Measurement of Protein Sulfenic Acid Content

Leslie B. Poole1

1 Wake Forest University School of Medicine, Winston‐Salem, North Carolina
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 17.2
DOI:  10.1002/0471140856.tx1702s38
Online Posting Date:  November, 2008
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Abstract

Protein sulfenic acids are reactive, reversibly oxidized cysteinyl residues with roles in redox catalysis and regulation. Detection and quantification of these species in proteins is accomplished through chemical modification by reagents such as 7‐chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole (NBD chloride), 2‐nitro‐5‐thiobenzoate (TNB), dimedone, or derivatives of dimedone, followed by UV‐visible spectroscopy or mass spectrometric analysis. Curr. Protoc. Toxicol. 38:17.2.1‐17.2.27. © 2008 by John Wiley & Sons, Inc.

Keywords: sulfenic acids; cysteine modification; cysteine oxidation; UV‐visible spectroscopy; mass spectrometry

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Sulfenic Acid Trapping and Detection Using NBD Chloride
  • Alternate Protocol 1: Sulfenic Acid Trapping and Detection Using NBD Fluoride
  • Basic Protocol 2: Quantitation of Sulfenic Acid Formation by Reaction With TNB
  • Basic Protocol 3: Modification and Detection of Protein Sulfenic Acids with Dimedone
  • Basic Protocol 4: Modification and Detection of Protein Sulfenic Acids with Biotin‐Linked or Fluorophore‐Linked Dimedone Derivatives
  • Basic Protocol 5: Determination of the Protein Sulfenic Acid Dissociation Constant (pKa)
  • Support Protocol 1: Preparation of Sulfenic Acid–Containing Protein
  • Support Protocol 2: Functional Analyses of the Modified Protein
  • Support Protocol 3: Tryptic Digestion of Modified Proteins
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Sulfenic Acid Trapping and Detection Using NBD Chloride

  Materials
  • Putative sulfenic acid–containing protein, purified and in a neutral pH buffer (pH 6.5 to 7.5)
  • Neutral pH buffer: 25 mM potassium phosphate buffer, pH 7.0 (see appendix 2A) or equivalent/1 mM EDTA, or other pH 7 buffer
  • 100 mM NBD chloride (7‐chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole) in DMSO (see recipe)
  • Acetonitrile
  • Formic acid
  • Anaerobic quartz cuvette (if required) and regular quartz cuvette
  • Ultrafiltration unit of appropriate MWCO (e.g., Centricon, Millipore; Apollo, Orbital Biosciences; Vivaspin, Vivascience)
  • UV‐visible scanning or diode‐array spectrophotometer
  • Electrospray ionization mass spectrometer (ESI‐MS) or access to a fee‐for‐service facility
  • Software for comparing observed and predicted mass (e.g., Calculate pI/MW tool; http://www.expasy.ch)
  • Additional reagents and equipment for preparing proteins containing sulfenic acid ( protocol 7) and determining protein concentration by a non‐NBD‐affected assay (e.g., Detergent‐Compatible Protein Assay Kit, Bio‐Rad)

Alternate Protocol 1: Sulfenic Acid Trapping and Detection Using NBD Fluoride

  Materials
  • Putative sulfenic acid–containing protein, purified and in a neutral pH buffer (pH 6.5 to 7.5)
  • Neutral pH buffer: 25 mM potassium phosphate buffer, pH 7.0 to 7.5 (see appendix 2A)/1 mM EDTA, or other pH 7.0 to 7.5 buffer
  • 4 mM 2‐nitro‐5‐thiobenzoic acid (TNB) solution (see recipe)
  • 100 mM dithiothreitol (DTT; see recipe)
  • Anaerobic quartz cuvette (if required) and regular quartz cuvette
  • UV‐visible scanning or diode‐array spectrophotometer
  • Ultrafiltration unit of appropriate MWCO (e.g., Centricon, Millipore; Apollo, Orbital Biosciences; Vivaspin, Vivascience)
  • Additional reagents and equipment for preparing proteins containing sulfenic acid ( protocol 7), determining protein concentration by a non‐TNB‐affected assay (e.g., Detergent‐Compatible Protein Assay Kit, Bio‐Rad; optional), and performing functional assays ( protocol 8; optional)

