Comparison of 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH) and 2,2'azobis(2,4-dimethylvaleronitrile) (AMVN) as free radical initiators: A spin-trapping study

Spin trapping with 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) and its hydrophobic analogue 2,2-dimethyl-4-phenyl-2H-imidazole 1-oxide (DMPIO) was used to identify and to monitor the concentration of participating radical species in oxidation reactions initiated by ate compounds: water-soluble 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH) and lipophilic 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN), Incubation of AAPH with spin traps in aqueous media produced alkoxyl radical spin adducts with hyperfine splitting constants being a(N) = 14.62 G, (beta)a(H) = 15.29 G, (gamma)a(H) = 0.72 G and a(N) = 13.46 G, (beta)a(H) = 12.53 G, for DMPO and DMPIO, respectively. In contrast, formation of AMVN-derived peroxyl radicals was detected by both direct EPR and spin trapping in DMSO (dimethylsulfoxide) solutions, In the presence of either rabbit skeletal sarcoplasmic reticulum (SR) membranes (10 g dm(-3) of SR protein) or egg phosphatidylcholine liposomes (10 g dm(-3) of lipid) preloaded with AMVN no spin adduct formation was observed, for both DMPO and DMPIO spin traps, indicating that AMVN-derived radical species do not escape the lipid environment. Only a small portion of AAPH-derived alkoxyl radicals was trapped by DMPIO in the presence of SR membranes, Spectral characteristics of the DMPIO spin adduct indicate its location at the lipid-water interface. At the same time, there was virtually no effect of SR on the rate of formation and steady-state level of the DMPO-spin adduct formed in the aqueous phase. From these data we suggest that the bulky cytosolic domains of the SR Ca2+-ATPase protect the membrane surface from radicals generated in the bulk (aqueous) solvent, Other evidence also demonstrates different mechanisms for free radical formation by AAPH and AMVN azo-initiators.