Subunit Structure and Physical Properties of the Hemocyanin of the Giant Isopod Bathynomus giganteus

The hemocyanin of the deep sea dwelling isopod Bathynomus giganteus has been isolated. From the determination of the sedimentation coefficient (s_20,W = 16.6 S) and the diffusion coefficient (D_20,w = 3.1 X 10^-7 cm²/s at 1.12 mg/mL), a molecular weight of 4.7 X 10⁵ was calculated. The 16S molecule is the largest aggregate observed even under conditions of temperature, pressure, and concentration simulating in vivo conditions. The dissociation of the 16S molecule to 6S monomers is dependent on pH and divalent cation concentration. The sedimentation coefficient and calculated molecular weight are consistent with a hexameric native molecule composed of six monomers of molecular weight about 70000-80000, as has been observed for other hemocyanins. The constituent subunits are heterogeneous on both sodium dodecyl sulfate (two bands, 70200 and 71800 molecular weight) and nondenaturing alkaline electrophoresis (three bands). In the presence of 10 mM CaCl₂, the hexamer is stable to above pH 9.0. Upon dialysis vs. buffer containing 10 mM ethylenediaminetetraacetate, the hemocyanins begin to dissociate into monomers at pH 8.0. The pH-dependent hexamer-monomer dissociation is slowly equilibrating and completely reversible and obeys the law of mass action. The reaction can be described by a cooperative mechanism with one proton binding per monomer. The oxygen binding properties of the hexamer hemocyanin are characterized by a large positive Bohr effect (P₅₀'s at pH 9 and 7 are 5.6 and 57.5, respectively) and moderate cooperativity (n_H = 1.8-3.0). Divalent cations do not appreciably affect either the oxygen affinity or the cooperativity of the hexamer. Complete dissociation to monomers yields noncooperative binding, as expected. The oxygen binding curves of partial dissociation mixtures are well represented by the weighted sum of the curves for “pure monomer” and “pure hexamer” conditions. Comparison of the oxygen binding properties of this deep sea animal with coastal and intertidal species suggests that physiological requirements are more important than phylogenetic relationships in determining the properties of the oxygen- transport protein of a particular species.