Basic Protocol 2: Quantitation of Sulfenic Acid Formation by Reaction With TNB

  Materials
  • Putative sulfenic acid–containing protein sample, purified and in a neutral pH buffer (pH 6.5 to 7.5)
  • Neutral pH buffer: 25 mM potassium phosphate buffer, pH 7.0 (see appendix 2A)/1 mM EDTA, or other pH 7 buffer
  • 100 mM 5,5‐dimethyl‐1,3‐cyclohexanedione (dimedone): prepared by adding 14 mg dimedone (Sigma‐Aldrich) to 1 ml DMSO; store in aliquots up to several months at −20°C
  • Acetonitrile
  • Formic acid
  • Anaerobic cuvette
  • Ultrafiltration unit of appropriate MWCO (e.g., Centricon, Millipore; Apollo, Orbital Biosciences; Vivaspin, Vivascience)
  • Electrospray ionization mass spectrometer (ESI‐MS) or access to a fee‐for‐service facility
  • Software for comparing observed and predicted mass (e.g., Calculate pI/MW tool; http://www.expasy.ch)
  • Additional reagents and equipment for preparing proteins containing sulfenic acid ( protocol 7) and determining the identity of the labeled peptide ( protocol 9; optional)

Basic Protocol 3: Modification and Detection of Protein Sulfenic Acids with Dimedone

  Materials
  • Putative sulfenic acid–containing protein sample, purified and in a neutral pH buffer (pH 6.5 to 7.5)
  • Neutral pH buffer: 25 mM potassium phosphate buffer, pH 6.5 to 7.5 (see appendix 2A)/1 mM EDTA, or other pH 7 buffer
  • 250 mM DCP‐based affinity and fluorescent reagents (see recipe; see Fig. and Poole et al., for structures):
    • Biotinylated DCP‐linked probe (DCP‐Bio1, DCP‐Bio2, DCP‐Bio3)
    • Fluorescent‐labeled DCP‐linked probe (DCP‐FL1, DCP‐FL2, DCP‐Rho1, DCP‐Rho2)
  • 50 mM and 0.1 M ammonium bicarbonate buffer (not pH adjusted)
  • Acetonitrile
  • Formic acid
  • Acetone or trichloroacetic acid (optional)
  • Avidin‐conjugated (e.g., streptavidin, monoavidin, or neutravidin) Sepharose or agarose beads
  • Phosphate‐buffered saline ( appendix 2A)
  • SDS sample buffer (containing 2‐mercaptoethanol; e.g., see appendix 3F)
  • Anaerobic cuvette or pear‐shaped flask
  • Ultrafiltration unit of appropriate MWCO (e.g., Centricon, Millipore; Apollo, Orbital Biosciences; Vivaspin, Vivascience)
  • Electrospray ionization mass spectrometer (ESI‐MS) or access to a fee‐for‐service facility
  • PD‐10 columns (GE Healthcare) or Bio‐Gel P6 spin columns (Bio‐Rad), optional
  • Software for comparing observed and predicted mass (e.g., Calculate pI/MW tool; http://www.expasy.ch)
  • Gel documentation system with filters for fluorescein or rhodamine B
  • Additional reagents and materials for preparing proteins containing sulfenic acid ( protocol 7), determining the identity of the labeled peptide (optional; protocol 9), purifying proteins (Bollag and Edelstein, ), performing SDS‐PAGE ( appendix 3F or Bollag and Edelstein, ), and performing immunoblotting (see unit 2.3)

Basic Protocol 4: Modification and Detection of Protein Sulfenic Acids with Biotin‐Linked or Fluorophore‐Linked Dimedone Derivatives

  Materials
  • Citrate/phosphate buffer (pH from 3 through 7.6)/1 mM EDTA (see recipe)
  • Protein, purified and in 10 mM neutral pH buffer (e.g., 10 mM potassium phosphate buffer pH ∼7; see appendix 2A)
  • UV‐visible spectrophotometer or stopped‐flow spectrophotometer (recommended)
  • 3‐ to 5‐ml syringes
  • Additional reagents and equipment for preparing proteins containing sulfenic acid ( protocol 7) and determining the identity of the labeled peptide (optional; protocol 9)

