EPR Spin Trapping of Free Radicals Produced by Bleomycin and Ascorbate

Abstract

In the presence of ascorbate, bleomycin (BLM) is converted to a redox-inactive form that is incapable of inducing DNA strand scission. We have employed EPR spin trapping with 5,5-dimethylpyrroline-1-oxide (DMPO) to examine free radical production during this process. The introduction of ascorbate to an Fe(III)BLM-DMPO system results in the formation of three EPR observable free radicals. One of these radicals is the resonance-stabilized ascorbate free radical (a^H = 1.8 G) that is not spin trapped by DMPO; the other two are the result of DMPO spin trapping. These radicals appear to be two carbon-centered radicals, DMPO/•CR₁, (a^N₁ = 15.75 G, a^H₁ = 22.30 G, a^N/a^H = 0.706) and DMPO/•CR₂ (a^N₂ = 15.20 G, a^H₂ = 19.20 G, a^N/a^H = 0.79). Although it is not possible to identify the exact structures of the carbon-centered radicals that are spin trapped, the hyperfine splittings, as well as the a^N/a^H values, are characteristic of the presence of electron-withdrawing groups, such as the oxygen atom when attached to the carbon atom. In fact, these parameters are characteristic of DMPO spin trapping results obtained when sugars are subjected to oxidative insult from HO.. Thus, these BLM-ascorbate produced radicals may well be derived from the sugar moiety of BLM.

Key Words:

• ascorbate
• bleomycin
• electron paramagnetic resonance
• free radical
• spin trapping