Successful preparation of cobalt(II)-substituted hemocyanin

The cobalt(II)-substituted hemocyanin was first prepared by dialysis of apohemocyanin against Tris-HCl buffer (pH 8.0) containing cobalt(II) ion. The amount of cobalt(II) introduced into apohemocyanin reached 47% of the total sites for copper ion in native hemocyanin, being estimated as nearly complete formation of the half-filled cobalt(II)-hemocyanin (Co(II)-Hc). The Co(II)-Hc did not bind oxygen molecule even under O2-atmosphere. The spectral data indicated that the Co(II) is in tetrahedral geometry (high-spin state).