**2. Results**

In this study, Adk activity was determined by a direct and continuous spectrophotometric technique without coupled enzymes. In the enzymatic reaction catalyzed by Adk, the formation of two ADPs from ATP and AMP is accompanied by the generation of hydrogen ions. Bromothymol blue is an excellent acid–base indicator as it forms a highly conjugated structure while protonated in acid solution, resulting in an obvious color change from blue to yellow. The absorbance of bromothymol blue at 614 nm is associated with the hydrogen ion concentration in solution. Thus, Adk activity can be monitored in real time by the absorbance of bromothymol blue at 614 nm in solution, which can be detected by a sensitive spectrophotometer. The principle of this assay is illustrated in Figure 1.

**Figure 1.** The principle of the spectrophotometric assay for adenylate kinase (Adk) activity.

The effects of substrates and the pH indicator on the assay were analyzed using an orthogonal design, which is key to establishing the most optimal assay for Adk activity. The factors and levels affecting Adk activity assay are shown in Table 1.


**Table 1.** Factors and levels affecting Adk activity assay.
