REACTION OF AFLATOXIN B-1 EXO-8,9-EPOXIDE WITH DNA - KINETIC-ANALYSISOF COVALENT BINDING AND DNA-INDUCED HYDROLYSIS

The exo isomer of aflatoxin B-1 (AFB(1)) 8,9-epoxide appears to be the only product of AFB(1) involved in reaction with DNA and reacts with the N-7 atom of guanine via an S(N)2 reaction from an intercalated sta te, Although the epoxide hydrolyzes rapidly in H2O (0.6 s(-1) at 25 de grees C), very high yields of DNA adduct result, Experimental binding data were fit to a model in which the epoxide forms a reversible compl ex with calf thymus DNA (K-d = 0.43 mg ml(-1), or 1.4 mM monomer equiv alents) and reacts with guanine with a rate of 35 s(-1). Stopped-flow kinetic analysis revealed attenuation of fluorescence in the presence of DNA that was dependent on DNA concentration, Kinetic spectral analy sis revealed that this process represents conjugation of epoxide with DNA, with an extrapolated rate maximum of 42 s(-1) and half-maximal ve locity at a DNA concentration of 1.8 mg ml(-1) (5.8 mM monomer equival ents), The rate of hydrolysis of the epoxide was accelerated by calf t hymus DNA in the range of pH 6-8, with a larger enhancement at the low er pH (increase of 0.23 s(-1) at pH 6.2 with 0.17 mg DNA ml(-1)), The same rate enhancement effect was observed with poly[dA-dT].poly[dA-dT] , in which the epoxide can intercalate but not form significant levels of N-7 purine adducts, and with single-stranded DNA, The increased ra te of hydrolysis by DNA resembles that reported earlier for epoxides o f polycyclic hydrocarbons and is postulated to involve a previously su ggested localized proton field on the periphery of DNA. The epoxide pr eferentially intercalates between base pairs, and the proton field is postulated to provide acid catalysis to the conjugation reaction.