Fluorescein is a complex fluorophore in the sense that it displays four prototropic forms (cation, neutral, monoanion and dianion) in the pH range 1-9. In experiments with fluorescein-labeled proteins we have sometimes observed complex nanosecond emission kinetics, which could be due to conversion of the excited monoanion into the excited dianion through an excited state proton exchange with a proton acceptor in the labeled protein. However, the literature is ambiguous on whether this possible excited state proton reaction of fluorescein does occur in practice. In this article we describe a general steady-state fluorescence method for evaluating excited state proton reactions of simple as well as complex pH-sensitive fluorophores and apply it to evaluate excited state proton reactions of fluorescein. The method depends on finding a buffer that can serve as an excited state proton donor-acceptor but does not significantly perturb ground state proton equilibrium and especially does not form ground (or excited state complexes) with the fluorophore. Our results show that the excited monoanion-dianion proton reaction of fluorescein does occur in the presence of phosphate buffer, which serves as a proton donor-acceptor that does not significantly perturb ground state proton equilibria. The reaction becomes detectable at phosphate buffer concentrations greater than 20 mM and the reaction efficiency increases with increase in phosphate buffer concentrations. The reaction is most clearly demonstrated by adding phosphate buffer to a solution of fluorescein at constant pH 5.9 with preferential excitation of the monoanion. Under these conditions, the excited monoanion converts to the dianion during its lifetime. The conversion is detected experimentally as an increase in dianion and decrease in monoanion fluorescence intensities with increase in phosphate buffer concentration. The absorption spectrum is not significantly perturbed by the increase in phosphate buffer concentration. To quantitate the reaction, we have recorded titration graphs of fluorescence intensity versus pH for fluorescein solutions at low ( 5 mM) and high buffer (1 M) concentrations with preferential excitation of the monoanion and preferential detection of the dianion emission. We have also developed theoretical expressions that relate fluorescence intensity to pH in terms of the concentration of the four prototrophic forms of fluorescein, extinction coefficients, fluorescence efficiencies and ground and excited state p k . The theoretical expressions give very good fits to the experimental data and allow evaluation of fundamental parameters such as pK, and fluorescence efficiencies. The analysis of the experimental data shows that the excited monoanion-dianion reaction does not significantly occur at 5 mM phosphate buffer concentration. However, at 1 M buffer concentration the reaction is sufficiently fast that it practically achieves equilibrium during the lifetimes of the excited fluorescein monoanion and dianion. The p&* of the excited monoanion-dia...

The aim of the study was to evaluate the effect of melatonin supplementation on antioxidant capacity and DNA damage in high intensity interval training (HIIT) athletes. A 2-week randomised, double-blinded, placebo-controlled trial with two groups was conducted. Placebo (PG) and melatonin (MG) (20 mg/d) athletes were monitored over a two-week period of HIIT and strength training. The total antioxidant capacity (TAC) and the glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities were analysed in blood samples. DNA damage was measured in isolated lymphocytes by comet assay prior to and immediately after exercise. The supplementation increased plasma melatonin levels in the melatonin-treated group (p<0.05) after two weeks of intervention. Analysis of antioxidant status indicated higher (p<0.05) TAC and GPx in MG than PG post-intervention. No differences were found in SOD enzyme activity. DNA damage was diminished in MG (p<0.05) compared to PG in post-training conditions. Antioxidant status was associated with DNA damage (r=-0.679; p=0.047) in the melatonin-treated athletes. The present study suggest that melatonin supplementation improves antioxidant status and may prove to have beneficial effects preventing DNA damage induced by high intensity training.

We have made a comparative study of the dediazoniation of p-hydroxy and p-nitrobenzenediazonium ions. The electron-withdrawing and donating properties of the -NO2 and -OH groups strongly determine the reactivity of both compounds, thus exerting different influences upon the dediazoniation reaction. We describe here how the decomposition of p-hydroxy and p-nitrobenzenediazonium ions in a neutral aqueous medium follows a different pattern in the presence of the metal-chelator diethylenetriaminepentaacetic acid (DTPA). The decomposition rate of p-hydroxybenzene diazonium decreases whilst the decomposition of the p-nitrobenzenediazonium ion is enhanced. The experimental data are discussed with reference to a common scheme of interference for both benzenediazonium ions in the light of the radical-scavenging capacity of DTPA.Key words: p-hydroxybenzenediazonium ion, p-nitrobenzenediazonium ion, di-ethylenetriaminepentaacetic acid, dediazoniation, radical scavenging, artifacts.

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derstood [3]. The nitrosation of certain aromatics is nevertheless of considerable biomedical interest in view of the proven carcinogenic or mutagenic properties of the resulting products. This is the case of phenol and its derivatives [4-7]: the phenol derivative tyramine, which occurs in cheese, meat extract, beer, and soybean products [8-10] has been identified as one of the precursors largely responsible for the mutagenic activity of certain Japanese soy sauces treated with nitrite [11,12]; and bamethan [1-(4-hydroxyphenyl)-2-butylaminoethan-ol], a phenolic drug used for long-term oral treatment of cardiovascular disease, is both nitrosatable and a directly acting mutagen [13]. In view of their status as nitrosatable pre

The kinetics of the reactions between sodium nitrite and phenol or m‐, o‐, or p‐cresol in potassium hydrogen phthalate buffers of pH 2.5–5.7 were determined by integration of the monitored absorbance of the C‐nitroso reaction products. At pH > 3, the dominant reaction was C‐nitrosation through a mechanism that appears to consist of a diffusion‐controlled attack on the nitrosatable substrate by NO+/NO2H2+ ions followed by a slow proton transfer step; the latter step is supported by the observation of basic catalysis by the buffer which does not form alternative nitrosating agents as nitrosyl compounds. The catalytic coefficients of both anionic forms of the buffer have been determined. The observed order of substrate reactivities (o‐cresol ≈ m‐cresol > phenol ≫ p‐cresol) is explained by the hyperconjugative effect of the methyl group in o‐ and m‐cresol, and by its blocking the para position in p‐cresol. Analysis of a plot of ΔH# against ΔS# shows that the reaction with p‐cresol differs from those with o‐ and m‐cresol as regards the formation and decomposition of the transition state. The genotoxicity of nitrosatable phenols is compared with their reactivity with NO+/NO2H2+. © 1997 John Wiley & Sons, Inc.

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