Cytofluorometric determination of protein-bound thiols and DNA in cell nuclei

The aim of the present study was to establish a cytofluorometric method for the simultaneous determination of protein-bound sulfhydryl-groups (PSH) and DNA in isolated cell nuclei. DNA was stained with ethidiumbromide and PSH with N-iodoacetyl-N(5-sulfo-1-naphthyl) ethylendiamine (AEDANS). Disulfide groups of nuclear proteins were determined by the same method after reduction with sodium borohydride or thioglycollic acid. The method was established by using nuclei of human lymphocytes, which then served as a biological standard for further investigations of the nuclei of different mammalian cell types: nuclei from mouse liver cells and nuclei from the cells of two human melanoma cell lines. For non-proliferating lymphocytes distinct DNA- and PSH-values could be measured. The PSH-values detected in the nuclei of the other cell types were higher by comparison and varied within the cell cycle; i.e., PSH increased during the S-phase and was almost doubled during the cell generation cycle from G1- to G2-phase. Cell line and cell cycle-dependent variations of nuclear disulfides could also be detected. These results are discussed with respect to their radiobiological implications. In conclusion, thiol groups may represent one factor determining the radiosensitivity of cells, but they are not the only decisive one.