Detection of Nitrosated Proteins

Ying‐Yi Zhang1, Joseph Loscalzo1

1 Boston University School of Medicine, Boston, Massachusetts
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 10.3
DOI:  10.1002/0471140856.tx1003s00
Online Posting Date:  May, 2001
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Abstract

Three methods are provided in this unit to detect nitrosated proteins. The Saville assay is the most commonly used and has the advantages that it is less sensitive to interference than the quinine sulfate‐based fluorescent assay, uses simple chemical reagents, and requires a spectrophotometer. The diaminonaphthalene assay (a fluorescence assay) and chemiluminescence assay are used when the experiment requires high sensitivity. Mass spectrometry is employed for precise structural determinations.

     
 
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Table of Contents

  • Basic Protocol 1: Saville Assay to Detect Nitrosated Proteins
  • Alternate Protocol 1: Microtiter‐Plate Saville Assay to Detect Nitrosated Proteins
  • Basic Protocol 2: Spectrophotometric Assay to Detect Nitrosated Proteins
  • Basic Protocol 3: Quinine Fluorescence Quenching Assay to Detect Nitrosated Proteins
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Saville Assay to Detect Nitrosated Proteins

  Materials
  • Sample to be tested (nitroso compounds, nitrosated proteins, cell homogenates)
  • 100 µM NaNO 2 (freshly prepared)
  • 0.5% (w/v) ammonium sulfamate (ASM; Aldrich) in water (store at room temperature; stable 1 year or more)
  • 2 N HCl
  • Hg/sulfanilamide (Hg/SN) solution (see recipe)
  • N‐1‐(Naphthyl)ethylenediamine dihydrochloride (NEDD) solution (see recipe)

Alternate Protocol 1: Microtiter‐Plate Saville Assay to Detect Nitrosated Proteins

  • 40 µM NaNO 2 (freshly prepared)
  • Hg/SN/NEDD solution (see recipe)
  • 96‐well microtiter plate
  • Microtiter plate reader

Basic Protocol 2: Spectrophotometric Assay to Detect Nitrosated Proteins

  Materials
  • Appropriate reference solution (see step annotation)
  • Sample to be tested

Basic Protocol 3: Quinine Fluorescence Quenching Assay to Detect Nitrosated Proteins

  Materials
  • Sample to be tested
  • 100 µM S‐nitrosoglutathione (GSNO) solution (see recipe)
  • 10 µM quinine sulfate
  • 0.1 M H 2SO 4
  • Spectrofluorometer
  • Cuvettes or microcuvettes
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Figures

Videos

Literature Cited

Literature Cited
   Alpert, C., Ramdev, N., George, D., and Loscalzo, J. 1997. Detection of S‐nitrosothiols and other nitric oxide derivatives by photolysis‐chemiluminescence spectrometry. Anal. Biochem. 245:1‐7.
   Conboy, J.J. and Hotchkiss, J.H. 1989 Photolytic interface for high‐performance liquid chromatography–chemiluminescence detection of non‐volatile N‐nitroso compounds. Analyst. 114:155‐159
   Cook, J.A., Kim, S.Y., Teague, D., Krishna, M.C., Pacelli, R., Mitchell, J.B., Vodovotz, Y., Nims, R.W., Christodoulou, D., Miles, A.M., Grisham, M.B., and Wink, D.A. 1996. Convenient colorimetric and fluorometric assays for S‐nitrosothiols. Anal. Biochem. 238:150‐158.
   Downes, M.J., Edwards, M.W., Elsey, T.S., and Walters, C.L. 1976. Determination of a non‐volatile nitrosamine by using denitrosation and a chemiluminescence analyser. Analyst 101:742‐748.
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   Stamler, J.S., Jaraki, O., Osborne, J., Simon, D.I., Keaney, J., Vita, J., Singel, D., Valeri, C.R., and Loscalzo, J. 1992. Nitric oxide circulates in mammalian plasma primarily as an S‐nitroso adduct of serum albumin. Proc. Natl. Acad. Sci. U.S.A. 89:7674‐7677.
   Upchurch, G.R., Welch, G.N., and Loscalzo, J. 1996. S‐nitrosothiols: Chemistry, biochemistry, and biologic actions. Adv. Pharmacol. 34:343‐349.
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   Zhang, Y.‐Y., Xu, A.‐M., Nomen, M., Walsh, M., Keaney, J.F. Jr., and Loscalzo, J. 1996. Nitrosation of tryptophan residues(s) in serum albumin and model dipeptides: Biochemical characterization and bioactivity. J. Biol. Chem. 271:14271‐14279.
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