Reassembly of Lumbricus terrestris hemoglobin: A study by matrix-assisted laser Desorption/Ionization mass spectrometry and 3D reconstruction from frozen-hydrated specimens

Dodecamers and four types of linker chains (L1-L4) were purified from dissociated hemoglobin of the earthworm Lumbricus terrestris. Various preparations comprising dodecamer of globin chains and linker chains were allowed to reassemble at neutral pH. They produced various oligomers that were purified by gel filtration, analyzed in matrix-assisted laser desorption/ionization mass spectrometry and submitted to 3D reconstruction from isolated particles observed in cryoelectron microscopy. Despite the impossibility to completely free the L2, L3, and L4 preparations from L1, the following conclusions were obtained. First, hemoglobin molecules indistinguishable from native hemoglobin at 25 Å resolution were obtained in the absence of linker chains L2, L3, or L4. Second, the 3D reconstruction volumes of reassembled hemoglobins containing dodecamers and L1+L3 or dodecamers and L1+L4 demonstrate that reassembly of native-like structures can be obtained from at most two linker chains and dodecamers. Third, the 3D reconstruction volumes of native and reassembled hemoglobins containing dodecamers and (1) L1, L2, and L4, (2) L1, L3, and L4, (3) L1 and L4, and (4) L1 and L3 were highly similar. Since these structures comprise two types of substructures (one involved in the c3a, c3b, and c4 linking units of the hollow globular substructure and the other in the c5 connection and the toroid), it seems highly probable that the minimal number of linker chains required to reassemble native-like hemoglobin is at most two.