Modification of Cysteine
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- 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.
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
Materials
Basic Protocol 1: Alkylation of a Protein of Known Size and Composition with Haloacyl Reagents or N‐Ethylmaleimide
Materials
Alternate Protocol 1: Alkylation of a Protein of Unknown Size and Composition with Haloacyl Reagents or N‐Ethylmaleimide
Materials
Alternate Protocol 2: Alkylation of ≤50 µg Protein with Haloacyl Reagents or N‐Ethylmaleimide
Materials
Basic Protocol 2: Alkylation with 3‐Bromopropylamine
Materials
Basic Protocol 3: Alkylation with N‐(Iodoethyl)‐Trifluoroacetamide
Materials
Basic Protocol 4: Alkylation with 4‐Vinylpyridine
Materials
Basic Protocol 5: Alkylation with Acrylamide
Materials
Basic Protocol 6: Sulfitolysis
Materials
Basic Protocol 7: Oxidation with Performic Acid
Materials
Basic Protocol 8: Air Oxidation to a Disulfide
Materials
Basic Protocol 9: Alkylation of Cysteine in Protein Applied to Sequencer Membranes
Materials
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Figures
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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 | |
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Chan, W. 1968. A method for the complete S‐sulfonation of cysteine residues in proteins. Biochemistry. 12:4247‐4253. | |
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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. | |
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Wynn, R. and Richards, F.M. 1995. Chemical modification of protein thiols: Formation of mixed disulfides. Methods Enzymol. 251:351‐356. | |
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