*2.3. Effect of H<sup>+</sup> on the Absorbance of Bromothymol Blue*

To determine the sensitivity of bromothymol blue on the Adk activity assay, we measured the response of bromothymol blue to hydrogen ion under the most optimal condition, AB3C1D. The absorbance spectrum of bromothymol blue was scanned from 450 to 800 nm in the presence of various concentrations of HCl. The results showed that the absorbance of bromothymol blue at 614 nm gradually declined with the increase in hydrogen ion concentration (Figure 2b). The absorbance change can be linearly fitted as the function of hydrogen ion concentration with the following equation (Figure 2c):

$$\mathbf{y} = 0.138 \,\mathrm{\*}\,\mathrm{x} - 0.066$$

The results suggested that the absorbance of bromothymol blue at 614 nm had a good response to pH change in the assay, and the absorbance change was positively correlated with the hydrogen ion concentration.

**Figure 2.** The maximum absorption wavelength of the reaction mixture and its relationship with the hydrogen ion concentration. (**a**) The absorption spectra of nine different combinations in the presence of 5 mM MgAC2; (**b**) effect of hydrogen ion concentration on the absorption of the reaction system; (**c**) the correlation of the absorbance change of bromothymol blue at 614 nm with hydrogen ion concentration.

#### *2.4. Effect of Adk Contents on the Reaction Velocity*

The effect of Adk contents on the reaction velocity was determined as described in the Materials and Methods section. The results showed that, with the increase in Adk contents, the reaction velocity increased, and the reaction time required to reach equilibrium shortened (Figure 3a). In the first 5 s, the absorbance of bromothymol blue at 614 nm (Abs614) declined linearly with time; thus, the slope of the reaction in the first 5 s was defined as the initial reaction velocity. The plot in Figure 3b shows that the absorbance change of bromothymol blue at 614 nm could be linearly fitted as the function of Adk contents.

**Figure 3.** The effect of Adk contents on the assay. (**a**) Effect of different Adk contents on the assay; (**b**) the correlation of the absorbance change of bromothymol blue at 614 nm with different Adk contents.

#### *2.5. Effect of Temperature and KCl on Adk Activity*

The effect of temperature on Adk activity was investigated to characterize the thermostability of Adk. The results showed that Adk activity was almost unaffected under 45 ◦C. However, when the temperature was increased to 60 ◦C, Adk quickly lost its activity (Figure 4a). Adk from the muscle has a half-life of 30 min in 0.1 N hydrochloric acid at 100 ◦C [14]. Our results indicate that the thermostability of Adk from *Bombyx mori* (BmAdk) is much lower than that of Adk from the muscle.

Allan Hough et al. proved that KCl can almost completely inhibit myokinase activity [15]. Here, the effect of KCl on Adk activity was assessed with the assay. The results showed that low concentration of KCl (<5 mM) had a slight inhibitory effect on Adk activity. With the increase in KCl concentration, the inhibitory effect of KCl on Adk activity became more and more obvious. About 70 mM KCl resulted in 50% loss of Adk activity (Figure 4b). Compared with the "three-minute" method [15], the inhibitory effect of KCl on Adk activity could be assessed more easily with our developed assay.

**Figure 4.** The thermostability of Adk and the inhibition of KCl on Adk activity. (**a**) Effect of temperature on Adk activity; (**b**) effect of KCl concentration on Adk activity.

#### **3. Materials and Methods**

#### *3.1. Chemicals and Materials*

ATP and AMP were purchased from Aladdin (Shanghai, China) in the form of sodium salt. Magnesium acetate was from Sigma (St. Louis, MO, USA). Bromothymol blue sodium salt, glycine, and other reagents all came from Sangon Biotech Corp. (Shanghai, China). Plastic cuvettes were purchased from Centome Corp. (Chengdu, China).