Basic Protocol 5: Determination of the Protein Sulfenic Acid Dissociation Constant (pKa)

  Materials
  • Protein solution
  • Argon or oxygen‐free nitrogen (optional)
  • Neutral pH buffer: 25 mM potassium phosphate (pH 7.0)/1 mM EDTA (or other buffer at pH 7 or lower)
  • Peroxide of choice, e.g., 8 mM hydrogen peroxide (H 2O 2) or cumene hydroperoxide (see recipes)
  • 100 mM dithiothreitol (DTT; see recipe)
  • Anaerobic cuvette assembly (Williams et al., ) or small pear‐shaped flask with stopcock

Support Protocol 1: Preparation of Sulfenic Acid–Containing Protein

  Materials
  • Modified protein (see Basic Protocols protocol 11, protocol 32, protocol 43, or protocol 54) and unmodified protein (optional)
  • 50 mM N‐ethylmaleimide (NEM; optional; see recipe)
  • 10 M urea or 8 M guanidine hydrochloride (see recipes)
  • 100 mM Tris⋅Cl buffer, pH 8.0 ( appendix 2A) or 100 mM HEPES, pH 7.6 (see recipe)
  • 100 mM calcium chloride (1.11 g anhydrous CaCl 2 in 100 ml water; store up to several weeks at room temperature)
  • TPCK‐treated trypsin solution (see recipe)
  • Acetic acid
  • 10 mM EGTA
  • 100 mM DTT
  • 95° or 60°C water bath (optional)
  • HPLC equipped with a C18 reversed‐phase column, solvents for peptide isolation: e.g., 0.1% (v/v) trifluoroacetic acid, 70% (v/v) acetonitrile with 0.08% trifluoroacetic acid
  • Electrospray ionization mass spectrometer (ESI‐MS) or access to a fee‐for‐service facility
  • Additional reagents and equipment for performing MALDI‐TOF mass spectrometry (optional), SDS‐PAGE ( appendix 3F; optional), or Tris‐tricine‐PAGE ( appendix 3F; optional)
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Figures

