Imidazole stabilizes NAD(P)H and improves reductase assays under challenging conditions

The reduced form of nicotinamide adenine dinucleotide (NAD(P)H) is prone to hydrolysis and ring-opening degradation under high-temperature and acidic conditions, resulting in a loss of reducing power and substantial inaccuracies in enzymatic assays. In this study, we systematically investigated the stabilizing effect of imidazole on NADH and NADPH under elevated temperature (30-60 °C) and acidic (pH 3-5) conditions, and further evaluated its applicability in reductase activity assays. Incubation experiments with varying imidazole concentrations (0-300 mM) demonstrated a pronounced concentration-dependent stabilization of NAD(P)H. Enzymatic assays revealed that imidazole enhanced the apparent activities of lactate dehydrogenase (LDH) and imine reductase (IRED) under both acidic and high-temperature conditions. Moreover, imidazole addition broadened the optimal pH and temperature range of these enzymes, markedly improving the accuracy and reproducibility of activity measurements. Collectively, these findings identify imidazole as an efficient stabilizing additive for NAD(P)H, enabling more reliable assessment of NAD(P)H-dependent reductase activities under high-temperature or acidic conditions.