Zofia Maskos

The hydroxylation of tryptophan

Products of the chemical hydroxylation of tryptophan by Fenton and Udenfriend reactions are similar to those obtained by ionizing radiation. When tryptophan is exposed to either of these systems, a mixture of four hydroxytryptophans, oxindole-3-alanine, and N-formylkynurenine is formed. This observation indicates that the hydroxyl radical attacks the aromatic nucleus as well as the 2 and 3 positions of the pyrrole ring. During gamma-radiolysis of nitrous oxide-saturated tryptophan solution and in the absence of oxygen or ferric edta, the hydroxyl radical adduct (or hydroxycyclohexadienyl radical) of tryptophan undergoes dimerization and polymerization, which results in a yellow product with maximal absorbance at 425 nm. In the presence of ferric edta, or in a Fenton system, the hydroxyl radical adduct disproportionates, and hydroxylated derivatives are formed. The yields of the hydroxytryptophans are proportional to the concentration of ferric edta to a limiting yield of 54% of the theoretical yield, which is taken to be one hydroxylated product per two hydroxyl radicals. Under these conditions, 4-, 5-, 6-, and 7-hydroxy-derivatives of tryptophan are found in the proportion 4:2:2:3, respectively. The presence of dioxygen during gamma-radiolysis increases the yield of N-formylkynurenine, but does not affect the total yield of hydroxytryptophans. Similarly, tryptophan subjected to the Udenfriend reaction yields 4-, 5-, 6-, and 7-hydroxytryptophan and N-formylkynurenine in approximately equal amounts.

The hydroxylation of the salicylate anion by a fenton reaction and Γ-radiolysis: A consideration of the respective mechanisms

The yield of 2,3- and 2,5-dihydroxybenzoates (dHBs) from the reaction of .OH radicals with salicylate (SA) ions has been measured as a function of pH and in the presence of oxidants. Under steady-state radiolysis conditions, the production of these products occurs via the reactions .OH + SA----HO-SA. (radical adduct) HO-SA. H+.OH+----2-carboxyphenoxyl radical (SA.) + H2O HO-SA. + SA.----2,3-/2,5-dHB + SA The addition of the oxidants O2, Fe3+ edta, or Fe(CN)63- increases the relative yield of 2,5-dHB/2,3-dHB from about 0.2 to 1. A model to account for this effect is presented. Steady-state radiolyses of 3- and 4-hydroxybenzoate give dihydroxybenzoate products consistent with the phenol group being an ortho-para director in the electrophilic attack of the hydroxyl radical on the aromatic ring. A comparison of product distributions from the reaction of ferrous edta with hydrogen peroxide using salicylate as a scavenger strongly suggests that the same hydroxyl radical adducts are formed as in the radiation experiments.

Reactions of iron(II) nucleotide complexes with hydrogen peroxide

The rate constants for the reactions of hydrogen peroxide with ferrous complexes of ATP, ADP, UTP, citrate and pyrophosphate were measured at pH 7.2. These ligands are potential chelators of iron(II) in the low‐molecular weight iron pool that may catalyze oxidative degradation of tissues. The second‐order rate constants range from 5.5 × 103 M−1s−1 (UTP) to 1 × 105 M−1s−1 (pyrophosphate) at pH 7.2. The kinetic dependences of the ATP reaction are consistent with a mechanism involving a ‘ferryl’ (FeIV) transient which decays to the hydroxyl radical and ferric ATP.

Variant MoFe proteins of Azotobacter vinelandii: effects of carbon monoxide on electron paramagnetic resonance spectra generated during enzyme turnover.

The resting state of wild-type nitrogenase MoFe protein exhibits an S=3/2 electron paramagnetic resonance (EPR) signal originating from the FeMo cofactor, the enzyme’s active site. When nitrogenase turns over under CO, this signal disappears and one (sometimes two) of three new EPR signals, which also arise from the FeMo cofactor, appears, depending on the CO concentration. The appearance and properties of these CO-inducible EPR signals, which were also generated with variant MoFe proteins (αR96Q, αR96K, αQ191K, αR359K, αR96K/αR359K, αR277C, αR277H, and ΔnifV) that are impacted around the FeMo cofactor, have been investigated. No new CO-induced EPR signals arise from any variant, suggesting that no new CO-binding sites are produced by the substitutions. All variant proteins, except αR277H, produce the lo-CO signal; all, except αQ191K, produce the hi(5)-CO signal; but only two (αR96Q and ΔnifV) exhibit the hi-CO signal. FeMo cofactor’s environment clearly dictates which CO-induced EPR signals are generated; however, none of these EPR signals correlate with CO inhibition of H2 evolution observed with some of these variants. CO inhibition of H2 evolution is, therefore, due to CO binding to a different site(s) from those responsible for the CO-induced EPR signals. Some resting-state variants have overlapping S=3/2 EPR signals, whose intensities simultaneously decrease under turnover conditions, indicating that all FeMo cofactor conformations are catalytically active. Moreover, these variants produce a similar number of hi(5)-CO signals after turnover under CO to the number of resting-state S=3/2 signals. The FeMo cofactor associated with the hi(5)-CO signal likely contains two bridging CO molecules.

