The fluorogenic coumarin cargo provides a convenient fluorescence readout for monitoring the cyclization-elimination cascade at lower concentrations ([SIE2]0 = 10 µM). Because coumarin is weakly fluorescent in PBS buffer, we conducted this set of experiments in a pH 6.5 MES buffer and monitored coumarin release by fluorescence spectroscopy. The t1/2 of free SIE2 was determined to be 1.5 h by fitting the time-dependent coumarin release results to a first-order rate expression (Figure , right). The introduction of 1×, 2×, 4×, and 10×CB to the SIE2 solution extended the t1/2 of coumarin release to 3.5 h, 6.0 h, 10.8 h, and 82.8 h respectively, corresponding to approximately 2-, 4-, 7-, and 55-fold decrease in reaction rates compared to the free SIE2. The CB-dependent reactivity trends from these fluorescence-monitored experiments agree with the NMR-measured trends described in the earlier section, while the slightly faster release rates in the fluorescence-monitored group are attributed to the micromolar concentration used in fluorescence studies and the replacement of pH 6.5 phosphate buffer by pH 6.5 MES.