A New Strategy for As(V) Biosensing Based on the Inhibition of the Phosphatase Activity of the Arsenate Reductase from Thermus thermophilus

Abstract

Arsenic (As) pollution is a widespread problem worldwide. In recent years, biosensors based on enzymatic inhibition have been developed for arsenic detection, making the study of the effect of inhibitors on the selected enzymatic activity crucial for their setup. The arsenate reductase of Thermus thermophilus HB27, TtArsC, reduces As(V) into As(III), but is also endowed with phosphatase activity. This work investigates the inhibitory effects of As(V) and As(III) on phosphatase activity by taking advantage of a simple colorimetric assay; the results show that both of them are non-competitive inhibitors affecting the Vmax but not the K_M of the reaction. However, their Ki values are different from each other (15.2 ± 1.6 μM for As(V) and 394.4 ± 40.3 µm with As(III)), indicating a higher inhibitory effect by As(V). Moreover, the inhibition-based biosystem results to be selective for As(V) since several other metal ions and salts do not affect TtArsC phosphatase activity; it exhibits a sensitivity of 0.53 ± 0.03 mU/mg/μM and a limit of detection (LOD) of 0.28 ± 0.02 μM. The good sensitivity and specificity for As(V) point to consider inhibition of TtArsC phosphatase activity for the setup of a novel biosensor for the detection of As(V).

Keywords: Thermus thermophilus; arsenic; biosensor; thermostable arsenate reductase.

Figure 1

Graphical representation of Michaelis–Menten kinetics at different concentrations of (a) As(V) and (c) A(III) and their relatives Lineweaver–Burk double reciprocal plots in presence of (b) As(V) and (d) As(III).

Figure 2

(a) Dose-response curve of the inhibition of TtArsC phosphatase activity at increasing As(V) concentration was (reported in panel b). (b) TtArsC phosphatase activity assay in a multiwell plate at increasing concentrations of As(V).

Figure 3

(a) Retained activity of TtArsC in presence of 10, 50, and 100 μM of heavy metals; (b) Retained activity of TtArsC in presence of a fixed concentration of As(V) (10 μM) and increasing concentrations of other heavy metals (10, 50, and 100 μM). Heavy metals are reported as follows: As(V) in dark blue, As(III) in red, Cd(II) in green, Hg(II) in purple, Ni(II) in orange, Co(II) in black, and Cu(II) in brown. Statistical analysis was performed through the ordinary one-way ANOVA on GraphPad Prism 7.00; significant differences with respect to (a) TtArsC activity without metals and (b) to TtArsC activity in presence of As(V) (10 μM) are indicated as follows: * p < 0.05.

Figure 4

TtArsC phosphatase activity performed in ddH₂O, three drinkable waters (DW), and in the presence of As(V) 5 and 10 μM.