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. 2021 Jun 7;1(7):998–1013. doi: 10.1021/jacsau.1c00160

Figure 2.

Figure 2

Reversible and dynamic interaction between DNIC-1 and endogenous proteins. (a) Schematic illustration for reversible interaction between dinuclear DNIC-1 and endogenous proteins leading to the assembly of protein-bound DNICs. (b) EPR spectra for DNIC-1 in PBS (pH 7.4) with (black) or without (olive) the presence of BSA. EPR spectrum for reaction of DNIC-1 with NEM-modified BSA in PBS (pH 7.4) is shown in blue, whereas the EPR spectrum of BSA is shown in red. (c) Formation and decay of BSA-bound DNIC derived from the reaction of DNIC-1 and BSA in PBS (pH 7.4) at 37 °C, whereas the decay of BSA-bound DNIC was fitted to pseudo-first-order kinetics (red line). Data show the mean ± SD from three independent experiments. (d) Time-dependent change of UV–vis spectra for the PBS solution in the basolateral compartment of a dialysis tube, of which DNIC-1 and BSA in PBS (pH 7.4) were added in the apical compartment. (e) Decompositions of DNIC-1 with the presence of BSA (black) or l-cysteine (blue) in PBS (pH 7.4) under an aerobic condition at 37 °C, which were fitted to pseudo-first-order kinetics. Data show the mean ± SD from three independent experiments. (f) Half-life for DNIC-1 with the presence of l-cysteine (or BSA) or in the isolated plasma at 37 °C. Data represent the mean ± SD (n = 3). (g) Equivalent of released NO after degradation of BSA-bound DNIC derived from the reaction of DNIC-1 and BSA in PBS (pH 7.4). The data are the mean values ± the SD pooled from three independent experiments. (h) EPR spectra for isolated plasma with (black) or without (red) the presence of DNIC-1. (i) Decay of protein-bound DNIC derived from the addition of DNIC-1 into the isolated plasma, which is fitted to pseudo-first-order kinetics (red line). Data represent the mean ± SD (n = 3). (j) EPR spectra for the reaction of DNIC-1 and porcine stomach mucin in 100 mM phosphate buffer at pH 7.4 (black), pH 6.8 (red), and pH 2.7 (blue). EPR spectrum of porcine stomach mucin in 100 mM phosphate buffer at pH 7.4 is shown in magenta. (k) Time-dependent change of UV–vis spectra for the 100 mM phosphate buffer (pH 7.4) in the basolateral compartment of a dialysis tube, of which DNIC-1 and porcine stomach mucin in 100 mM phosphate buffer (pH 7.4) were added in the apical compartment.