Enzymatic assay The usual in vitro
Enzymatic assay. The usual in vitro test for the measure of the activity of DbH involves ascorbate as a cosubstrate (e.g., 5mM) and tyramine as a substrate (e.g., 10mM). When using N-aryl-N′-hydroxyguanidines instead of ascorbate, the hydroxylase activity of DbH was measured by HPLC as the amounts of octopamine formed at end of reaction. An usual incubation mixture (final volume 50μL) contained tyramine (10mM), N-aryl-N′-hydroxyguanidine (4mM), and DbH. Incubations were performed in 50mM phosphate buffer for 8min at 28°C under stirring, and stopped by the addition 2μL ortho-phosphoric acid containing an internal reference (4-methoxybenzamide in 30% CH3CN). Samples were further diluted by the addition of phosphate buffer and injected onto a 250mm Agilent Lichrospher RP-18 column (pore size 5μm). The elution solvents were 5mM H3PO4 as eluant A and HPLC-grade CH3CN as eluant B. The gradient program was the following: 0–6min, isocratic 96% A; 6–16min linear gradient from 4% to 40% B; 16 to 30min, isocratic 40% B. Continuous flow rate was 1mL/min. Products were detected by measuring the BMS 195614 at 240nm. Retention time and peak area calibration for the amounts of octopamine formed in the incubations were determined by analyzing solutions of commercial octopamine under the same conditions.
Identification of the products formed during the incubation of N-(4-methoxyphenyl)-N′. Separation and detection of the metabolites of N-(4-methoxyphenyl)-N′-hydroxyguanidine 1 were performed on an Ultrasphere Beckman column (4.6×150mm, pore size 5μm). Elution solvents were (C) 1mM ammonium acetate buffered to pH 3.0 using formic acid and acetonitrile. The elution gradient was the following: 0–6min, isocratic at 96% C; 6–20min, linear gradient from 96% to 60% C, under a constant flow rate of 1mL/min. Products were detected by following the absorption at 240nm. Peaks were analyzed by electrospray ionization carried out at 5000V and fragments were analyzed at 20eV collision energy.
Discussion Our results demonstrate that N-aryl-N′-hydroxyguanidines constitute a new family of reducing cosubstrates for the copper-containing enzyme DbH and that N-(4-methoxyphenyl)-N′-hydroxyguanidine 1 can substitute for ascorbate in the hydroxylase activity assays. Moreover, experiments performed in the presence of modified N-hydroxyguanidines demonstrated that the whole function is absolutely required for their ability to act as cosubstrates of DbH. The first requirement is the presence of a free N–OH group, as a N–OCH3 group led to an inactive compound. This suggests that hydrogen-atom abstraction is necessary for activity. The iminoxyl radical resulting from the one-electron oxidation of an N-hydroxyguanidine is therefore probably involved during catalysis by DbH . A recent publication by Cho et al.  suggests that iminoxyl radicals are formed during oxidation of N-hydroxyguanidines by CuSO4, which is in agreement with our proposal. Moreover, the two other nitrogen atoms of the guanidine moiety are also required, which suggests they are involved in the recognition by the active site of DbH. The present study is the first one that uses N-aryl-N′-hydroxyguanidines in the presence of copper-containing enzymes, while these molecules have been widely used as substrates for iron-containing proteins over the past 15 years. Interest in these compounds arose from the demonstration by Stuehr et al.  and that N-hydroxy-l-arginine was an intermediate in the transformation of l-arginine into NO by NOS. The main discrepancy between the activities observed with DbH and that of iron-containing systems towards N-hydroxyguanidines lies in the fact that these molecules directly exchange electrons with DbH, as shown by their ability to substitute for reducer ascorbate, while iron-containing systems require the additional presence of a redox partner to oxidize these molecules , , . The cosubstrate ability of N-hydroxyguanidines was extended to that of another copper-containing enzyme that possesses numerous similarities with DbH, PHM . Using HPLC detection for measuring hydroxylation of substrate dansyl-TyrValGly by this enzyme, we observed that both N-(4-methoxyphenyl)-N′-hydroxyguanidine 1 and chlorophenyl derivative 6 could substitute for ascorbate as reducing cosubstrate for PHM (P. Slama, unpublished results).