Fluorogenic tagging of protein 3-nitrotyrosine with 4-(aminomethyl)benzenesulfonate (ABS) in tissues: a useful alternative to immunohistochemistry for fluorescence microscopy imaging of protein nitration

Protein tyrosine nitration is a common biomarker of biological aging and diverse pathologies associated with the excessive formation of reactive oxygen and nitrogen species. Recently, we suggested a novel fluorogenic derivatization procedure for the detection of 3-nitrotyrosine (3-NT) using benzylamine derivatives to convert specifically protein or peptide bound 3-NT to a highly fluorescent benzoxazole product. In the current study, we applied this procedure to fluorogenic derivatization of protein 3-NT in sections from adult rat cerebellum in order to: (i) test this method in imaging nitrated proteins in fixed brain tissue sections, and (ii) compare the chemical approach to immunohistochemical labeling with anti-3-NT antibodies. Immunofluorescence analysis of cerebellar sections using anti-3-NT antibodies showed differential levels of immunostaining in the molecular, Purkinje, and granule cell layers of the cerebellar cortex; in agreement with previous reports, the Purkinje cells were most highly labeled. Importantly, fluorogenic derivatization reactions of cerebellar proteins with 4-(aminomethyl)benzenesulfonic acid (ABS) and K3Fe(CN)6 at pH 9, following sodium dithionite (SDT) reduction of 3-NT to 3-aminotyrosine (3-AT), showed a very similar pattern of relative intensity of cell labeling and improved resolution when compared with antibody labeling. Our data demonstrate that ABS-derivatization may be either a useful alternative or a complimentary approach to immunolabeling in imaging protein nitration in cells and tissues, including under conditions of dual labeling with antibodies to cell proteins, thus allowing for cellular co-localization of nitrated proteins and any protein of interest

Inactivation of rabbit muscle glycogen phosphorylase b by peroxynitrite revisited: does the nitration of Tyr613 in the allosteric inhibition site control enzymatic function?

There is increasing evidence that sequence-specific formation of 3-nitrotyrosine (3-NT) may cause functional changes in target proteins. Recently, the nitration of Tyr residues in glycogen phosphorylase b (Ph-b) was implicated in the age-associated decline of protein function (Sharov et al., Exp. Gerontol. 41, 407–416; 2006); in another report, the nitration of one specific residue, Tyr613, located in the allosteric inhibition site was hypothesized as a rationale for peroxynitrite inactivation (Dairou et al., J. Mol. Biol. 372, 1009–1021; 2007). In the present study, we have optimized the analysis of in-gel Ph-b digests by high performance liquid chromatography-electro spray ionization-tandem mass spectrometry, in order to achieve a quantitative analysis of nitration of individual Tyr residues at a high coverage of Tyr-containing sequences (92%). Our data do not confirm the role of Tyr613 nitration in the control of enzymatic function. Furthermore, we show here that the enzymatic activity of Ph-b does not directly correlate with the protein nitration levels, and that the modification of Cys and, potentially, other amino acid residues can better rationalize Ph-b inactivation by peroxynitrite

A Methodology for Simultaneous Fluorogenic Derivatization and Boronate Affinity Enrichment of 3-Nitrotyrosine Containing Peptides

