The role of linkers in the reassembly of the 3.6 MDa hexagonal bilayer hemoglobin from Lumbricus terrestris

The extent and kinetics of reassembly of the four groups of linkers L1-L4 w ith 213 kDa subassemblies of twelve globin chains D, (bac)(3)(d)(3), isolat ed from the similar to 3.6 MDa hexagonal bilayer (HBL) hemoglobin (Hb) of L umbricus terrestris, was investigated using gel filtration. The reassembled HBL's were characterized by scanning transmission electron microscopic (ST EM) mass mapping and their subunit content determined by reversed-phase chr omatography. Ln reassembly by method (A), the linkers isolated by RP-HPLC a t pH similar to 2.2 were added to D at neutral pH; in method (B), the linke rs were renatured at neutral pH and then added to D. With method (A) the pe rcentage of HBL reassembly varied from greater than or equal to 13 % in the absence of Ca(II) to less than or equal to 75 % in 1-10 mM Ca(II). Reassem bly to HBL structures whose linker contents, STEM images and masses were si milar to the native Hb was observed with all the linkers (greater than or e qual to 75 %), with ternary and binary linker combinations (40-50%) and wit h individual linkers producing yields increasing in the following order: L1 =1-3%, L2 approximate to L3 = 10-20% and L4 = 35-55%. The yield was two- to eightfold lower with method (B), except in the case of linkers L1-L3. Alth ough the reassembly kinetics were always biphasic, with t(1/2) = 0.3-3.3 ho urs and 10-480 hours, the ratio of the amplitudes fast:slow was 1:0.6 with method (A) and 1:2.5 with method (B). These results are consistent with a s cheme in which the slow HBL reassembly is dependent on a slow conversion of linker conformation at neutral pH from a reassembly incompetent to a reass embly competent conformation. Although all the linkers self-associate exten sively at neutral pH, forming complexes ranging from dimers to >18-mers, th e size of the complex does not affect the extent or rate of reassembly. The oxygen binding affinity of reassembled HBLs was similar to that of the nat ive Hb, but their cooperativity was lower. A model of HBL reassembly was pr oposed which postulates that binding of linker dimers to two of the three T subunits of D causes conformational alterations resulting in the formation of complementary binding sites which permit lateral self-association of D subassemblies, and thus dictate the formation of a hexagonal structure due to the S-fold symmetry of D. (C) 1999 Academic Press.