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 disso ciated hemoglobin of the earthworm Lumbricus terrestris. Various preparatio ns comprising dodecamer of globin chains and linker chains were allowed to reassemble at neutral pH. They produced various oligomers that were purifie d by gel filtration, analyzed in matrix-assisted laser desorption/ionizatio n mass spectrometry and submitted to 3D reconstruction from isolated partic les observed in cryoelectron microscopy. Despite the impossibility to compl etely free the L2, L3, and L4 preparations from L1, the following conclusio ns were obtained. First, hemoglobin molecules indistinguishable from native hemoglobin at 25 Angstrom resolution were obtained in the absence of linke r chains L2, L3, or L4. Second, the 3D reconstruction volumes of reassemble d hemoglobins containing dodecamers and L1 + L3 or dodecamers and L1 + L4 d emonstrate that reassembly of native-like structures can be obtained from a t most two linker chains and dodecamers. Third, the 3D reconstruction volum es 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 highl y similar. Since these structures comprise two types of substructures (one involved in the c3a, c3b, and c4 linking units of the hollow globular subst ructure and the other in the c5 connection and the toroid), it seems highly probable that the minimal number of linker chains required to reassemble n ative-like hemoglobin is at most two. (C) 2000 Academic Press.