The N-acetylation of arsanilic acid in vitro by mammalian enzymes

The N-acetylation of arsanilic acid was assayed in vitro by modifying a lit erature method for acetylation of p-aminobenzoic acid. Conditions included final concentrations of 1.0 mM dithiothreitol, 1.0 mM EDTA, 0.45 mM acetyl coenzyme A, an acetyl coenzyme A regenerating system using bacterial phosph otransacetylase and acetyl phosphate, 5.0 mM arsanilate substrate, and 25 m M sodium/potassium phosphate buffer, pH 7.4, in a total volume of 0.5 ml. I ncubation was at 37 degrees C, with 0.5- to 2-mg N-acetyltransferase enzyme protein from a preparation of guinea pig liver. The reaction was terminate d by heat precipitation. The resulting supernatant was put through a 4 mm 0 .45 mu m polysulfone membrane syringe filter. The filtrate could then be in jected directly onto the HPLC. With arsanilic acid as substrate, the produc t N-acetylarsanilic acid (NAA) was identified by its retention time (33 min ) in the HPLC system of the laboratory. The 33-min fraction collected from the HPLC was scanned and gave the characteristic UV spectrum of NAA, with p eaks at 203 and 256 nm. In addition, the product comigrated in the HPLC sys tem with standard NAA. Under comparable assay conditions, the N-acetylation of arsanilate by the guinea pig enzyme preparation is about 24% the rate o f that of the model substrate p-aminobenzoic acid. Typical activity for ars anilate acetylation was 0.5 nmol/min/mg enzyme protein. Using the same assa y system and HPLC detection method, the supernatant from bacterial lysates containing recombinant human N-acetyltransferase 1 exhibited acetylation ac tivity toward arsanilate of 720 nmol/min/mg enzyme protein.