INHIBITION OF 2-AMINO-3-METHYLIMIDAZO[4,5-F]QUINOLINE (IQ) DNA-BINDING BY CHLOROPHYLLIN - STUDIES OF ENZYME-INHIBITION AND MOLECULAR-COMPLEX FORMATION

Chlorophyllin (CHL), a copper/sodium salt of chlorophyll used in the treatment of geriatric patients, is an anti-mutagen that has been demonstrated to inhibit carcinogen-DNA binding in vivo. To study the mechanism of inhibition, the microsomal metabolism of 2-amino-3-methylimidazo[4,5-f]-quinoline (IQ) and the kinetics of IQ-DNA binding were investigated in the presence and absence of CHL. In time-course studies, CHL produced > 80% inhibition of IQ - DNA binding and blocked the metabolism of IQ, such that 80% of the initial dose of carcinogen was recovered unmetabolized from the incubations after 1 h. Kinetic constants were determined for the in vitro DNA binding reaction, with the reaction rate measured as 'pmol IQ bound/mg DNA/min/mg microsomal protein'. Without altering V(max), the K(m) of the IQ-DNA binding reaction was increased by CHL, and the replot of K(m)/V(max), versus CHL concentration yielded a straight line with an inhibitor constant of 58.3-mu-M CHL. Spectrophotometric studies provided evidence in vitro for the formation of a non-covalent complex between CHL and IQ. The CHL-IQ complex had a stoichiometric ratio of 2:1 (mole ratio method) and an apparent dissociation constant from the Benesi-Hildebrand plot of 1.41 X 10(-4)M at pH 7.4. These results are discussed in the context of a CHL inhibitory mechanism involving enzyme inhibition and molecular complex formation.