Chemical Research in Toxicology  2014  27  483-489:


l-Tryptophan Radical Cation Electron Spin Resonance Studies: Connecting Solution-Derived Hyperfine Coupling Constants with Protein Spectral Interpretations

Henry D. Connor, Bradley E. Sturgeon, Carolyn Mottley§, Herbert J. Sipe, Jr. and Ronald P. Mason Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, P.O. Box 12233, Research Triangle Park, North Carolina 27709 J. Am. Chem. Soc., 2008, 130, 6381-6387


Abstract Image  Fast-flow electron spin resonance (ESR) spectroscopy has been used to detect a free radical formed from the reaction of l-tryptophan with Ce4+ in an acidic aqueous environment. Computer simulations of the ESR spectra from l-tryptophan and several isotopically modified forms strongly support the conclusion that the l-tryptophan radical cation has been detected by ESR for the first time. The hyperfine coupling constants (HFCs) determined from the well-resolved isotropic ESR spectra support experimental and computational efforts to understand l-tryptophan's role in protein catalysis of oxidation-reduction processes. l-Tryptophan HFCs facilitated the simulation of fast-flow ESR spectra of free radicals from two related compounds, tryptamine and 3-methylindole. Analysis of these three compounds' β-methylene hydrogen HFC data along with equivalent l-tyrosine data has led to a new computational method that can distinguish between these two amino acid free radicals in proteins without dependence on isotope labeling, electron-nuclear double resonance, or high-field ESR. This approach also produces geometric parameters (dihedral angles for the β-methylene hydrogens) that should facilitate protein site assignment of observed l-tryptophan radicals as has been done for l-tyrosine radicals.


Fast-Flow EPR Spectroscopic Observation of the Isoniazid, Iproniazid, and Phenylhydrazine Hydrazyl Radicals by Herbert J. Sipe, Jr., Adrian R. Jaszewski, and Ronald P. Mason, Chemical Research in Toxicology, 2004, 17, 226-233.

Hydrazyl radical intermediates have been suggested as important intermediates in the biochemistry of hydrazides and hydrazines. Although spin-trapping studies have intercepted those species previously, there has been no report of the direct observation of the unstable hydrazyl radicals of isoniazid and iproniazid. We have employed the fast-flow technique in electron paramagnetic resonance (EPR) spectroscopy to measure spectra for the short-lived hydrazyl radicals of a family of hydrazides, including the pharmacologically important compounds isoniazid and iproniazid, as well as for a series of phenylhydrazines. Our investigations of the phenylhydrazine radical and the related chloro-substituted analogues have allowed definitive assignments of the hyperfine coupling constants of that toxicologically important free radical. Theoretical values of hyperfine coupling constants, calculated by density functional formalism, provided a guide to assignments for the hydrazyl species and confirmed the experimentally based assignments for phenylhydrazyl radical.


The Fate of the Oxidizing Tyrosyl Radical in the Presence of Glutathione and Ascorbate: IMPLICATIONS FOR THE RADICAL SINK HYPOTHESIS, Bradley E. Sturgeon, Herbert J. Sipe, Jr., David P. Barr, Jean T. Corbett, Jose´ G. Martinez, and Ronald P. Mason. Journal of Biological Chemistry, 1998, 273, 30116-30121.

Cellular systems contain as much as millimolar concentrations of both ascorbate and GSH, although the GSH concentration is often 10-fold that of ascorbate. It has been proposed that GSH and superoxide dismutase (SOD) act in a concerted effort to eliminate biologically generated radicals. The tyrosyl radical Tyrz) generated by horseradish peroxidase in the presence of hydrogen peroxide can react with GSH to form the glutathione thiyl radical (GSz). GSz can react with the glutathione anion (GS2) to form the disulfide radical anion (GSSG.). This highly reactive disulfide radical anion will reduce molecular oxygen, forming superoxide and glutathione disulfide (GSSG). In a concerted effort, SOD will catalyze the dismutation of superoxide, resulting in the elimination of the radical. The physiological relevance of this GSH/SOD concerted effort is questionable. In a tyrosyl radical-generating system containing ascorbate (100 mM) and GSH (8 mM), the ascorbate nearly eliminated oxygen consumption and diminished GSz formation. In the presence of ascorbate, the tyrosyl radical will oxidize ascorbate to form the ascorbate radical. When measuring the ascorbate radical directly using fast-flow electron spin resonance, only minor changes in the ascorbate radical electron spin resonance signal intensity occurred in the presence of GSH. These results indicate that in the presence of physiological concentrations of ascorbate and GSH, GSH is not involved in the detoxification pathway of oxidizing free radicals formed by peroxidases.


In Vitro Free Radical Metabolism of Phenolphthalein by Peroxidases. Herbert J. Sipe, Jr., Jean T. Corbett, Ronald P. Mason. Drug Metabolism and Disposition, 1997, 25, 468-480.

Phenolphthalein, a widely used laxative, is the active ingredient in more than a dozen commercial nonprescription formulations. Fast-flow EPR studies of the reaction of phenolphthalein with horseradish peroxidase (HRP) and hydrogen peroxide permit the direct detection of two free radicals. One has EPR parameters characteristic of phenoxyl radicals. The other has a broad unresolved spectrum, possibly arising from free radical polymeric products of the initial phenoxyl radical. EPR spin-trapping studies of incubations of phenolphthalein with lactoperoxidase, reduced glutathione (GSH), and hydrogen peroxide with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) demonstrate stimulated production of DMPO/lSG compared with an identical incubation lacking phenolphthalein. In the absence of DMPO, measurements with a Clarktype oxygen electrode show that molecular oxygen is consumed by a sequence of reactions initiated by the glutathione thiyl radical. Enhanced production of DMPO superoxide radical adduct is also found in a system of phenolphthalein, NADH, and lactoperoxidase. In this system the phenolphthalein phenoxyl radical abstracts hydrogen from NADH to generate NADl, which is not spin trapped by DMPO, but reacts with molecular oxygen to produce the superoxide radical detected by EPR. In the absence of DMPO, the oxygen consumption is measured using the Clark-type electrode. Production of ascorbate radical anion is also enhanced in a system of phenolphthalein, ascorbic acid, hydrogen peroxide, and lactoperoxidase. Ascorbate inhibits oxygen consumption when phenolphthalein is metabolized in the presence of either glutathione or NADH by reducing radical intermediates to their parent molecules and forming the relatively stable ascorbate anion radical. The detection of enhanced free radical production in these three systems, a consequence of futile metabolism (or redox cycling), suggests that phenolphthalein may be a significant source of oxidative stress in physiological systems. Parallel EPR and oxygen consumption studies with phenolphthalein glucuronide give analogous results, but with lesser enhancement of free radical production.