Enzymatic activity assays are essential and critical for the study of enzyme kinetics. Adenylate kinase (Adk) plays a fundamental role in cellular energy and nucleotide homeostasis. To date, assays based on different principles have been used for the determination of Adk activity. Here, we show a spectrophotometric analysis technique to determine Adk activity with bromothymol blue as a pH indicator. We analyzed the effects of substrates and the pH indicator on the assay using orthogonal design and then established the most optimal assay for Adk activity. Subsequently, we evaluated the thermostability of Adk and the inhibitory effect of KCl on Adk activity with this assay. Our results show that this assay is simple, rapid, and precise. It shows great potential as an alternative to the conventional Adk activity assay. Our results also suggest that orthogonal design is an effective approach, which is very suitable for the optimization of complex enzyme reaction conditions.
