Fig. 4.

Fig. 4a. The kinetics of integral intensities decay of the EPR spectra of 50 μM biradical $R_2^{•}SS{R}_2^{•}$ (□), concentration is multiplied by factor 2 to account for two monoradical subunits) and 100 μM monoradicals, $R_2^{•}\mathit{SSG}$ and $R_2^{•}SH$ (■), solutions in 0.1 M Na-phosphate buffer, pH 7.4, 1 mM DTPA, after addition of 2.5 mM ascorbic acid. The solution of the monoradicals was obtained by mixing 0.5 mM of $R_2^{•}SS{R}_2^{•}$ with 1.5 mM of GSH in 10 mM aqueous NaOH and incubating for about 10 min to complete the biradical disulfide splitting monitored by EPR, then a 10 μl aliquot was diluted in 0.1 M Na-phosphate buffer, pH 7.4, 1 mM DTPA, and ascorbate was added to initiate the kinetics. Lines are the best exponential fits yielding the bimolecular rate constants for reduction of the biradical, (1.7±0.2) M⁻¹ s⁻¹, and monoradicals, (1.5±0.2) M⁻¹ s⁻¹. The bell-shaped curves represent the kinetics of the low-field EPR peak intensity, I_m, of the solution of the biradical $R_2^{•}SS{R}_2^{•}$ in 0.1 M Na-phosphate buffer, pH 7.4, 1 mM DTPA, after addition of (○) 2.5 mM and (●) 0.625 mM ascorbic acid.

Fig 4b. The dependence of the time point, t_max, corresponding to the maximum $I_m^{max}$ of the bell-shaped kinetics of the low-field EPR peak intensity of the solution of the biradical $R_2^{•}SS{R}_2^{•}$ in 0.1 M Na-phosphate buffer, pH 7.4, 1 mM DTPA, on the inverse concentration of reducing agent, ascorbate. The linear regression yields the value of k_r equal to (2.2±0.2) M⁻¹ s⁻¹.