FIGURE 3.

The metal-dependent dimerization of AnhE. Protein samples were applied to a Superdex 75 10/300 GL column pre-equilibrated with 20 mm HEPES, pH 7.5, 50 mm NaCl. Elution profiles were as follows: 0.1 mm AnhE before and after incubation with an equimolar amount of CoCl₂ (A); 0.5 mm AnhE before and after incubation with an equimolar amount of CoCl₂ (B); and 0.1 mm AnhE in the presence of 0.1 mm ZnCl₂, NiCl₂ andCuCl₂ (C). D, standard curve for the molecular mass determination was established using lysozyme (15,000), chymotrypsinogen (25,000), carbonic anhydrase (32,000), β-lactoglobulin (36,000), and bovine serum albumin (68,000). The calculated sizes of the two indicated AnhE species were 16,200 and 29,000 and were taken to represent monomer and dimer, respectively.