Binding of radioprotective thiols and disulfides in Chinese hamster V79 cell nuclei

Binding of thiols of varying charge (Z) in nuclei prepared in suspension was determined to assess the extent to which histones, Mg2+ spermine and chromatin structure influence counter-ion condensation of cationic thiols and co-ion depletion of anionic thiols at DNA. The nuclei were prepared in suspension buffer, washed and incubated in buffer containing thiol and graded amounts of Mg2+ and spermine. The nuclei were separated from the incubation medium by centrifugation through silicone oil, and the thiols were determined in the nuclear pellet and in the incubation buffer by labeling with monobromobimane and HPLC. Measurements of the water content of nuclei indicated that chromatin was fully condensed in buffer containing 5 mM MgCl2 and 115 mM KCl. Under these conditions nuclei incubated in 1 mM substrate had concentrations of 0.80 +/- 0.21 mM glutathione (Z = -1), 1.05 +/- 0.12 mM 2-mercaptoethanol (Z = 0), 0.95 +/- 0.15 mM cysteine (Z = 0), 0.75 +/- 0.29 mM cysteamine (Z = +1), 2.5 +/- 0.3 mM WR-1065 (Z = +2), 3.4 +/- 0.5 mM WR-35980 (Z = +3) and 12 +/- 2 mM WR-33278 (disulfide of WR-1065, Z = +4), respectively. Spermine up to 1 mM in the presence of 5 mM Mg2+ had little effect upon the binding of these thiols and disulfide, but did suppress the binding of 0.1 mM WR-33278, the results indicating that WR-33278 and spermine compete for the same sites with comparable affinity. From the results observed and the assumption that deviations from the bulk solution concentration (1 mM) result from counter-ion condensation within 3 nm of DNA, we estimate that WR-1065 (Z = +2), WR-35980 (Z = +3) and WR-33278 (Z = +4) were concentrated near DNA 6-, 8- and 20-fold, respectively, in the presence of histones, 5 mM Mg2+ and 1.0 mM spermine.