Modification of Cysteine

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Abstract
Table of Contents
Materials
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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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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.

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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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Modification of Cysteine

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited