Determination of the complex stability of zinc with carbonic anhydrase in sea-water

Abstract

Carbonic anhydrase (CA) is inactive unless associated with zinc, with possible substitution by cobalt. In this work, the complexation of zinc by CA was determined in sea-water using cathodic stripping voltammetry (CSV) with ligand competition. The zinc was found to be released from the CA over a period of 3 h when equilibrated with a competing complexing ligand and the complex was re-formed with the CA when zinc was added. A value of 8.90 ± 0.27 was found for logK′_ZnCA where K′_ZnCA is the conditional stability constant for the complex of Zn²⁺ with CA in pH 8 sea-water. A value for the molecular weight of CA was calculated from its equivalent ligand concentration (in nM) obtained by titrations with zinc at various CA concentrations (1–4 mg l⁻¹). The value found (34740 g mol⁻¹) for the molecular weight is consistent with values found previously by other methods (29000–31000 g mol⁻¹) confirming that the stoichiometry of the complex between zinc and CA is 1∶1. This work confirms that the zinc–CA complex is reversible and that the interaction between zinc and CA can be determined using CSV with ligand competition.