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

Abstract

The extent and kinetics of reassembly of the four groups of linkers L1-L4 with 213 kDa subassemblies of twelve globin chains D, (bac)3(d)3, isolated from the approximately 3.6 MDa hexagonal bilayer (HBL) hemoglobin (Hb) of Lumbricus terrestris, was investigated using gel filtration. The reassembled HBL's were characterized by scanning transmission electron microscopic (STEM) mass mapping and their subunit content determined by reversed-phase chromatography. In reassembly by method (A), the linkers isolated by RP-HPLC at pH approximately 2.2 were added to D at neutral pH; in method (B), the linkers were renatured at neutral pH and then added to D. With method (A) the percentage of HBL reassembly varied from >/=13% in the absence of Ca(II) to </=75% in 1-10 mM Ca(II). Reassembly to HBL structures whose linker contents, STEM images and masses were similar to the native Hb was observed with all the linkers (>/=75%), with ternary and binary linker combinations (40-50%) and with individual linkers producing yields increasing in the following order: L1=1-3%, L2 approximately 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. Although the reassembly kinetics were always biphasic, with t1/2=0.3-3.3 hours 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 scheme in which the slow HBL reassembly is dependent on a slow conversion of linker conformation at neutral pH from a reassembly incompetent to a reassembly competent conformation. Although all the linkers self-associate extensively at neutral pH, forming complexes ranging from dimers to >18-mers, the 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 native Hb, but their cooperativity was lower. A model of HBL reassembly was proposed 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 3-fold symmetry of D.