the formation of peroxynitrite (ONOO ) in reaction with superoxide radical anion (O2 ), or NO2 and N2O3 (strong nitrosating agents) in the reaction with molecular oxygen. to understand the role and the fate of NO in biological system, wide range of diamine-based fluorogenic probes have been developed. Although, they are widely used, the exact mechanism of their action remains controversial. in literature, two mechanism of diamine probes transformation are described. First postulate formation of one-electron oxidation product, e.g. radical cation, in the reaction with NO2 followed by the formation of triazole derivative in the

recombination reaction with NO. Second, direct formation of triazole in reaction with N2O3. Here we present the study on the course of N-nitrosation of 2,3-diaminonaphtalene (DAN) and rhodamine spirolactam-based probe (RhPDA) in NO/O2 generating system. Fluorescence spectroscopy was used to study the N-nitrosation yield of DAN and RhPDA in the presence of PAPA NONOate ( NO donor) and SIN-1 (ONOO generator). in both cases conversion was observed. Moreover, in the presence of, PAPA NONOate and SIN-1 together, the yield of the conversion was greatly enhanced. the influence of azide anion, glutathione and hydrogen bicarbonate on the conversion of both probes was also studied. Our results suggest that both pathways play an important and different role in the mechanism of DAN and RhPDA nitrosation.

Radiolytic and Quantum Mechanical Study on the One-electron Oxidation Products of Hydroethidine Bartosz Michalowski1, Debski Dawid1, Jan Adamus1, Andrzej Marcinek1, and Adam Sikora1 1Institute of Applied Radiation Chemistry, Lodz, Poland, University of Technology, Lodz, Poland 2-hydroxyethidium (2-OH-E+), one of the products of hydroethidine (HE) oxidation, is an unique marker of superoxide radical anion (O2 ). Although, it can be used rather as qualitative than quantitative probe, it is still worthwhile to examine the mechanism of its oxidation, formation of 2-OH-E+ and factors affecting it. Here we present, the results of radiolytic and quantum mechanical (QM) study on the one-electron oxidation of HE. the hydroethidine radical cation (HE +) was characterized with the use of low-temperature glass technique. the reactivity of HE toward selected chloromethylperoxyl radicals was also studied. We showed that those radicals oxidize HE to its radical cation. Based on the relationship between determined second-order rate constant of HE/RO2 reaction and peroxyl radicals reduction potentials we were able to evaluate the rate constants of HE reaction with HO2 , 2k=(5±2)x106 M-1s-1,and O2

2k=(1.2±0.5)x104 M-1s-1, assuming that the HO2

is the main oxidizing species in superoxide generating system. the reactivity toward GSH derived radicals was also studied. Pulse radiolysis was used to show that glutathionyl radicals (GS ), oxidize HE to HE + with the rate constant at pH 7.4 of 2k=2.0x109M-1s-1. Moreover, high effect of glutathione on the formation of 2-OH-E+ in O2 generating system was observed. DFT quantum mechanical calculations were also performed to characterize the one-electron oxidation products of HE.

Thiyl Radicals and Post-Translational Modification Thomas Nauser1 and Willem H. Koppenol1 1ETH Zürich, Switzerland H and OH add to aromatic amino acids, but addition reactions by other radicals have not attracted much attention. in pulse radiolysis experminents, we have shown that thiyl radicals add intra-molecularly to phenylalanine in model peptides [1]. We now extended our investigations to the other aromatic amino acids which are more electron-rich, should therefore show a higher propensity to form such adducts and would be natural precursors to known posttranslational modifications. We observe inermediates by time-resolved UV-Vis spectroscopy. Histidine exhibits only negligible absorptivity down to 250 nm and therefore does not interfere with detection, so we chose to start our investigations here. . . . . For histidine, known examples of related posttranslational modifications include tyrosinase, hemocyanin and catechol oxidase. Controls showed early on, that the mechanism is general, not limited to oxidising radicals and relevant already at low, millimolar, concentrations of the aromatic substrate. [1] T. Nauser et al., Chem.Comm., 3400-3402, 2005.

Comparison of Peroxiredoxin Sensitivity towards Inactivation by Peroxide Substrates Kimberly J. Nelson1, Stacy T Knutson2, Derek Parsonage1, Jacquelyn S. Fetrow2, P. Andrew Karplus3, and Leslie B. Poole1 1Wake Forest School of Medicine, United States, 2Wake Forest University, United States, 3Oregon State University, United States Peroxiredoxin (Prxs) proteins are widely distributed, highly expressed, and highly efficient at the detoxification of hydrogen peroxide, organic peroxides, and peroxynitrite. Many Prx also are susceptible to being oxidatively inactivated by their peroxide substrates through formation of a sulfinic acid on the catalytic cysteine. Inactivation of to play roles in eukaryotic signaling by allowing hydrogen peroxide to locally build up and oxidize specific target proteins in response to growth factor and cytokine signaling pathway (the floodgate hypothesis). Prx inactivation is also likely to be involved in stress response by increasing the intracellular pool of reduced thioredoxin and some hyperoxided Prxs have been shown to exhibit a chaperone-like activity. the level of sensitivity to such hyperoxidation varies depending both on the enzyme involved and the type of peroxide substrate. Here we present three distinct approaches that can be used to obtain quantitative or semiquantitative estimates of Prx sensitivity and define a simple way of quantifying sensitivity (Chyp1%) that corresponds to the peroxide concentration at which 1% of the Prx molecules would be inactivated per turnover. This has allowed us to easily compare the degree of sensitivity of various Prx proteins and to identify structural features that influence the degree of sensitivity within the Prx1 (AhpC) subgroup.

C

NH NH

N

R1

OR2

+ R S

C

NHR2

NH

N

HS

R

R1

O

reaction 1

doi: 10.1016/j.freeradbiomed.2013.10.616

doi: 10.1016/j.freeradbiomed.2013.10.617

doi: 10.1016/j.freeradbiomed.2013.10.618

doi: 10.1016/j.freeradbiomed.2013.10.619