Elucidating the Electrochemical Mechanism of NG-Hydroxy-L-arginine

Abstract

N^G-Hydroxy-L-arginine (NOHA) is a stable intermediate product in the urea cycle that can be used to monitor the consumption of L-arginine by nitrous oxide synthase (NOS) to produce nitric oxide (NO) and L-citrulline. Research has implicated the urea cycle in many diseases and NO has cultivated interest as a potential biomarker for neural health. Electrochemical detection is an established, cost-effective method that can successfully detect low levels of analyte concentrations. As one of the few electrochemically active species in the urea cycle, NOHA shows promise as a biomarker for monitoring disruptions in this biochemical process. In this study, we show that NOHA has an oxidation peak at +355 mV vs Ag/AgCl at a glassy carbon electrode. In addition, cyclic voltammetry studies with structural analogs - alanine and N-hydroxyguanidine - allowed us to approximate the oxidation wave at +355 mV vs Ag/AgCl to be a one electron process. Diffusivity of NOHA was found using linear scan voltammetry with a rotating disk electrode and approximated at 5.50×10^-5 cm²/s. Ample work is still needed to make a robust biosensor, but the results here characterize the electrochemical activity and represent principle steps in making a NOHA biosensor.

Figure 1.

A NOHA serial dilution from 0.5 to 148 μM in PBS. (A,B) Oxidation peak of NOHA observed at +355mV vs Ag/AgCl and a reduction peak at +188 mV vs Ag/AgCl at (A) high concentrations and (B) low concentrations. (C,D) NOHA calibration curve for serial dilution from 0.5 to 148 μM in PBS. Both the (C) oxidation peak current at +355 mV vs Ag/AgCl and (D) reduction peak current at +188mV vs Ag/AgCl were calibrated in a linear and logarithmic fit respectively. Data was collected in triplicate and error bars represent standard deviation. Reproduced, in part, with permission from Ref (19). Copyright 2018, The Electrochemical Society.

Figure 2.

Cyclic voltammatograms of alanine from 0.5 to 142 μM revealed no faradaic electrochemical activity supporting that alanine is not electrochemically active.

Figure 3.

(A) Cyclic voltammatograms of N-hydroxyguanidine from 0.5 to 142 μM revealed an oxidation wave at +355 mV vs. Ag/AgCl and (B) a calibration curve of the oxidation current versus concentration of N-hydroxyguanidine.

Figure 4.

(A) Rotating disk electrode experiments were done from 900–4900 rpm at a constant concentration of 160 μM of NOHA in 7.3 pH PBS. (B) Calibration curve for RDE experiments from 900–4900 rpm comparing the limiting current with the square root of the angular velocity shows a linear relationship.

Figure 5.

(A) Cyclic voltammatograms of NOHA in an amino acid solution from 0 to 148 μM revealed an oxidation peak at +360 mV vs. Ag/AgCl. (B) A calibration curve of the oxidation current versus concentration of NOHA shows a linear relationship. The specificity of the oxidation of NOHA is 4.76 nA/uM in the presence of an amino acid solution.

Figure 6.

(A) Fast scan cyclic voltammatograms of NOHA in an amino acid solution from 50 nM to 150 μM revealed an oxidation peak at +250 mV vs. Ag/AgCl. (B) A calibration curve of the oxidation current versus concentration of NOHA shows a logarithmic relationship.

Scheme 1.

Oxidation of L-arginine to L-citrulline involving NOHA as a key intermediate.

Scheme 2.

Two possible electron oxidation products of NOHA.

Scheme 3.

Oxidation of alanine did not occur (top) but oxidation of N-hydroxyguanidine exhibited an oxidation event at 355 mV.