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Modification of Cysteine

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1121
  • Abstract
  • Table of Contents
  • Materials
  • Figures
  • Literature Cited

Abstract

 

This unit describes a number of methods for modifying cysteine residues of proteins and peptides by reduction and alkylation procedures. A general procedure for alkylation of cysteine residues in a protein of known size and composition with haloacyl reagents or N ?ethylmaleimide (NEM) is presented, and alternate protocols describe similar procedures for use when the size and composition are not known and when only very small amounts of protein are available. Alkylations that introduce amino groups using bromopropylamine and N ?(iodoethyl)?trifluoroacetamide are also presented. Two procedures that are often used for subsequent sequence analysis of the protein, alkylation with 4?vinylpyridine and acrylamide, are described, and a specialized procedure for 4?vinylpyridine alkylation of protein that has been adsorbed onto a sequencing membrane is also presented. Reversible modification of cysteine residues by way of sulfitolysis is described, and a protocol for oxidation with performic acid for amino acid compositional analysis is also provided. Gentle oxidation of cysteine residues to disulfides by exposure to air is detailed. Support protocols are included for recrystallization of iodoacetic acid, colorimetric detection of free sulfhydryls, and desalting of modified samples.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Alkylation of a Protein of Known Size and Composition with Haloacyl Reagents or N‐Ethylmaleimide
  • Alternate Protocol 1: Alkylation of a Protein of Unknown Size and Composition with Haloacyl Reagents or N‐Ethylmaleimide
  • Alternate Protocol 2: Alkylation of ≤50 µg Protein with Haloacyl Reagents or N‐Ethylmaleimide
  • Basic Protocol 2: Alkylation with 3‐Bromopropylamine
  • Basic Protocol 3: Alkylation with N‐(Iodoethyl)‐Trifluoroacetamide
  • Basic Protocol 4: Alkylation with 4‐Vinylpyridine
  • Basic Protocol 5: Alkylation with Acrylamide
  • Basic Protocol 6: Sulfitolysis
  • Basic Protocol 7: Oxidation with Performic Acid
  • Basic Protocol 8: Air Oxidation to a Disulfide
  • Basic Protocol 9: Alkylation of Cysteine in Protein Applied to Sequencer Membranes
  • Support Protocol 1: Recrystallization of Iodoacetic Acid
  • Support Protocol 2: Colorimetric Quantitation of Free Sulfhydryls
  • Support Protocol 3: Desalting the Sample after Cysteine Modification
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Alkylation of a Protein of Known Size and Composition with Haloacyl Reagents or N‐Ethylmaleimide

  Materials
  • Protein, lyophilized
  • Tris/guanidine buffer, pH 8.0 (see recipe ) or 0.1 M Tris·Cl, pH 8.0
  • 50 mM dithiothreitol (DTT; 7.7 mg/ml)
  • Nitrogen
  • Alkylating agent (see recipe )
  • 2‐mercaptoethanol (2‐ME; 14.4 M)
  • 0.5‐ to 2.5‐ml microcentrifuge tube
  • Additional reagents and equipment for desalting modified protein (see protocol 14 )

Alternate Protocol 1: Alkylation of a Protein of Unknown Size and Composition with Haloacyl Reagents or N‐Ethylmaleimide

  Materials
  • Protein, lyophilized
  • 20 mM and 2 M dithiothreitol (DTT) in 0.5 M Tris·Cl (pH 8.2) /6 M guanidine·HCl
  • 0.5 M 3‐bromopropylamine (see recipe )
  • Nitrogen
  • 0.5‐ to 2.5‐ml microcentrifuge tube
  • Additional reagents and equipment for desalting modified protein (see protocol 14 )

Alternate Protocol 2: Alkylation of ≤50 µg Protein with Haloacyl Reagents or N‐Ethylmaleimide

  Materials
  • Protein, lyophilized
  • 0.2 M N ‐ethylmorpholine acetate (pH 8.1)/6 M guanidine·HCl
  • 50 mM dithiothreitol (DTT; 7.7 mg/ml)
  • Nitrogen
  • Aminoethyl‐8 Reagent (Pierce Chemical)
  • Methanol
  • 2 N acetic acid
  • 0.5‐ to 2.5‐ml microcentrifuge tube
  • Additional reagents and equipment for desalting modified protein (see protocol 14 )

Basic Protocol 2: Alkylation with 3‐Bromopropylamine

  Materials
  • 1 M Tris·Cl (pH 8.5 )/4 mM EDTA
  • 8 M guanidine·HCl
  • Protein, lyophilized
  • 10% (v/v) 2‐mercaptoethanol (2‐ME)
  • Argon
  • 4‐vinylpyridine, fresh
  • 0.5‐ to 2.5‐ml microcentrifuge tube
  • Additional reagents and equipment for desalting modified protein (see protocol 14 )

Basic Protocol 3: Alkylation with N‐(Iodoethyl)‐Trifluoroacetamide

  Materials
  • Protein, lyophilized
  • 0.3 M Tris·Cl, pH 8.3
  • 50 mM dithiothreitol (DTT; 7.7 mg/ml; optional)
  • 6 M acrylamide
  • 10% (v/v) 2‐mercaptoethanol (2‐ME)
  • 0.5‐ to 1.5‐ml microcentrifuge tube
  • Additional reagents and equipment for desalting modified protein (see protocol 14 )
CAUTION: Acrylamide monomer is neurotoxic. A mask should be worn when weighing acrylamide powder. Gloves should be worn while handling the solution, and the solution should not be pipetted by mouth.