We synthesized and characterized a new tagging reagent, (3R,4S)-1-(4-(aminomethyl)phenylsulfonyl)pyrrolidine-3,4-diol (APPD), for the selective fluorogenic derivatization of 3-nitrotyrosine (3-NT) residues in peptides (after reduction to 3-aminotyrosine) and affinity enrichment. The synthetic 3-NT-containing peptide, FSAY(3-NO2)LER, was employed as a model for method validation. Further, this derivatization protocol was successfully tested for analysis of 3-NT-containing proteins exposed to peroxynitrite in the total protein lysate of cultured C2C12 cells. The quantitation of 3-NT content in samples was achieved through either fluorescence spectrometry or boronate affinity chromatography with detection by specific fluorescence (excitation and emission wavelengths of 360 and 510 nm, respectively); the respective limits of detection were 95 and 68 nM (19 and 13 pmol total amount) of 3-NT. Importantly, the derivatized peptides show a strong retention on a synthetic boronate affinity column, containing sulfonamide-phenylboronic acid, under mild chromatographic conditions, affording a route to separate the derivatized peptides from large amounts (milligrams) of non-derivatized peptides, and to enrich them for fluorescent detection and MS identification. Tandem MS analysis identified chemical structures of peptide 3-NT fluorescent derivatives and revealed that the fluorescent derivatives undergo efficient backbone fragmentations, permitting sequence-specific identification of protein nitration at low concentrations of 3-NT in complex protein mixtures

Designing Formulation Strategies for Enhanced Stability of Therapeutic Peptides in Aqueous Solutions: A Review

Over the past few decades, there has been a tremendous increase in the utilization of therapeutic peptides. Therapeutic peptides are usually administered via the parenteral route, requiring an aqueous formulation. Unfortunately, peptides are often unstable in aqueous solutions, affecting stability and bioactivity. Although a stable and dry formulation for reconstitution might be designed, from a pharmaco-economic and practical convenience point of view, a peptide formulation in an aqueous liquid form is preferred. Designing formulation strategies that optimize peptide stability may improve bioavailability and increase therapeutic efficacy. This literature review provides an overview of various degradation pathways and formulation strategies to stabilize therapeutic peptides in aqueous solutions. First, we introduce the major peptide stability issues in liquid formulations and the degradation mechanisms. Then, we present a variety of known strategies to inhibit or slow down peptide degradation. Overall, the most practical approaches to peptide stabilization are pH optimization and selecting the appropriate type of buffer. Other practical strategies to reduce peptide degradation rates in solution are the application of co-solvency, air exclusion, viscosity enhancement, PEGylation, and using polyol excipients

Sarcoendoplasmic Reticulum Ca2+ ATPase. A Critical Target in Chlorine Inhalation–Induced Cardiotoxicity

Autopsy specimens from human victims or experimental animals that die due to acute chlorine gas exposure present features of cardiovascular pathology. We demonstrate acute chlorine inhalation–induced reduction in heart rate and oxygen saturation in rats. Chlorine inhalation elevated chlorine reactants, such as chlorotyrosine and chloramine, in blood plasma. Using heart tissue and primary cardiomyocytes, we demonstrated that acute high-concentration chlorine exposure in vivo (500 ppm for 30 min) caused decreased total ATP content and loss of sarcoendoplasmic reticulum calcium ATPase (SERCA) activity. Loss of SERCA activity was attributed to chlorination of tyrosine residues and oxidation of an important cysteine residue, cysteine-674, in SERCA, as demonstrated by immunoblots and mass spectrometry. Using cardiomyocytes, we found that chlorine-induced cell death and damage to SERCA could be decreased by thiocyanate, an important biological antioxidant, and by genetic SERCA2 overexpression. We also investigated a U.S. Food and Drug Administration–approved drug, ranolazine, used in treatment of cardiac diseases, and previously shown to stabilize SERCA in animal models of ischemia–reperfusion. Pretreatment with ranolazine or istaroxime, another SERCA activator, prevented chlorine-induced cardiomyocyte death. Further investigation of responsible mechanisms showed that ranolazine- and istaroxime-treated cells preserved mitochondrial membrane potential and ATP after chlorine exposure. Thus, these studies demonstrate a novel critical target for chlorine in the heart and identify potentially useful therapies to mitigate toxicity of acute chlorine exposure.This work was supported by the CounterACT Program, National Institutes of Health, Office of the Director, and the National Institute of Environmental Health Sciences grant U54 ES015678 (C.W.W.), and by Children’s Hospital of Colorado/Colorado School of Mines Pilot Award G0100394 and a Children’s Hospital of Colorado Research Institute’s Pilot Award (S.A.)