Electrochemical Detection of N^G-Hydroxy-L-Arginine

Abstract

NG-hydroxy-L-arginine (NOHA) is a stable intermediate product in the consumption of L-arginine in the urea cycle by nitric oxide synthase (NOS) to produce nitric oxide (NO) and L-citrulline. Research has shown that the urea cycle is disrupted in various diseases. As one of the few electrochemically active species in the urea cycle, NOHA shows promise as a marker for detection of various diseases. Electrochemical detection is an established, cost-effective method that is able to successfully detect low levels of analyte concentrations. NOHA, to the best of our knowledge, has not been electrochemically detected previously. Using cyclic voltammetry with a glassy carbon electrode, we have found that NOHA has an oxidation peak at 355 mV with a sensitivity of 5.4 nA/μM. We also investigated detecting NOHA with differential pulse voltammetry, which shows similar sensitivity and oxidation peaks. While there is significant work ahead to understand the kinetics of NOHA detection, the results here represent the first steps in making a NOHA biosensor.

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“…The substrate of these enzymes is the amino acid l-arginine (L-arg), and the reaction requires molecular O 2 , flavin adenine dinucleotide, flavin mononucleotide, Ca 2+ , calmodulin, (6R)-5,6,7,8-tetrahydro-L-biopterin (BH 4 ) and NAD(P)H as cofactors [10] (Figure 1). The conversion of L-arg to l-citrulline with the production of NO takes place in two steps: the first step is the hydroxylation of l-arginine to N G -hydroxy-l-arginine, which is an intermediate metabolite [11], and the second step involves an electron oxidation of the N G -hydroxy-l-arginine that binds to two moles of O 2 and 1.5 mol of reducing equivalent, NAD(P)H, resulting in the formation of a mole of NO. Both steps are catalyzed by the heme iron complex present in the enzyme [9].…”

“…The O 2 − required may come from the electron transport chain in the mitochondria, from peroxisomes, NAD(P)H oxidases, xanthine oxidases (ORX), cytochrome p450 and/or the cyclooxygenase (COX) pathways [12]. [11], and the second step involves an electron oxidation of the N G -hydroxy-L-arginine that binds to two moles of O2 and 1.5 mol of reducing equivalent, NAD(P)H, resulting in the formation of a mole of NO. Both steps are catalyzed by the heme iron complex present in the enzyme [9].…”