Sigmaplot v12

Human ALDH1A1, ALDH1A2, ALDH1A3, ALDH2, ALDH1B1, ALDH3A1, ALDH4A1, ALDH5A1 and rat ALDH1L1 were prepared and purified as previously described. ^{27 (link), 63 (link)–66 (link)}Inhibition of ALDH activity by compounds and IC₅₀ curves were determined by measuring the formation of NAD(P)H spectrophotometrically at 340 nm (molar extinction coefficient of 6200 M⁻¹ cm⁻¹) on the Beckman DU-640 and Spectramax 340PC spectrophotometers using purified recombinant enzyme. Reaction components for ALDH1A and ALDH2 assays consisted of 100–200 nM enzyme, 200 μM NAD⁺, 100 μM propionaldehyde, and 1% DMSO in 25 mM BES buffer, pH 7.5. For ALDH1B1, the assay included 500 μM NAD⁺ and 200μM propionaldehyde. For ALDH3A1, the assay included 300μM NADP⁺, 20nM enzyme, and 300μM benzaldehyde. For ALDH4A1 and ALDH5A1, the assay included 1.5mM NAD⁺, 100nM enzyme, with 20mM propionaldehyde for ALDH4A1 and 2mM propionaldehyde for ALDH5A1. For rat ALDH1L1, the assay included 0.5mM NADP⁺, 200nM enzyme ad 4mM propionaldehyde. All assays were performed at 25°C and were initiated by addition of substrate after a 2 min incubation period. IC₅₀ curves were collected for compounds which substantially inhibited ALDH1A activity at 20 μM compound. Data were fit to the four parameter EC₅₀ equation using SigmaPlot (v12) and the values represent the mean/SEM of three independent experiments (each n=3).