FOOTPRINTING PROTEINS ON DNA WITH PEROXONITROUS ACID

The peroxonitrite anion (ONOO-) is a stable species in alkaline solution that quickly generates a strong oxidant at neutral pH. A convenient procedure for the preparation of ONOOK has been developed based on the procedure of Keith & Powell [(1969) J. Chem. Soc. A, 90], which when added to a sample of duplex DNA buffered at neutral pH rapidly generates a strong oxidant capable of nonspecifically cleaving the DNA present. We show that this solution containing ONOOK can be used to hydroxyl radical footprint the binding the cI-repressor (cI) of phage lambda with the right operator, O(R). In addition, we show that the individual-site binding isotherms determined by quantitative DNase I, Fe-EDTA and ONOOK footprinting are identical within experimental error. The identical isotherms obtained with the three different reagents with greatly differing sampling times indicates that the sampling time of the footprinting probe need not be short relative to the kinetic dissociation constants that govern protein-DNA interactions.