Protein Modifications by Activated Carcinogens

Abstract

Abstract The postulated fragmentation of RNase by N-acetoxy-2-fluorenylacetamide (N-acetoxy-2-FAA), an activated metabolite of the carcinogen, N-hydroxy-2-fluorenylacetamide, has been investigated. The interaction of N-acetoxy-2-FAA with RNase resulted in the formation of two new proteins separable from RNase by electrophoresis and ion exchange chromatography. These proteins contained no new NH2-terminal amino acids, and their amino acid composition was identical with that of native RNase, indicating that the protein had not been modified by cleavage of the peptide chain. The alternative possibility that the formation of the additional proteins was due to a decrease in the positive charge of RNase was examined by the determination of the incorporation of 3H and 14C from N-[3H]acetoxy-[9-14C]2-FAA into RNase. The ratio of bound 3H to bound 14C, which measures the relative extent of acetylation and arylamidation in the modified proteins, suggested that acetylation rather than arylamidation was the predominant reaction modifying RNase. The conclusion that the acetyl group of N-[3H]-acetoxy-[9-14C]2-FAA had been transferred to RNase was confirmed (a) by the recovery of [3H]acetic acid from the hydrolysates of the modified protein and (b) by the isolation of e-N-acetyl-l-lysine from enzymatic hydrolysates of the modified protein. The isolation of the N-acetylated amino acid accounted in part for the decrease in the positive charge of RNase after reaction with N-acetoxy-2-FAA. The results indicate that N-acetoxy-2-FAA is an acetyl donor and modifies proteins primarily by acetylation rather than arylamidation.