Deaminase activity was determined for A3A protein by assaying active enzyme against linear DNA substrates and measuring the product formation using 1H NMR. Active A3A protein (50 nM) was assayed against linear DNA substrates (200 µM) in buffer with 50 mM MES pH 6.0, 100 mM NaCl, 0.5 mM TCEP, and 5% D2O. Experiments were performed on 9-mer substrates containing the target sequences AA(A/G/T)TC(A/G/T)AAA and at 40 °C to prevent the DNA from oligomerizing due to high concentration. Experiments were performed using a Bruker Avance III NMR spectrometer operating at a 1H Larmor frequency of 600 MHz and equipped with a cryogenic probe. Product concentration was estimated from peak integrals with Topspin 3.5 software (Bruker Biospin Corporation, Billerica, MA) using an external standard. Activity was determined from the initial rate of product formation via first-order exponential fitting of the progress curve. Rate errors were estimated by Monte Carlo simulation using 100 synthetic data sets and taking the residuals of the initial fit to the experimental data as the concentration error.