The Role of the Dodecamer Subunit in the Dissociation and Reassembly of the Hexagonal Bilayer Structure of Lumbricus terrestris Hemoglobin (∗)

The dissociation of the ▪3500-kDa hexagonal bilayer (HBL) hemoglobin (Hb) of Lumbricus terrestris upon exposure to Gdm salts, urea and the heteropolytungstates [SiW▪O▪]▪ (SiW), [NaSb▪W▪O▪]▪ (SbW) and [BaAs▪W▪O▪]▪ (AsW) at neutral pH was followed by gel filtration, SDS-polyacrylamide gel electrophoresis, and scanning transmission electron microscopy. Elution curves were fitted to sums of exponentially modified gaussians to represent the peaks due to undissociated oxyHb, D (▪200 kDa), T+L (▪50 kDa), and M (▪25 kDa) (T = disulfide-bonded trimer of chains a-c, M = chain d, and L = linker chains). OxyHb dissociation decreased in the order Gdm•SCN > Gdm•Cl > urea > Gdm•OAc and AsW > SbW > SiW. Scanning transmission electron microscopy mass mapping of D showed ▪10-nm particles with masses of ▪200 kDa, suggesting them to be dodecamers (a+b+c)▪d▪. OxyHb dissociations in urea and Gdm•Cl and at alkaline pH could be fitted only as sums of 3 exponentials. The time course of D was bell-shaped, indicating it was an intermediate. Dissociations in SiW and upon conversion to metHb showed only two phases. The kinetic heterogeneity may be due to oxyHb structural heterogeneity. Formation of D was spontaneous during HBL reassembly, which was minimal (≤ 10%) without Group IIA cations. During reassembly, maximal (▪60%) at 10 mM cation, D occurs at constant levels (▪15%), implying the dodecamer to be an intermediate.