Biologically Relevant Small Radicals

Biologically relevant small radicals are at the focus of the working group 4 (WG4) of the COST Action CM0603 (Free Radicals in Chemical Biology, CHEMBIORADICAL). This article surveys the areas of research being undertaken by the partners in WG4. The character of the radicals is described together with experimental techniques utilized to follow their structure and reactivity. Specifically, C-, S-, N- and O-centered radicals of small size, and their interaction with different biomolecules are described. Processes at the molecular level exemplifying important biological signaling and damaging pathways are introduced

Zymographic assay of plant diamine oxidase on entrapped peroxidase polyacrylamide gel electrophoresis. A study of stability to proteolysis

A zymographic assay of diamine oxidase (DAO, histaminase, EC 1.4.3.6), based on a coupled peroxidase reaction, and its behavior at proteolysis in simulated gastric and intestinal conditions, are described. The DAO activity from a vegetal extract of Lathyrus sativus seedlings was directly determined on sodium dodecyl sulfate polyacrylamide electrophoretic gels containing entrapped horseradish peroxidase, with putrescine as substrate of histaminase and ortho-phenylenediamine as co-substrate of peroxidase. The accumulation of azo-aniline, as peroxidase-catalyzed oxidation product, led to well-defined yellow-brown bands on gels, with intensities corresponding to the enzymatic activity of DAO. After image analysis of gels, a linear dependency of DAO content (Coomassie-stained protein bands) and of its enzymatic activity (zymographic bands) with the concentration of the vegetal extract was obtained. In simulated gastric conditions (pH 1.2, 37 °C), the DAO from the vegetal extract lost its enzymatic activity before 15 min of incubation, either in the presence or absence of pepsin. The protein pattern (Coomassie-stained) revealed that the DAO content from the vegetal extract was kept almost constant in the simulated intestinal fluid (containing pancreatin or not), with a slight diminution in the presence of pancreatic proteases. After 10 h of incubation at 37 °C, the DAO enzymatic activity from the vegetal extract was 44.7% in media without pancreatin and 13.6% in the presence of pancreatin, whereas the purified DAO retained only 4.65% of its initial enzymatic activity in the presence of pancreatin

Kinetics of the reactions of catechins with hypochlorite, peroxynitrite, and amino acid–derived peroxyl‐ radicals

Catechins, one of the class of flavonoids, are known as very efficient antioxidants. Here we investigated the kinetics of the reactions of three catechins, namely, catechin, epigallocatechin, and epigallocatechin gallate (EGCG) with some oxidants, which are formed in vivo under oxidative stress, hypochlorite, peroxynitrite, and amino acid peroxyl radicals. Stopped-flow spectrophotometry and pulse radiolysis technique with absorption detection were used to observe the formation of intermediate products of oxidized catechins. We found that catechins react with hypochlorite with the rate constant of the order of 10(5)-10(6) M-1 s(-1) at pH 7.4. Experimental kinetic traces of the reaction of EGCG with valine peroxyl radicals were fitted using chemical simulation, and the rate constant of this reaction was found to be 5 x 10(5) M-1 s(-1). The rate constants of the formation of unstable catechin quinones in the reaction with peroxynitrite were comparable to that of spontaneous peroxynitrite isomerization, which indicates that catechins are oxidized indirectly by peroxynitrite. Biological consequences of these reactions are discussed

Spectroscopic, Structural, and Functional Characterization of the Alternative Low-Spin State of Horse Heart Cytochrome c

The alternative low-spin states of Fe3+ and Fe2+ cytochrome c induced by SDS or AOT/hexane reverse micelles exhibited the heme group in a less rhombic symmetry and were characterized by electron paramagnetic resonance, UV-visible, CD, magnetic CD, fluorescence, and Raman resonance. Consistent with the replacement of Met80 by another strong field ligand at the sixth heme iron coordination position, Fe3+ ALSScytc exhibited 1-nm Soret band blue shift and ɛ enhancement accompanied by disappearance of the 695-nm charge transfer band. The Raman resonance, CD, and magnetic CD spectra of Fe3+ and Fe2+ ALSScytc exhibited significant changes suggestive of alterations in the heme iron microenvironment and conformation and should not be assigned to unfold because the Trp59 fluorescence remained quenched by the neighboring heme group. ALSScytc was obtained with His33 and His26 carboxyethoxylated horse cytochrome c and with tuna cytochrome c (His33 replaced by Asn) pointing out Lys79 as the probable heme iron ligand. Fe3+ ALSScytc retained the capacity to cleave tert-butylhydroperoxide and to be reduced by dithiothreitol and diphenylacetaldehyde but not by ascorbate. Compatible with a more open heme crevice, ALSScytc exhibited a redox potential ∼200 mV lower than the wild-type protein (+220 mV) and was more susceptible to the attack of free radicals