QUANTIFICATION OF RADIATION-INDUCED HYDROXYL RADICALS WITHIN NUCLEOHISTONES USING A MOLECULAR FLUORESCENT-PROBE

We present a method that specifically records (OH)-O-. formation within histones and possibly at other sites in irradiated nucleohistone. The approach uses the radiation-induced fluorescence emissions from a chromatin-conjugated (OH)-O-. detector, SECCA (a succinylated derivative of coumarin), that is converted to a fluorescent derivative, 7-hydroxy-SECCA (7-OH-SECCA), after interaction with (OH)-O-. in neutral aqueous solutions. It is shown that (a) the fluorescent product 7-OH-SECCA cannot be generated by direct radiation effects after gamma or neutron irradiation of SECCA; (b) when SECCA-labeled histone is complexed with DNA to form nucleohistone, the physical properties of the modified nucleohistone are similar to those of unlabeled nucleoprotein; and (c) after irradiation of SECCA-labeled nucleohistone, a linear induction of the fluorescence signal is observed within the radiation doses examined (0.3-30 Gy). Since the sample remains available for further studies after registration of the optical signal, the current approach should permit the investigator to correlate in a single sample the localization and frequency of (OH)-O-. formation with the results of other assays.