A spectrophotometric method to determine the amount of CO bound to hemocyanin

Abstract

The amount of CO bound to hemocyanin has been determined by titration with human hemoglobin (Hb). The spectrophotometric method makes use of the high affinity of Hb for CO, which (at pH 9) allows a complete transfer of the ligand from hemocyanin to hemoglobin. Taking advantage of the large spectral changes at the level of the heme, this method allows the rapid determination of the CO bound to hemocyanin, using relatively small amounts of protein (less than 5 mg). Application of this method shows (i) that CO binds to hemocyanins with a stoichiometry of 1:1, in agreement with one earlier observation with 14C-labeled carbon monoxide (1), and (ii) that decrease of the CO-copper luminescence of hemocyanin observed upon addition of KCN is quantitatively related to the displacement of CO from the active site. © 1983.