The erythrocruorin of Eisenia fetida. II. Properties of the principal subunit.

The erythrocruorin of Eisenia fetida can be dissociated partially into its principal subunits, the putative one-twelfths of the molecule and smaller subunits, by three different methods: freezing and thawing (Af), exposure to alkaline pH (Aa) and aging (Ao). The isolated subunits possess a relative molecular mass of 310,000 +/- 20,000 by gel filtration and their SDS-polyacrylamide gel electrophoretic patterns are identical to (Af) or slightly different from (Aa, Ao) that of the erythrocruorin. The absorption spectra and Hill constant h of the principal subunit Af correspond to the values of the whole erythrocruorin in the state of low cooperativity, which results from freezing and thawing, exposure to alkaline pH, and aging. Electron microscopic studies of the principal subunits and of the 'treated' erythrocruorin showed that their dimensions had increased relative to the native erythrocruorin: a diameter of 9 nm vs. 8.5 nm and (26.4 +/- 0.4) X (18.3 +/- 0.4) nm vs. 25.0 X 16.5 nm, respectively. Erythrocruorin reconstituted from the Af subunits possessed the dimensions (26.6 +/- 0.4) X (18.6 +/- 0.4) nm. Based on the subunit model of Eisenia erythrocruorin proposed previously it is suggested that there can exist an association-dissociation equilibrium between the principal subunits, smaller subunits, and the erythrocruorin when it is in the state of low cooperativity, but not when it is in its native, high-cooperativity state.