Saito K, Shinohara A, Kamataki T, Kato R
Anal Biochem. 1986 Feb 1;152(2):226-31. doi: 10.1016/0003-2697(86)90402-1.
A simple fluorometric assay for N-hydroxyarylamine O-acetyltransferase is described and compared with a nucleic acid-binding assay. The assay method is based on the finding that the highly mutagenic 2-hydroxyamino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (N-OH-Glu-P-1) was reduced to the corresponding amine, 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1), through N-acetoxy-Glu-P-1 as the reactive intermediate in the presence of N-hydroxyarylamine O-acetyltransferase, acetyl-CoA, and a sulfhydryl compound. The formation of Glu-P-1 was determined by its characteristic fluorescence intensity at 445 nm with excitation wavelength at 376 nm. The reductive reaction was inhibited by the addition of tRNA, DNA, and poly(G), to which the enzymatic product, N-acetoxy Glu-P-1, bound effectively due to its electrophilic nature. Since the fluorometric assay for the O-acetyltransferase is rapid, simple, and sensitive as compared with the nucleic acid-binding method using radioisotope-labeled N-hydroxyarylamine, this method is applicable to the general assay for the formation of reactive N-acetoxy-Glu-P-1.
本文描述了一种用于N-羟基芳基胺O-乙酰基转移酶的简单荧光测定法，并将其与核酸结合测定法进行了比较。该测定方法基于以下发现：在N-羟基芳基胺O-乙酰基转移酶、乙酰辅酶A和一种巯基化合物存在的情况下，具有高度致突变性的2-羟基氨基-6-甲基二吡啶并[1,2-a:3',2'-d]咪唑（N-OH-Glu-P-1）通过N-乙酰氧基-Glu-P-1作为反应中间体被还原为相应的胺，即2-氨基-6-甲基二吡啶并[1,2-a:3',2'-d]咪唑（Glu-P-1）。通过在激发波长为376nm时测定其在445nm处的特征荧光强度来确定Glu-P-1的形成。添加tRNA、DNA和聚（G）会抑制还原反应，由于其亲电性质，酶促产物N-乙酰氧基Glu-P-1会与这些物质有效结合。由于与使用放射性同位素标记的N-羟基芳基胺的核酸结合方法相比，该O-乙酰基转移酶的荧光测定法快速、简单且灵敏，因此该方法适用于活性N-乙酰氧基-Glu-P-1形成的常规测定。
