Abstract
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 nm3, 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-c and chain d. Equilibrium sedimentation data were fitted best with a trimer-dodecamer model, ln K4 = 7 (association K in liters3/g3) at 1 degrees C and 4 at 25 degrees C, providing DeltaH = -20 kcal/mol and DeltaS = 4.4 eu/mol. Oxydodecamer dissociation at pH 8.0, in urea, GdmCl, heteropolytungstate K8[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 P50 = 8.5, 11.5-11.9 and 11.9-13.5 torr, and n50 = 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. P50 was decreased 3- and 2-fold in approximately 100 mM Ca2+ and Mg2+, respectively, with concomitant but smaller increases in cooperativity.
Highlights
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 nm3, with no difference between the oxy and deoxy states
A few models were found to exhibit scattering equivalent to the dodecamer, i.e. fulfilling the following requirements within the mean square deviation, 1) same radius of gyration, 2) same scattering curve up to h ϭ 1.9 nmϪ1, 3) same maximum diameter and shape of p(r) functions, and 4) similar volume
The Molecular Shape of Dodecamer from SAXS—The data shown in Fig. 1 and Table I demonstrate that the molecular shapes of the oxy and deoxy forms of dodecamer from Hb dissociation in urea are the same within the experimental error of the SAXS method
Summary
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, 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– c and chain d. Equilibrium oxygen binding measurements at pH 6.5– 8.5, provided P50 ؍8.5, 11.5–11.9 and 11.9 –13.5 torr, and n50 ؍ 5.2–9.5, 3.2– 4.9, and 1.8 –2.7 for blood, Hb, and dodecamer, respectively, at pH 7.5, 25 °C. Dissociation of the oxyHb at neutral pH in the presence of urea, Gdm salts, and other chaotropes at concentrations that do not denature the subunits, provides a 213-kDa dodecamer complex, consisting of T and M subunits [10, 11]. We report the results obtained regarding the molecular shape and physical homogeneity of the oxydodecamer in solution at neutral pH, its dissociation, and its equilibrium oxygen binding
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