Figure 3.

CcScsC wt binds copper(I) with high affinity. (a) Co-elution of CcScsC wt with one molar equivalent of copper(I) from a desalting column. The protein concentration in each fraction was determined by an Ellman test. Copper(I) concentrations were determined colorimetrically using bathocuproine disulfonic acid (BCA). (b) Titration of CcScsC wt into a mixture of 27–30 µM copper(I) and either 75 µM (n = 3) or 150 µM (n = 2) BCA. The [Cu^IBCA₂]³⁻ complex absorbs light at 562 nm and thus a decrease in A ₅₆₂ indicates competition between the protein and BCA to bind copper(I). The titration curves were fitted as described in Section 2 to yield log K _d = 13.1 ± 0.1 M (solid lines). Simulated curves corresponding to a tenfold tighter (dotted lines) or tenfold weaker (dashed lines) affinity are displayed as references.