DNA BREAKING ACTIVITY OF THE CARBON-CENTERED RADICAL GENERATED FROM 2,2'-AZOBIS(2-AMIDINOPROPANE) HYDROCHLORIDE (AAPH)

When supercoiled plasmid DNA was incubated with 2,2'-azobis (2-amidino propane)hydrochloride (AAPH) at pH 7.4 in the presence and absence of oxygen, the DNA single strands were effectively cleaved. The breaking in the presence of oxygen was not inhibited by superoxide dismutase an d catalase, but inhibited by mannitol, ethanol, butyl hydroxyanisole, thiol compounds, tertiary amines and spin trapping agents N-tert-butyl -alpha-phenylnitrone (PBN) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO) . The breaking in the absence of oxygen was inhibited by ethanol, a te rtiary amine and PBN. By electron spin resonance spin-trapping with PB N, the carbon-centered radical was detected both in the presence and t he absence of oxygen. Hydroxyl radical was detected by use of DMPO onl y in the presence of oxygen. The DNA breaking activity of AAPH was fou nd to be due primarily to the aliphatic carbon-centered radical. While the reactivity of carbon-centered radicals have received little atten tion, the aliphatic carbon-centered radical generated from AAPH was fo und to be highly reactive to break the DNA strands.