Sonia Poli

Oxidation of catechols and catecholamines by horseradish peroxidase and lactoperoxidase: ESR spin stabilization approach combined with optical methods

Abstract Oxidation of catechols and catecholamines by horseradish peroxidase (HRP) and lactoperoxidase (LPO) has been studied by electron spin resonance (ESR) and electronic spectroscopies. The ESR technique has been used as an ESR spin stabilization approach by complexation of o -semiquinone free radicals with Zn II ions. ESR spectra and parameters of these free radical complexed forms are in good agreement with those obtained previously for complexed and uncomplexed species. The K m values obtained with the two methods show stereoselective effects towards the chiral substrates l - and d -dopa from HRP and LPO. Furthermore, these enzymes display opposite stereochemical interactions, in agreement with the analogous effects observed on l - and d -tyrosine by electronic and NMR binding studies.

Use of Isotopes and LC-MS-ESI-TOF for Mechanistic Studies of Tienilic Acid Metabolic Activation

Tienilic Acid (TA) is a uricosuric drug marketed in 1978 and which caused a number of rare immunoallergic hepatitis. It was withdrawn in US in 1980, in France in 1992. Early batches of tienilic acid also contained 0.1–0.5% tienilic acid isomer. Tienilic acid isomer (TAI), has been shown to be metabolised by Cytochrome P450 into a reactive thiophene 1-oxide which either binds to proteins, or can be trapped by sulfur nucleophiles (Valadon et al. 1996). Tienilic acid is metabolized by human cytochrome P450 2C9 into 5-hydroxytienilic acid (a major metabolite representing 70% of the dose excreted in human urine) but it also forms (a) reactive metabolite(s) which binds covalently to CYP 2C9 and it is a mechanism based inhibitor of CYP 2C9. Adding glutathione to incubations decreases the covalent binding, but only to 1 mol/mol P450. However the reactive metabolite of tienilic acid is still unknown. Recently Koenigs et al. have shown using ESI-LC-MS that CYP 2C9 binds ≈ 2 mol of TA in absence of GSH and only one in presence of 3 mM GSH (Koenigs et al. 1999).

Purification, characterization and catalytic activity of anionic zucchini peroxidase

The isolation and purification, by preparative electrofocusing, of the major anionic (ZPOA) and cationic (ZPOC) isoenzymes, collected from young zucchini squash, are reported. The Mr and sugar content are similar to those found previously for the major isoenzymes from the ripe fruits and in the range commonly observed for plant peroxidases. The amount of the two cationic enzymes was very low compared with that of anionic ZPOA. The anionic enzyme has been characterized by electronic, circular dichroism, proton NMR and electron paramagnetic resonance spectroscopy. The spectra are qualitatively similar to those of the corresponding anionic horseradish peroxidase (HRPA) derivatives, with minor differences attributable to the particular protein environment around the heme. The kinetics of the enzymatic oxidation of a series of phenols by H2O2 have been studied. ZPOA shows a parallel behavior to HRPA, but it is systematically more active than HRPA, indicating that the zucchini enzymes have a marked tendency to carry out oxidation of this type of compounds.

Electron paramagnetic resonance studies on VO(IV)-D-aspartic acid and VO(IV)-D-aspartic acid α-benzylester complexes

A detailed electron paramagnetic study has been done on oxovanadium(IV)-D-aspartic acid and oxovanadium(IV)-D-aspartic acid α-benzylester complexes, molar ratio 1:1 and 1:2 in the physiological pH range. Their isotropic and anisotropic spin Hamiltonian parameters have been calculated to second order and the superimposed mixed species spectral patterns have been simulated. The complexes display an approximate C4v geometry whose relative hyperfine coupling constant values are connected with the ligand field strength on the equatorial plane. Furthermore it seems that aspartic acid, in some circumstances, binds oxovanadium(IV) acting as tridentate ligand, and aspartic acid α-benzylester can coordinate the metal-ion giving complexes with six-membered chelate rings (α-NH2/β-COO− donor groups). Our EPR results are in agreement with those of UV-visible and CD spectral measurements.