Modification of Amino Groups

Kieran F. Geoghegan1

1 Structural Biology and Biophysics, Pfizer Worldwide Research, Groton, Connecticut
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 15.2
DOI:  10.1002/cpps.17
Online Posting Date:  November, 2016
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Chemical modification of amino groups in proteins serves a diversity of preparative and analytical purposes. The most prominent is to attach nonpeptide groups with useful properties to proteins. Examples of these groups include biotin for affinity capture and fluorescent dyes for detectability. A widely applied chemistry, and one for which many reagents are available, is reaction of the activated ester of a carboxylic acid (often a succinimidyl ester) with amino groups at mildly basic pH. Reductive alkylation using a carbonyl compound and a hydride‐donating reducing agent is another valued reaction with multiple applications. Most proteins contain more than one amino group, so the extent of reaction desired must be considered in advance and the result assessed experimentally after the fact. The distinctive environment of the α‐amino group of a polypeptide sets it apart from the ϵ‐amino groups of lysine side chains, and can afford useful specificity. © 2016 by John Wiley & Sons, Inc.

Keywords: amino group; chemical modification; crystallization; reductive alkylation; succinimidyl ester

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Amidation Using a Succinimidyl Ester
  • Basic Protocol 2: Addition of Fluorescein Isothiocyanate to Amino Groups
  • Basic Protocol 3: Succinylation of Amino Groups
  • Basic Protocol 4: Reductive Methylation of Amino Groups
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Amidation Using a Succinimidyl Ester

  • 0.1 to 10 mM succinimidyl ester reagent in acetonitrile, dimethylformamide (DMF), or dimethylsulfoxide (DMSO); or 0.1 to 10 mM sulfosuccinimidyl ester reagent in H 2O
  • Protein solution: 0.1 to 2 mM protein in 0.1 M sodium bicarbonate, pH not adjusted
  • Additional reagents and equipment for gel‐filtration chromatography (unit 8.3) or dialysis (unit 4.4 and appendix 3B) and for mass spectrometry (see Chapter 16)

Basic Protocol 2: Addition of Fluorescein Isothiocyanate to Amino Groups

  • Protein, lyophilized or as a 5 to 20 mg/ml solution in 0.1 M sodium bicarbonate, pH 9.0
  • 0.1 M sodium bicarbonate, pH 9.0 (pH adjusted with sodium hydroxide)
  • Fluorescein isothiocyanate (FITC)
  • Dimethylformamide (DMF) or dimethylsulfoxide (DMSO)
  • 1.5 M hydroxylamine·HCl, pH 8.5, prepared fresh
  • Gel‐filtration column containing chromatography medium (e.g., PD‐10 column containing Sephadex G‐25; GE Healthcare Life Sciences) and equilibrated in PBS ( appendix 2E)

Basic Protocol 3: Succinylation of Amino Groups

  • Succinic anhydride
  • 1 to 10 mg protein/ml in 0.1 M sodium bicarbonate, pH 8.3
  • 1 M NaOH
  • 1 M hydroxylamine·HCl, pH 7.0
  • pH 6 to 10 dye‐indicator pH sticks
  • Gel‐filtration column containing chromatography medium (e.g., PD‐10 column containing Sephadex G‐25, Pharmacia Biotech)
  • Additional reagents and equipment for gel‐filtration chromatography (unit 8.3) or dialysis (unit 4.4 and appendix 3B) and for mass spectrometry (see Chapter 16) or isoelectric focusing (unit 10.2)

Basic Protocol 4: Reductive Methylation of Amino Groups

  • 1 to 10 mg protein/ml in 0.1 M sodium citrate, pH 6.0
  • 37% formaldehyde solution (Sigma‐Aldrich)
  • Dimethylamine borane (borane dimethylamine complex) (H(CH 3) 2 N:BH 3, Sigma‐Aldrich)
  • Methanol
  • Additional reagents and equipment for dialysis (unit 4.4 and appendix 3B) or gel‐filtration chromatography (unit 8.3) and for mass spectrometry (Chapter 16) or amino acid analysis (unit 3.2)
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Key References
  Koniev, O. and Wagner, A. 2015. See above.
  An excellent overview of chemical modification of proteins, and good on all aspects of amino group modification.
  The Molecular Probes Handbook—A Guide to Fluorescent Probes and Labeling, 11th ed. 2010. (ThermoFisher Scientific).
  Earlier editions of this commercially supported resource were authored by R. P. Haugland. It is a long‐standing and valuable resource for all aspects of chemical modification related to the incorporation of dyes, biotin, and other reporter groups into proteins and other biomolecules.
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