Fig. 3. Cu²⁺ selectivity and reversibility of GCS proteins. (a and b) Fluorescence responses of 1 μM GCS-1 (a) and GCS-2 (b) to various metal ions. White bars represent fluorescence responses with 5 μM of the respective metal ions. Black bars represent fluorescence responses after the addition of 5 μM Cu²⁺. (c) Fluorescence responses of 1 μM GCS protein with 5 μM Cu⁺ including 250 μM of l-ascorbic acid to prevent oxidation of Cu⁺ to Cu²⁺. Fluorescence spectra were collected within 5 min after Cu⁺ addition. (d) Fluorescence responses of 1 μM GCS proteins with 5 μM Cu²⁺ followed by the addition of 50 μM EDTA. All responses were normalized against the fluorescence intensity of 1 μM GCS protein lacking a metal ion. The error bars correspond to the standard error of the mean of three independent measurements.