Protein modifications by activated carcinogens II. The acetylation of ribonuclease by N-acetoxy-3-fluorenylacetamide

Abstract

The reaction of N-[ 3H]acetoxy-3-fluorenylacetamide ( N-[ 3H]acetoxy-3-FAA), a potent carcinogen for the rat, with RNAase yielded three modified proteins separable from RNAase by ion exchange chromatography on Bio-Gel CM-30 with a gradient of increasing sodium ion concentration. Only minor amounts of RNAase were recovered. The modified proteins were labeled with 3H to a varying degree, and their order of elution was inversely related to the extent of labeling. The modification of the proteins was the result of the transfer of the acetyl group from N-[ 3H]acetoxy-3-FAA to RNAase. The evidence for this conclusion was (a) the release of 84–86% of the radioactivity as [ 3H] acetic acid from the two major proteins upon acid hydrolysis and (b) the isolation of ε- N-[ 3H]acetyl- l-lysine from enzymatic hydrolysates of these proteins. A comparison of the present data with those previously reported for the acetylaton of RNAase by the isomeric carcinogen, N-acetoxy-2-FAA, showed that N-acetoxy-3-FAA is the more potent acetylating agent. The present study in conjunction with the previous results, suggests that structural alteration of cellular nucleophiles by acylation may be a biochemical mechanism underlying the biological activity of N-acetoxy-3-FAA and related activated carcinogens.