Basic Protocol 4: Alkylation with 4‐Vinylpyridine

  Materials
  • Protein, lyophilized
  • Sodium sulfite reagent buffer (see recipe )
  • 4 M urea/ 1 M Tris·Cl, pH 7.5
  • 2‐mercaptoethanol (2‐ME)
  • 0.5‐ to 2.5‐ml microcentrifuge tube
  • Additional reagents and equipment for desalting modified protein (see protocol 14 )

Basic Protocol 5: Alkylation with Acrylamide

  Materials
  • Formic acid
  • 30% hydrogen peroxide
  • Protein, lyophilized and chilled
  • 48% (v/v) hydrobromic acid
  • Ice bath

Basic Protocol 6: Sulfitolysis

  Materials
  • Protein or peptide, lyophilized
  • 0.1 M ammonium bicarbonate
  • Additional reagents and equipment for reversed‐phase HPLC (unit 11.6 ) and desalting oxidized protein (see protocol 14 )

Basic Protocol 7: Oxidation with Performic Acid

  Materials
  • Protein
  • Reduction/alkylation cocktail (see recipe )
  • Sequencer reagents (Perkin‐Elmer or equivalent)
  • Biobrene coated glass fiber disk, precycled
  • PE‐ABD sequencer, cartridge (Perkin‐Elmer) or equivalent

Basic Protocol 8: Air Oxidation to a Disulfide

  Materials
  • Iodoacetic acid (IAA)
  • Diethyl ether
  • Petroleum ether, ice cold
  • Buchner funnel
  • Whatman No. 1 filter paper or equivalent
  • Amber bottle with Teflon‐lined lid

Basic Protocol 9: Alkylation of Cysteine in Protein Applied to Sequencer Membranes

  Materials
  • 0.1 M sodium phosphate, pH 8.0 ( appendix 2E )
  • Reagent solution: 4 mg/ml Ellman reagent/ 0.1 M sodium phosphate, pH 8.0
  • Cysteine standard stock solution (see recipe )
  • Unknown sample
  • 13 × 100‐mm clear test tubes
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Figures

  •   Figure 15.1.1 Modification of cysteine. Interrelationship of the cysteine‐modifying reactions described in this unit. Abbreviations: BP, Basic Protocol; R, alkyl group.
    View Image

Videos

Literature Cited

Literature Cited
   Barkholt, V. and Jensen, A.L. 1989. Amino acid analysis: Determination of cysteine plus half‐cystine in proteins after hydrochloric acid hydrolysis with a disulfide compound as additive. Anal. Biochem. 177:318‐322.
   Brune, D.C. 1992. Alkylation of cysteine with acrylamide for protein sequence analysis. Anal. Biochem. 207:285‐290.
   Chan, W. 1968. A method for the complete S‐sulfonation of cysteine residues in proteins. Biochemistry. 12:4247‐4253.
   Chau, M.‐H. and Nelson, J.W. 1992. Cooperative disulfide bond formation in apamin. Biochemistry. 31:4445‐4450.
   Gerwin, B.I. 1967. Properties of the single sulfhydryl group of streptococcal proteinase. A comparison of the rates of alkylation by chloroacetic acid and chloroacetamide. J. Biol. Chem. 242:251.
   Hawke, D. and Yuan, P. 1987. S‐Pyridylethylation of cystine residues. Applied Biosystems User Bulletin Issue No. 28.
   Hempel, J., Nilsson, K., Larsson, K., and Jörnvall, H. 1986. Internal chain cleavage and product heterogeneity during Edman degradation of isosteric peptide analogs lacking the carbonyl function. FEBS Lett. 194:333‐337.
   Hirs, C.H.W. 1967. Performic acid oxidation. Methods Enzymol. 11:197‐199.
   Hoogerheide, J.G. and Campbell, C.M. 1992. Determination of cysteine plus half‐cystine in protein and peptide hydrolysates: Use of dithioglycolic acid and phenylisothiocyanate derivatization. Anal. Biochem. 201:146‐151.
   Jue, R.A. and Hale, J.E. 1993. Identification of cysteine residues alkylated with 3‐bromopropylamine by protein sequence analysis. Anal. Biochem. 210:39‐44.
   Jue, R.A. and Hale, J.E. 1994. Alkylation of cysteine residues with 3‐bromopropylamine: Identification by protein sequencing and quantitation by amino acid analysis. In Techniques in Protein Chemistry V (J.W. Crabb, ed.) pp. 179‐188. Academic Press, San Diego.
   Noiva, R. 1994. Enzymatic catalysis of disulfide formation. Protein Expression Purif. 5:1‐13.
   Schwartz, W.E., Smith, P.U., and Garfield, P.R. 1980. N‐(β‐iodoethyl) trifluoroacetamide: A new reagent for the aminoethylation of thiol groups in proteins. Anal. Biochem. 106:43‐48.
   Wynn, R. and Richards, F.M. 1995. Chemical modification of protein thiols: Formation of mixed disulfides. Methods Enzymol. 251:351‐356.
   Zhang, R. and Snyder, G.H. 1991. Factors governing selective formation of specific disulfides in synthetic variants of α‐Conotoxin. Biochemistry 30:11,343‐11,348.
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