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 mobility molecular analysis (GEMMA) to separate globular proteins (mass 6 to 670 kDa) and the excellent correlation found between the electrophoretic mobility diameter (EMD), or Millikan diameter, and the protein mass (S. L. Kaufman et al., 1996, Anal. Chem. 68, 1895-1904; 1996, Anal. Chem. 68, 3703), prompted the examination of a large protein complex, the 3.6-MDa, heteromultimeric, hexagonal bilayer hemoglobin (Hb) and its subunits from the earthworm Lumbricus terrestris. The native Hb 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 nm, identified as a dodecamer of globin chains ([a+b+c]3d3, 213 kDa), the disulfide-bonded trimer of globin chains ([a+b+c], 52.7 kDa), all 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. Reassembly 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/cm3. GEMMA also provided a profile of the Hb completely dissociated in 0.1% SDS; its deconvolution permitted a quantitative determination of the subunit stoichiometry, providing a globin to linker ratio of 3 to 1.
Kaufman
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TSI Incorporated, 500 Cardigan Road, St. Paul, Minnesota, 55126, USA. skaufman@tsi.com
Kuchumov
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Kazakevich
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Vinogradov
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D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004586:Electrophoresis / Q000379:methods*; D006454:Hemoglobins / Q000032:analysis* / Q000737:chemistry; D009835:Oligochaeta / Q000737:chemistry*
D006454:Hemoglobins