Molecular shape, dissociation, and oxygen binding of the dodecamer subunit of Lumbricus terrestris hemoglobin

Small angle x-ray scattering of the 213-kDa dodecamer of Lumbricus terrestris Hb yielded radius of gyration = 3.74 +/- 0.01 nm, maximum diameter = 10.59 +/- 0.01 nm, and volume = 255 +/- 10 nm(3), with no difference between the oxy and deoxy states. Sedimentation velocity studies indicate the dodecamer to have a spherical shape and concentration- and Ca2+-dependent equilibria with its constituent subunits, the disulfide-bonded trimer of chains a-e and chain d. Equilibrium sedimentation data were fitted best with a trimer-dodecamer model, In K-4 = 7 (association K in liters(3)/g(3)) at 1 degrees C and 4 at 25 degrees C, providing Delta H = -20 kcal/mol and Delta S = 4.4 eu/mol. Oxydodecamer dissociation at pH 8.0, in urea, GdmCl, heteropolytungstate K-8[SiW11O39] and of metdodecamer at pH 7, was followed by gel filtration. Elution profiles were fitted with exponentially modified gaussians to represent the three peaks. Two exponentials were necessary to fit all the dissociations except in [SiW11O39](-8). Equilibrium oxygen binding measurements at pH 6.5-8.5, provided P-50 = 8.5, 11.5-11.9 and 11.9-13.5 torr, and n(50) = 5.2-9.5, 3.2-4.9, and 1.8-2.7 for blood, Hb, and dodecamer, respectively, at pH 7.5, 25 degrees C, P-50 was decreased 3- and 2-fold in similar to 100 nM Ca2+ and Mg2+, respectively, with concomitant but smaller increases in cooperativity.