2-4-dinitrophenylhydrazine and 3-nitrotyrosine

We review an array of newly developed in situ detection methods that can be used for the qualitative and semi-quantitative measurement of various indices related to oxidative stress. The importance of in situ methods over bulk analysis cannot be overstated when considering the structural and cellular complexity of tissue and the effects of diseases thereof. Indeed, in situ detection allows detection of specific cell types affected or specific localization such that a process affecting only a small fraction of the tissue or cells can be readily visualized. Consequently, a positive signal in situ indicates real levels that cannot be masked by unrelated or compensatory responses in adjacent cells, and corrections can be easily made for the modifications to long-lived proteins during physiological aging. In fact, the damage to extracellular matrix proteins of major vessels, provides a cumulative record of long-term oxidative insult. Yet the same properties that make vessels ideal markers for aging limits their sensitivity to detect disease-specific changes unless in situ techniques are used.

Topics: Alzheimer Disease; Biomarkers; DNA Damage; Glycation End Products, Advanced; Histocytochemistry; Humans; Immunohistochemistry; Metals; Oxidation-Reduction; Oxidative Stress; Phenylhydrazines; Tyrosine

The generation of reactive oxygen and nitrogen species is an inevitable result of cellular metabolism and environmental influence. Such oxidation processes are always combined with the formation of various protein oxidation products. Environmental oxidants might either be activated inside the cell or act by themselves. Therefore, differences in the localization of oxidant formation might change the major compartment of oxidant action. Therefore, we employed NO donors (SNOC, DETA/NO, and Spe/NO) alone or in combination with the redox-cycling bipyridinium compound paraquat, the superoxide- and NO-releasing compound SIN-1, the relatively more lipophilic oxidants tert-butyl and cumene hydroperoxide, and peroxynitrite itself to test the ability of these compounds to generate oxidized and nitrated proteins in various cellular compartments. Combined treatment with oxidants and nitrating compounds led to the formation of protein carbonyls and nitrotyrosine with a severalfold higher concentration in the cytosol, compared to the nucleus. In fluorescence microscopy studies, the resulting protein modifications show a similar distribution of oxidized proteins and nitrotyrosine with highest concentrations in the perinuclear area. Studying the time- and concentration-dependent formation and degradation of protein carbonyls and nitrated proteins large similarities could be measured. Therefore, it can be concluded that formation, localization, and kinetics of protein carbonyl and nitrotyrosine formation parallel each other depending on the stress-inducing agent.

Topics: Carbon; Cell Line, Tumor; Cytosol; Humans; Immunohistochemistry; Microscopy, Fluorescence; Nitric Oxide Donors; Nitrogen; Oxygen; Peroxynitrous Acid; Phenylhydrazines; Reactive Nitrogen Species; Reactive Oxygen Species; Tyrosine

Protein oxidation has been implicated in a variety of degenerative diseases as well as in the aging process. This unit describes techniques for the quantification of various protein oxidation products, including protein carbonyls, loss of protein thiol groups, dityrosine and nitrotyrosine, and isoaspartate formation. Such oxidatively modified products may also be used as biomarkers for the assessment of oxidative stress during aging and/or disease.

Topics: Biochemistry; Borohydrides; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Immunoblotting; Iodoacetamide; Isoaspartic Acid; Mass Spectrometry; Oxidation-Reduction; Phenylhydrazines; Protein Carbonylation; Proteins; Reference Standards; Spectrophotometry; Sulfhydryl Compounds; Tritium; Tyrosine

The regulation of free intracellular calcium [Ca2+]i is altered in neurons from the aged brain, possibly due to reductions in the activity of Ca2+ transporters. The plasma membrane Ca(2+)-ATPase (PMCA) plays a critical role in Ca2+ homeostasis, and its kinetic properties change in aged rat brain. These changes could be due to oxidative modification of PMCA as a result of age-related chronic oxidative stresses. The present studies were undertaken to determine the sensitivity of the neuronal PMCA to in vitro exposure of synaptic plasma membranes (SPMs) to reactive oxygen species (ROS). We examined the effects of three oxidants including peroxyl radicals generated by azo-initiators, 2,2'-Azobis 2-amidinopropane dihydrochloride (AAPH) and 4,4'-Azobis 14-cyanovaleric acid (ACVA), hydrogen peroxide (H2O2), and peroxynitrite (ONOO-). Synaptic plasma membranes briefly exposed to these oxidants were analyzed for functional and structural alterations in PMCA. Although all three oxidants led to significant loss of PMCA activity, the effect of ONOO- was the most potent, followed by peroxyl radicals and H2O2. Kinetic analysis of PMCA activity after oxidant treatment showed decreases in Vmax without significant changes in K(act). Immunoblots revealed oxidant-induced cross-linking of PMCA molecules that were partially reversed under reducing conditions and completely reversed with addition of urea. The PMCA appears to be very sensitive to inhibition by ROS and hence may be a target of oxidative stress in the aging brain. Reduction in its activity may contribute to age-related alterations in neuronal [Ca2+]i regulation.

Topics: Age Factors; Amidines; Animals; Azo Compounds; Brain; Calcium; Calcium-Transporting ATPases; Hydrogen Peroxide; Kinetics; Male; Nitrates; Oxidative Stress; Phenylhydrazines; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Synaptic Membranes; Tyrosine; Urea; Valerates

Proteins are targets of reactive species and detection of oxidatively modified proteins is often used as an index of oxidative stress. Peroxynitrite is a strong oxidant formed by reaction of nitric oxide with superoxide. Using fatty acid-free bovine serum albumin as a model we examined peroxynitrite-mediated protein modifications. The reaction of protein with peroxynitrite resulted in the oxidation of tryptophan and cysteine, in the nitration of tyrosine, in the formation of dityrosine, in the production of 2,4 dinitrophenylhydrazine-reactive carbonyls and in protein fragmentation. The formation of 3-nitrotyrosine represents a specific peroxynitrite-mediated protein modification that is different from modifications mediated by reactive oxygen species.

Topics: Animals; Cysteine; Electrophoresis, Polyacrylamide Gel; Fluorescence; Hydrogen-Ion Concentration; Male; Nitrates; Oxidation-Reduction; Oxidative Stress; Phenylhydrazines; Rats; Rats, Sprague-Dawley; Serum Albumin, Bovine; Tryptophan; Tyrosine