Heavy metal-induced inhibition of Aspergillus niger nitrate reductase: applications for rapid contaminant detection in aqueous samples

Enzyme inhibition assays have the potential to rapidly screen and identify heavy metals in environmental samples. Inhibition of nitrate reductase (NR) was examined as a method for detecting toxic metals. The activity of NR (EC 1.6.6.2) from Aspergillus niger was assayed as a function of metal concentration in the presence of Cd²⁺, Cr³⁺, Cr⁶⁺, Cu²⁺, Ni²⁺, Pb²⁺, and Zn²⁺. NR exhibited sensitivity to these metals at concentrations below 10 μM. Various buffers were screened for their ability to protect NR activity from metal inhibition, and 3-(N-morpholino) propanesulfonic acid (MOPS) was selected as the buffering system for the NR assays as it exhibited the least interference with metal inhibition, thus providing increased assay sensitivity. The hypothesis that chelating agents could prevent the inhibition of NR activity by metal ions was also tested. Results indicated that 10 mM ethylenediaminetetraacetic acid (EDTA) could protect NR activity from inhibition by Cr³⁺, Cu²⁺, Cd²⁺, Ni²⁺, and Zn²⁺ at concentrations below 100 μM, but that the EDTA had no effect on NR inhibition by Cr⁶⁺. An amount of 10 mM nitrilotriacetic acid (NTA) prevented NR inhibition by Cd²⁺, Cu²⁺, Ni²⁺, Pb²⁺, and Zn²⁺ at metal concentrations below 100 μM. However, 10 mM NTA was unable to protect the enzyme from inhibition by either Cr³⁺ or Cr⁶⁺. These results indicated that through specific metal chelation, a NR-based method for individually quantifying Cr³⁺ and Cr⁶⁺ species in aqueous solutions could be developed. The ability to restore activity to NR which been previously inhibited by exposure to 100 μM Pb²⁺, Cd²⁺, Zn²⁺, Cu²⁺, and Cr³⁺ was explored to determine whether NR activity could be recovered by EDTA additions for use in consecutive metal inhibition assays. The results showed NR activity could not be regained after exposure to Cr³⁺ or Cu²⁺, but did partially recover activity after Cd²⁺, Pb²⁺, and Zn²⁺ exposure.