Fig. 1. Characterization of the Se-click reaction. (A) Se-click reaction applied to site-specific protein labeling. (B) The absorbance of Bodipy593 exhibits a 47 nm bathochromic shift after the Se-click reaction. (C) Kinetic measurement of the reaction rate between SeF and Bodipy593. During the reaction, absorption at 518 nm decreased accompanied by an increase at 565 nm. The extinction coefficient of SeF-Bodipy593 is larger than that of Bodipy593, so the absorbance increase at 565 nm is larger than the decrease at 518 nm. The second-order rate constant was 26.3 ± 0.4 M⁻¹ s⁻¹, generated by fitting the absorbance at 570 nm. Reaction conditions: 20 μM Bodipy593 and 500 μM SeF (reduced by 1 mM TCEP). (D) Kinetic measurement of the reaction rate between N-acetyl-cysteine (NAC) and Bodipy593. The rate constant was 0.0075 ± 0.0009 M⁻¹ s⁻¹ generated by fitting the absorbance at 560 nm (reaction conditions: 20 μM Bodipy593 and 100 mM NAC). All experiments were performed in HEPES buffer (pH = 7.4, 200 mM) with 10% CH₃CN as the co-solvent at room temperature. Data are shown as mean ± s.e.m. (C) and (D) show representative data of three independent experiments.