*3.4. Orthogonal Design*

The concentration of ATP, AMP, bromothymol blue, and glycine–NaOH buffer is vital for Adk activity assay. Consequently, these factors were considered in the orthogonal design to screen the most optimal conditions for Adk activity. Assuming there were three levels for each factor, and the interaction among the factors were not taken into account, the orthogonal experiment of four factors at three levels was designed.

#### **4. Discussion**

Adk plays a crucial role in maintaining a balance of cellular energy and nucleic acid metabolism. Human Adk isoenzymes specifically expressed in organs are regarded as important indicators of organ dysfunction [17] or differentiation stages [18]. The Adk activity assays that have been developed so far are largely dependent on the coupled secondary enzymes hexokinase and glucose-6-phosphate dehydrogenase or adenosine monophosphate deaminase [19]. In addition, the different conditions between the coupled enzymes and Adk affect the application of these assays [14]. Therefore, developing a convenient, rapid, sensitive, reliable, and economic assay for Adk activity is of great significance.

Here, we developed a spectrophotometric assay for Adk activity using bromothymol blue as a pH indicator without coupled enzymes. The effective range of bromothymol blue is pH 6.0–7.6. Adk is optimally active at pH 7.6 [14]. Hence, Adk was dissolved in pH 7.6 buffer to keep it active. The correspondence of pH and the absorbance of the system are listed in Table 5. Here, we set the initial absorbance of 1.05 as the beginning of the reaction, which represented pH 6.8 of the reaction system. When 5 μL buffer was added into the system (995 μL), there was almost no change in the pH of the system. At the same time, the assay system could ensure bromothymol blue had a sensitive and stable response to pH change caused by Adk-catalyzed reaction.

**Table 5.** Correspondence between pH and the absorbance of bromothymol blue at 614 nm.


The effects of ATP, AMP, bromothymol blue, and glycine–NaOH buffer on the Adk activity assay were determined by a rational orthogonal design. For a full factorial assay with four factors at three levels, the number of tests is up to 81 (34). However, our rational orthogonal design greatly reduced the unnecessary experiments and effectively achieved significant results (Table 2). The orthogonal experimental design is a rational design method for multifactor experiment, which selects representative points from a full factorial assay to represent the overall situation. Therefore, it is highly efficient for the design of multifactor experiments with optimal combination levels. By means of the orthogonal design, we greatly reduced the number of required experiments and achieved significant results with the least number of experiments.

The optimal temperature for the growth of *Bombyx mori* is about 25 ◦C. Adk from *Bombyx mori* was relatively stable below 45 ◦C, implying its significance on the growth and development of *Bombyx mori*. The Adk structure suggests that the hydrophobic core packing is important for the stability and activity of Adk [20]. AMP has a strong inhibition effect on the reaction with ADP as the substrate, thus resulting in a rapid decrease in the reaction rate with time [6,19]. Here, we found that high concentration of AMP could inhibit Adk activity, which is in line with a previous report. Slater et al. [21] found that the inhibitory effect of AMP was easily observed even when excessive hexokinase and glucose were present. The inhibition was ascribed to the affinity of ADP to Adk, which is lower than that of AMP with Adk. However, in this study, the substrates were ATP and AMP. Therefore, the inhibition of AMP on Adk activity may be attributed to the noncompetitive inhibition of AMP with respect to ATP [22].

To summarize, we developed a simple and rapid assay to determine Adk activity with bromothymol blue as an indicator instead of coupled enzymes. The assays that have been developed so far require the assistance of other enzymes to convert the reaction product to other detectable signals; thus, they consist of multiple reaction steps and are discontinuous. The assays are not accurate as they are easily subject to errors at each step. However, this new assay only relies on the protons produced in the reaction and the corresponding absorbance changes of bromothymol blue in the reaction solution. Small changes in pH can lead to significant changes in the absorbance of bromothymol blue at 614 nm. It does not need the assistance of any other enzyme. Bromothymol blue is less expensive than the coupled enzymes, and the assay can be done in just one step, thus reducing the chance of errors and improving reliability. Compared with the existing assays, it is more simple, sensitive, and precise. In addition, it can be applied to research the activation or inhibition of Adk as it is continuous. However, although this assay is simple and precise, the reaction substrate must be prepared freshly as carbon dioxide in the air may interfere with the assay. ATP spontaneously and slowly hydrolyzes in solution, which can also cause an interference on the assay. Nevertheless, it can be a good alternative to the conventional enzymes-coupled Adk activity assay extensively used in clinical and research laboratories.

**Author Contributions:** K.S., Y.W., Y.L., C.D., and H.H. conceived and designed the experiments; K.S. and Y.L. performed the experiments; K.S., Y.L., and Y.W. analyzed the data; R.C., T.G., and P.Z. contributed reagents/materials/analysis tools; K.S. and Y.W. wrote the draft; Y.W. and H.H. supervised the research; Q.X. and H.H. revised the manuscript.

**Funding:** This work was supported by the National Natural Science Foundation of China (31572465), the State Key Program of the National Natural Science of China (31530071), Fundamental Research Funds for the Central Universities (XDJK2018B010, XDJK2018C063), start-up grant from Southwest University (SWU112111), Graduate Research and Innovation Project of Chongqing (CYB17069), and the Open Project Program of Chongqing Engineering and Technology Research Center for Novel Silk Materials (silkgczx2016003).

**Conflicts of Interest:** The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.
