Erythrocruorin from the Aquatic Snail Helisoma trivolvis. Quaternary Structure and Arrangement of Subunits

The subunit structure of erythrocruorin from the planorbid snail Helisoma trivolvis was studied. The native protein was found to have a sedimentation coefficient of 34.7S and a molecular weight, as determined by sedimentation equilibrium, of 2.25 X 10⁶. Iron and heme determinations gave 0.270 and 3.21%, corresponding to minimal molecular weights of 20 700 and 19 200, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and absence of 2-mercaptoethanol gave single bands with mobilities corresponding to molecular weights of 1.9 X 10⁵ and ~4 X 10⁵, respectively. Sedimentation equilibrium in 6 M guanidine hydrochloride in the presence and absence of 2-mercaptoethanol gave molecular weights of 1.87 X 10⁵ and 3.82 X 10⁵, respectively. We conclude that a Helisoma erythrocruorin molecule is composed of 12 single polypeptide chain subunits, each carrying 10 hemes. In the molecule, the subunits are grouped in pairs, the members of each pair being held together by disulfide bonds. We propose a model for the molecule composed of 12 spherical subunits arranged in a shell structure with tetrahedral symmetry. Projections of the model are consistent with 10-membered ring and rhombic profiles observed in the electron microscopy of negatively stained H. trivolvis erythrocruorin [Terwilliger, N. B., Terwilliger, R. C., & Schabtach, E. (1976) Biochim. Biophys. Acta 453, 101–110] and with hexagonal ring structures seen in the electron micrographs of Planorbis corneus erythrocruorin [Wood, E. J., & Mosby, L. J. (1975) Biochem. J. 149, 437-445].