Oxyradicals and multivitamin tablets

Abstract Ingestion of a single multivitamin tablet leads to hydroxyl radical production equivalent to a radiation dose rate of 53 Gy/h.

Photo-lability of CO bound to Mo-nitrogenase from Azotobacter vinelandii

In the presence of CO and under turnover conditions, Mo-nitrogenase generates three different electron paramagnetic resonance (EPR) signals. One of the signals, lo-CO, is an S=1/2 signal and occurs under low CO concentrations. The other two signals, hi-CO (S=1/2) and hi(5)-CO (S=3/2) displace the lo-CO as the CO concentration is raised above 0.05 atm. Irradiation of hi-CO with visible light at 12 K converts it into lo-CO. Using a series of color filters, the corrected action spectrum is determined and shown to contain 2-3 broad maxima in the region 350-730 nm. The conversion of lo-CO back into hi-CO is accomplished by warming the sample to 77 K for 5 min. Using this temperature cycle, the rate constant for the re-association of CO with lo-CO to form hi-CO is determined in the range 12-90 K. From these data, the activation energy for this reaction is calculated to be 3.9 kJ/mol. Identical irradiation of either lo-CO or hi(5)-CO induces no spectral change, showing that both of these states are photo-stable. The photo-stability of hi(5)-CO demonstrates that it is structurally different from hi-CO.

The superoxide dismutase activities of two higher-valent manganese complexes, MnIV desferrioxamine and MnIII cyclam

A green manganese desferrioxamine complex is rapidly formed at room temperature upon stirring freshly precipitated manganese dioxide in a solution of the ligand. Spectral studies and low-temperature ESR indicate that this compound, which has been previously described as a manganese(III) complex, is better characterized as containing tetravalent manganese. The complex appears to form oligomers in solution. The extinction coefficient at 635 nm is 137 +/- 6 M-1 cm-1 (per manganese) at pH 7.8 and 88 +/- 4 M-1 s-1 at pH 6.6 after purification by chromatography. The superoxide dismutase activity was measured and compared to that of mononuclear manganese(III) 1,4,8,11-tetraazacyclodecane (cyclam). The catalytic rate constants for superoxide dismutase activity are 1.7 x 10(6) M-1 s-1 and 2.9 x 10(6) M-1 s-1 for the desferrioxamine and the cyclam complexes, respectively.

Role of the Filters in the Formation and Stabilization of Semiquinone Radicals Collected from Cigarette Smoke

The fractional pyrolysis of Bright tobacco was performed in nitrogen atmosphere over the temperature range of 240 - 510 °C in a specially constructed, high temperature flow reactor system. Electron paramagnetic resonance (EPR) spectroscopy was used to analyze the free radicals in the initially produced total particular matter (TPM) and in TPM after exposure to ambient air (aging). Different filters have been used to collect TPM from tobacco smoke: cellulosic, cellulose nitrate, cellulose acetate, nylon, Teflon and Cambridge. The collection of the primary radicals (measured immediately after collection of TPM on filters), the formation and stabilization of the secondary radicals (defined as radicals formed during aging of TPM samples on the filters) depend significantly on the material of the filter. A mechanistic explanation about different binding capability of the filters decreasing in the order: cellulosic < cellulose nitrate < cellulose acetate < nylon ~ teflon is presented. Different properties were observed for the Cambridge filter. Specific care must be taken using the filters for identification of radicals from tobacco smoke to avoid artifacts in each case.

Metabolism of 2-Nitrosofluorene and p-Nitrosophenol by Horse Liver Alcohol Dehydrogenase (HLADH)

Abstract Reductive metabolism of C-nitroso substrates to their corresponding hydroxyl amines is thought to be requisite to the activation of these compounds to mutagenic products. We have studied 2-nitrosofluorene (2-NOF) and p-nitrosophenol (p-NSP) as aldehyde substrate analogs for HLADH. The direct product of 2-NOF reduction is N-hydroxy-2-aminofluorene (N-OH-2-AF), which undergoes further reduction to 2-aminofluorene (2-AF) and rearrangement to 1- and 3-hydroxy-2-aminofluorene (1- and 3-OH-2-AF). The formation of these products is inhibited by pyrazole, indicating the involvement of active-site zinc ion in the role of a Lewis acid. It is suggested that the rearrangement reaction occurs via elimination of the hydroxyl group from the N-OH-2-AF and hydrolysis of the fluorenyl nitrenium-derived carbocations to yield the hydroxy 2-AF products. HLADH-dependent metabolism of p-NSP results in formation of p-aminophenol, which appears to proceed at pH above 6 via a spectral intermediate at ∽255 nm, but does not...