Quantification of radiation-induced hydroxyl radicals within nucleohistones using a molecular fluorescent probe.
We present a method that specifically records .OH formation within histones and possibly at other sites in irradiated nucleohistone. The approach uses the radiation-induced fluorescence emissions from a chromatin-conjugated .OH detector, SECCA (a succinylated derivative of coumarin), that is converted to a fluorescent derivative, 7-hydroxy-SECCA (7-OH-SECCA), after interaction with .OH 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 formation with the results of other assays.
Makrigiorgos
G M
GM
Department of Radiation Oncology, Harvard Medical School, Boston, Massachusetts 02115.
Folkard
M
M
Huang
C
C
Bump
E
E
Baranowska-Kortylewicz
J
J
Sahu
S K
SK
Michael
B D
BD
Kassis
A I
AI
D000818:Animals; D002417:Cattle; D003374:Coumarins / Q000737:chemistry; D004247:DNA / Q000737:chemistry / Q000528:radiation effects; D005456:Fluorescent Dyes / Q000737:chemistry; D006657:Histones / Q000737:chemistry* / Q000528:radiation effects*; D017665:Hydroxyl Radical / Q000032:analysis*; D013050:Spectrometry, Fluorescence
D003374:Coumarins; D005456:Fluorescent Dyes; D006657:Histones; D017665:Hydroxyl Radical; D004247:DNA