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. 2016 May 23;6:26385. doi: 10.1038/srep26385

Figure 2. Photophysical and enzymatic properties of caspase probes.

Figure 2

(a) Photophysical and kinetic properties of probes and free dyes. All measurements were taken in 50 mM HEPES, 50 mM NaCl, 0.1% CHAPS, 10 mM EDTA, 5% glycerol, 10 mM DTT, at pH = 7.2. (b) Time-dependent ratiometric fluorescence changes of C1RB (6 μM) incubated with caspase-1 (10 nM) over 2 h at room temperature. Data were obtained by plotting the normalized ratio of fluorescence intensity at 500 nm (F500 nm) over that at 450 nm (F450 nm). (Inset) time-dependent fluorescence emission spectra with λex = 350 nm. (c) Time-dependent “Turn-ON” effect of C1FS (6 μM) incubated with caspase-1 (10 nM) measured and normalized at 460 nm (F460 nm) over 2 h at room temperature, with the corresponding fluorescence emission spectra (inset). λex = 350 nm. (d) Normalized S/N ratio of each of the four caspase-1 probes (6 μM) incubated with 25 μg of BV2 lysates spiked with different concentrations of caspase-1 (37 °C, t = 2 h). S and N represent the fluorescence emission intensity of each reaction solution in the presence (S) and absence (N) of caspase-1. (e) Normalized S/N ratio of each of the 3 caspase-1 probes (C1RB/C1RS/C1FS; 6 μM) incubated with different caspases (t = 2 h, room temperature). (−) no caspase; (caspase-1) 1.5 nM; (caspase-3) 0.8 nM; (caspase-6) 0.2 μM; (caspase-8) 10 nM; (caspase-9) 0.5 μM. (f) Probe selectivity profiles with the corresponding commercial tetrapeptide-AMC as references. For each probe (6 μM), uniform conditions with different caspases were applied. Normalized, relative fluorescence (RFU) values were obtained after 2-h incubation at room temperature. For (d–f), λex = 360 ± 40 nm; λem = 528 ± 20 nm, except for tetrapeptide-AMC and C1FS where λem = 460 ± 40 nm.