Spectrophotomatic Assay for the Enzyme Catalyzed Reaction of 4-nitroquinoline 1-oxide with Glutathione

4-Nitroquinoline 1-oxide (4NQO( is a toxic and carcinogenic compound that has been reported to be subject to conjugation with glutathione (GSH). This reaction may proceed non-enzymatically or be catalyzed by GSH transferases. The non-enzymatic rate for this reaction has been reported to be very high. The purposes of this investigation were todevelop a spectrophotometric assay for the reaction of4NQO with GSH and to determine whether the rate for the enzyme catalyzed reaction was significant relative to the non-enzymatic reaction. The absorbance spectrum of 4NQO in phosphate buffer exhibited a maximum at 365 nm. Reaction of 4NQO with GSH was accompanied by a shift to 353 nm and an absorbance increase which was maximal at 350 nm. The formation ofproduct could be quantitated from the increase in absorbance at 350 nm, where the change in the millimolar extinction coefficient was 7.20 mM~ 1 cm" \ Although the non-enzymatic reaction of4NQO and GSH proceeded rapidly at or above pH 8, at physiological pH this reaction was largely enzyme dependent. Inan assay system containing 0.1 mM 4NQO, 1 mM GSH, and 0.1 Mpotassium phosphate, at 25 °C, the conjugation of 4NQO with GSH by mouse livercytosol was optimal at pH 6.5 7.5. At pH 6.5 and 1 mM GSH, a GSH transferase purified from mouse liver catalyzed the reaction of 4NQO with GSH witha maximum velocity of156 /imoles/min per mg of protein. The Km for 4NQO was 35 fiM.The high activity of liver cytosol in promoting the reaction of 4NQO with GSH and the high affinity of the purified GSH transferase for 4NQO suggest that enzymatic catalysis of this reaction may be of considerable significance in vivo.