Oxygen binding domains of Helisoma trivolvis hemoglobin

Abstract

The extracellular hemoglobin of the planorbid snail Helisoma trivolvis is an unusual heme protein. The form occurring in vivo is a decamer having a molecular weight of 1.75·10 6. Electron micrographs suggest that the quaternary structure of the decamer is a ring with ten-fold symmetry. The subunits are very large, having molecular weights of about 175 000. This is in striking contrast to the 15–17 000 dalton subunits of most hemoglobins. Gentle proteolysis with subtilisin cleaves Helisoma hemoglobin into heme-containing domains with molecular weights of 15–17 000 and approximately integral multiples of this value. The isolated domains have about one heme for each 17 000 daltons of protein and all bind oxygen reversibly. However, the positive Bohr effect and cooperativity of oxygen binding which are characteristic of the intact pigment are not present in preparations with molecular weights of 17 000, 31 000 or 60–75 000. Thus, the intact polypeptide seems to be necessary for full expression of homotropic and heterotropic interactions. Kinetic studies of the intact hemoglobin and its isolated domains are consistent with the oxygen equilibrium studies. The relatively high oxygen affinities of this pigment and its domains are associated with low oxygen dissociation velocity constants. These rate constants are pH sensitive in the intact pigment and relatively pH insensitive in the isolated domains.