2, 32.1 kDa; L3, 24.9 kDa; L4, 24 .1 kDa), and the monomer subunit (chain d, 17 kDa), respectively. Reas sembly of the Hb complex was observed on restoring the pH from >8 to 7 . The EMDs and the masses of the Hb and its subunits are in excellent agreement with the correlation found earlier, under the assumption of nearly spherical shape with an effective density around 0.7 g/cm(3). G EMMA also provided a profile of the Hb completely dissociated in 0.1% SDS; its deconvolution permitted a quantitative determination of the s ubunit stoichiometry, providing a globin to linker ratio of 3 to 1. (C ) 1998 Academic Press.