Videos

Literature Cited

   Aboderin, A.A. and Boedefeld, E. 1976. Reaction of chicken egg white lysozyme with 7‐chloro‐4‐nitrobenz‐2‐oxa‐1,3‐diazole. II. Sites of modification. Biochim. Biophys. Acta 420:177‐186.
   Birkett, D.J., Price, N.C., Radda, G.K., and Salmon, A.G. 1970. The reactivity of SH groups with a fluorogenic reagent. FEBS Lett. 6:346‐348.
   Bollag, D.M. and Edelstein, S.J. 1991. Protein Methods. Wiley‐Liss, Inc., New York.
   Chae, H.Z., Uhm, T.B., and Rhee, S.G. 1994. Dimerization of thiol‐specific antioxidant and the essential role of cysteine 47. Proc. Natl. Acad. Sci. U.S.A. 91:7022‐7026.
   Claiborne, A., Yeh, J.I., Mallett, T.C., Luba, J., Crane, E.J. III, Charrier, V., and Parsonage, D. 1999. Protein‐sulfenic acids: Diverse roles for an unlikely player in enzyme catalysis and redox regulation. Biochemistry 38:15407‐15416.
   Claiborne, A., Mallett, T.C., Yeh, J.I., Luba, J., and Parsonage, D. 2001. Structural, redox, and mechanistic parameters for cysteine‐sulfenic acid function in catalysis and regulation. Adv. Prot. Chem. 58:215‐276.
   Ellis, H.R. and Poole, L.B. 1997a. Novel application of 7‐chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole to identify cysteine‐sulfenic acid in the AhpC component of alkyl hydroperoxide reductase. Biochemistry 36:15013‐15018.
   Ellis, H.R. and Poole, L.B. 1997b. Roles for the two cysteine residues of AhpC in catalysis of peroxide reduction by alkyl hydroperoxide reductase from Salmonella typhimurium. Biochemistry 36:13349‐13356.
   Fuangthong, M. and Helmann, J.D. 2002. The OhrR repressor senses organic hydroperoxides by reversible formation of a cysteine–sulfenic acid derivative. Proc. Natl. Acad. Sci. U.S.A. 99:6690‐6695.
   Ghosh, P.B. and Whitehouse, M.W. 1968. 7‐chloro‐4‐nitrobenz‐2‐oxa‐1,3‐diazole: A new fluorigenic reagent for amino acids and other amines. Biochem. J. 108:155‐156.
   Jeong, W., Cha, M.K., and Kim, I.H. 2000. Thioredoxin‐dependent hydroperoxide peroxidase activity of bacterioferritin comigratory protein (BCP) as a new member of the thiol‐specific antioxidant protein (TSA)/alkyl hydroperoxide peroxidase C (AhpC) family. J. Biol. Chem. 275:2924‐2930.
   Miki, M. 1985. Chemical modification of tropomysosin with NBD‐chloride. J. Biochem. (Tokyo) 97:1067‐1072.
   Poole, L.B. and Ellis, H.R. 2002. Identification of cysteine sulfenic acid in AhpC of alkyl hydroperoxide reductase. Meth. Enzymol. 348:122‐136.
   Poole, L.B., Zeng, B.B., Knaggs, S.A., Yakubu, M., and King, S.B. 2005. Synthesis of chemical probes to map sulfenic acid modifications on proteins. Bioconjug. Chem. 16:1624‐1628.
   Poole, L.B., Klomsiri, C., Knaggs, S.A., Furdui, C.M., Nelson, K.J., Thomas, M.J., Fetrow, J.S., Daniel, L.W., and King, S.B. 2007. Fluorescent and affinity‐based tools to detect cysteine sulfenic acid formation in proteins. Bioconjug. Chem. 18:2004‐2017.
   Riddles, P.W., Blakeley, R.L., and Zerner, B. 1979. Ellman's reagent: 5,5′‐dithiobis(2‐nitrobenzoic acid)—A reexamination. Anal. Biochem. 94:75‐81.
   Senior, A.E., Gros, P., and Urbatsch, I.L. 1998. Residues in P‐glycoprotein catalytic sites that react with the inhibitor 7‐chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole. Arch. Biochem. Biophys. 357:121‐125.
   Tripolt, R., Belaj, F., and Nachbaur, E. 1993. Unexpectedly stable sulfenic acid: 4,6‐Dimethoxy‐1,3,5‐triazine‐2‐sulfenic acid; synthesis, properties, molecular and crystal structure. Z. Naturforsch. 48B:1212‐1222.
   Vila, A., Tallman, K.A., Jacobs, A.T., Liebler, D.C., Porter, N.A., and Marnett, L.J. 2008. Identification of protein targets of 4‐hydroxynonenal using click chemistry for ex vivo biotinylation of azido and alkynyl derivatives. Chem. Res. Toxicol. 21:432‐444.
   Williams, C.H. Jr., Arscott, L.D., Matthews, R.G., Thorpe, C., and Wilkinson, K.D. 1979. Methodology employed for anaerobic spectrophotometric titrations and for computer‐assisted data analysis. Meth. Enzymol. 62:185‐198.
Key References
   Ellis and Poole, 1997a. See above.
  Describes, for the first time, the use of the NBD chloride method for identification of the cysteine sulfenic acid of the C165S mutant of AhpC.
   Poole and Ellis, 2002. See above.
  Gives practical details about the use of several protocols, including NBD chloride, TNB, and dimedone‐based methods for identification of sulfenic acids.
   Willett, W.S. and Copley, S.D. 1996. Identification and localization of a stable sulfenic acid in peroxide‐treated tetrachlorohydroquinone dehalogenase using electrospray mass spectrometry. Chem. Biol. 3:851‐857.
  Describes the use of dimedone modification and LC/MS/MS to identify and localize sulfenic acids within proteins.
   Poole et al., 2007. See above
  Describes the synthesis and use of seven fluorescent or affinity‐tagged reagents based on the 1,3‐cyclohexanedione core of dimedone that can be used for detection and identification of sites of sulfenic acid modification on proteins.
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