The Catalytic Effect of Bovine Serum Albumin on the Ortho Rearrangement of the Potential Ultimate Carcinogen, N-(Sulfooxy)-2-(acetylamino)fluorene, Generated Enzymatically from N-Hydroxy-2-(acetylamino)fluorene and Evidence for Substrate Specificity of the Enzymatic Sulfonation of Arylhydroxamic Acids

This investigation examines the catalytic effect of bovine serum albumin on the ortho rearrangement of the possible ultimate carcinogen, N-(sulfooxy)-2-(acetylamino)fluorene, generated from N-hydroxy-2-(acetylamino)fluorene by the sulfotransferase(s) in the cytosol of rat liver. With various preparations of cytosol, 55–75% of the substrate, N-hydroxy-2-(acetylamino)-fluorene, was found to rearrange to the nonmutagenic and noncarcinogenic o-(sulfooxy) esters, 1- and 3-(sulfooxy)-2-(acetylamino)fluorene, in the presence of bovine serum albumin, while <1% of the substrate rearranged in its absence. In presence of bovine serum albumin the cytosolic reduction of N-(sulfooxy)-2-(acetylamino)fluorene to 2-(acetylamino)fluorene decreased by 60–90% and its solvolytic degradation to 4-hydroxy-2-(acetylamino)fluorene by 80–90%. The covalent interaction of enzymatically generated N-(sulfooxy)-2-(acetylamino)fluorene with the nucleophilic acceptors, N-acetyl-l-methionine and guanosine, was lowered by >90% by addition of bovine serum albumin. These measurements indicated that the albumin-catalyzed ortho rearrangement controls the rates of concurrent metabolic and degradative reactions of N-(sulfooxy)-2-(acetylamino)fluorene. The results are in agreement with previous findings of a catalytic effect of serum albumin on the ortho rearrangement of synthetic N-(sulfooxy)-2-(acetylamino)fluorene. In contrast to its catalytic effect on the formation of o-(sulfooxy) esters from N-(sulfooxy)-2-(acetylamino)fluorene, bovine serum albumin had no effect on the formation of o-(acetylamino)fluorenols. To assess the substrate specificity of bovine serum albumin, its effect on the rearrangement of N-hydroxy-2-(benzoylamino)fluorene, a carcinogenic analogue of N-hydroxy-2-(acetylamino)fluorene, was analyzed under conditions of cytosolic sulfonation. Bovine serum albumin did not significantly increase the minimal quantities of the o-(sulfooxy) esters of 1- and 3-hydroxy-2-(benzoylamino)fluorene formed in its absence. However, synthetic sulfonation of N-hydroxy-2-(benzoylamino)fluorene yielded major amounts (85%) of the o-(sulfooxy) esters. These findings indicate that N-(sulfooxy)-2-(benzoylamino)fluorene is unstable and undergoes rapid ortho rearrangement. The evidence suggests that N-hydroxy-2-(benzoylamino)fluorene is a poor substrate for cytosolic sulfonation. It is therefore not a substrate suitable for investigation of the specificity of bovine serum albumin in catalyzing the ortho arrangement of enzymatically generated N-(sulfooxy) esters of arylhydroxyamic acids.