BINDING OF RADIOPROTECTIVE THIOLS AND DISULFIDES IN CHINESE-HAMSTER V79-CELL NUCLEI

Binding of thiols of varying charge (Z) in nuclei prepared in suspensi on was determined to assess the extent to which histones, Mg2+, spermi ne and chromatin structure influence counter-ion condensation of catio nic thiols and co-ion depletion of anionic thiols at DNA. The nuclei w ere prepared in suspension buffer, washed and incubated in buffer cont aining thiol and graded amounts of Mg2+ and spermine. The nuclei were separated from the incubation medium by centrifugation through silicon e oil, and the thiols were determined in the nuclear pellet and in the incubation buffer by labeling with monobromobimane and HPLC. Measurem ents 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-mercaptoethan ol (Z = 0), 0.95 +/- 0.15 mM cysteine (Z = 0), 0.75 +/- 0.29 mM cystea mine (Z = +1), 2.5 +/- 0.3 mM WR-1065 (Z = +2), 3.4 +/- 0.5 mM WR-3598 0 (Z = +3) and 12 +/- 2 mM WR-33278 (disulfide of WR-1065, Z = +4), re spectively. Spermine up to 1 mM in the presence of 5 mM Mg2+ had littl e effect upon the binding of these thiols and disulfide, but did suppr ess the binding of 0.1 mM WR-33278, the results indicating that WR-332 78 and spermine compete for the same sites with comparable affinity. F rom the results observed and the assumption that deviations from the b ulk 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-fol d, respectively, in the presence of histones, 5 mM Mg2+ and 1.0 mM spe rmine. (C) 1996 by Radiation Research Society