FIGURE 1.

Titration of Co²⁺ into wild type and C57S hSOD1, monitored by UV-visible absorption spectroscopy at 300–800 nm. A, stepwise titrations into wild type SOD1 at pH 5.5 show an end point at two equivalents Co²⁺ per dimer. Spectrum a, 1 equivalent Co²⁺ per dimer; spectrum b, 1.25 equivalents Co²⁺ per dimer; spectrum c, 1.5 equivalents Co²⁺ per dimer; spectrum d, 1.75 equivalents Co²⁺ per dimer; spectrum e, 2 equivalents Co²⁺ per dimer. B, stepwise titrations into C57S SOD1 at pH 5.5 show an end point at one equivalent Co²⁺ per dimer. Spectrum a, 1 equivalent Co²⁺ per dimer; spectrum b, 1.25 equivalents Co²⁺ per dimer; spectrum c, 1.5 equivalents Co²⁺ per dimer. C, addition of excess Co²⁺ to C57S SOD1 yields one equivalent per dimer after washing. Six equivalent Co²⁺ per dimer were added to C57S SOD1 at once. After washing, spectrum was taken again. Spectrum a, the end point of stepwise Co²⁺ titration (spectrum c from Fig. 1B); spectrum b, excess Co²⁺ addition before washing; spectrum c, excess Co²⁺ addition after washing. D, stepwise titrations into C57S SOD1 at pH 7.4 show an end point at two equivalents Co²⁺ per dimer. Spectrum a, apo; spectrum b, 1 equivalent Co²⁺ per dimer; spectrum c, 2 equivalent Co²⁺ per dimer; spectrum d, difference b − a. Squares and diamonds symbolize the zinc-binding and copper-binding sites, respectively.