Using anti-5,5-dimethyl-1-pyrroline N-oxide (anti-DMPO) to detect protein radicals in time and space with immuno-spin trapping

Abstract

The detection of protein free radicals using the specific free radical reactivity of nitrone spin traps in conjunction with nitrone–antibody sensitivity and specificity greatly expands the utility of the spin trapping technique, which is no longer dependent on the quantum mechanical electron spin resonance (ESR). The specificity of the reactions of nitrone spin traps with free radicals has already made spin trapping with ESR detection the most universal, specific tool for the detection of free radicals in biological systems. Now the development of an immunoassay for the nitrone adducts of protein radicals brings the power of immunological techniques to bear on free radical biology. Polyclonal antibodies have now been developed that bind to protein adducts of the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). In initial studies, anti-DMPO was used to detect DMPO protein adducts produced on myoglobin and hemoglobin resulting from self-peroxidation by H 2O 2. These investigations demonstrated that myoglobin forms the predominant detectable protein radical in rat heart supernatant, and hemoglobin radicals form inside red blood cells. In time, all of the immunological techniques based on antibody–nitrone binding should become available for free radical detection in a wide variety of biological systems.