Measurement of NAD(H) concentration using BCA dye and applications to assays of dehydrogenases

Dehydrogenases are a widespread enzyme family that play essential roles in all metabolic processes. We observe that one of the substrates of many dehydrogenases, the coenzyme NAD(H), reacts with copper in the bicinchoninic acid assay to produce a color change that can be measured spectroscopically. NADH reacts across a lower concentration range in this assay (<1 μM to ~50 μM) compared to NAD ⁺ (>100 μM), which allows the bicinchoninic acid (BCA) assay to be used to measure dehydrogenase activity, as demonstrated in assays with human malate dehydrogenase 1.