FIG. 5.

Purification of recombinant Cmc1p and reconstitution of CuCmc1p. (A) Recombinant His₆-Cmc1p was affinity purified using a combination of metal affinity and size exclusion chromatography as described in Materials and Methods. Subsequent fractions were analyzed by SDS-PAGE and Coomassie blue staining. CL, cell lysate; FT, flow through the metal column; E, pool of elution fractions; TH, thrombin-cleaved recombinant Cmc1p. (B) Cu(I) fluorescence of CuCmc1p. An excitation of 310 nm exhibits an emission peak with a maximum at ∼560 nm, indicative of Cu(I)-thiolate complexes that are not solvent accessible. The addition of 5 mM KCN, a Cu(I) chelator, to CuCmc1p significantly eliminates the observed emission. (C) Titration of reduced apoCmc1p with Cu(I). Maximal emission is obtained at one equivalent of copper.