Modification of Amino Groups

Kieran F. Geoghegan1

1 Pfizer, Inc., Groton, Connecticut
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 15.2
DOI:  10.1002/0471140864.ps1502s04
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit describes group‐specific modifications of amino groups. These reactions remain valid tools for early‐stage evaluation of structure‐function relationships, but are now valued even more for their applications in the preparation of bioconjugates, affinity columns, biosensors, and tagged macromolecules. Protocols are provided here for reaction of amino groups with succinimidyl esters and isothiocyanates. These methods are broadly useful for the stable coupling to proteins of groups with useful, non‐native functional properties. These include biotin for detection or recovery, fluorescent groups for biophysics or cytochemistry, cross‐linking reagents for making bioconjugates, or metal‐chelators that permit proteins to be loaded with radioisotopes for medical imaging or antitumor therapy. These applications require accurate product characterization, which preferably is performed by mass spectrometry, as described in this unit as a support procedure. A protocol employing succinic or acetic anhydrides to change the charge state of protein amino groups is provided here, as is a procedure for reductive alkylation that leaves their charge unaltered but converts primary amines to secondary or tertiary amines.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Amidation using a Succinimidyl Ester
  • Basic Protocol 2: Addition of Fluorescein Isothiocyanate
  • Basic Protocol 3: Succinylation
  • Basic Protocol 4: Reductive Methylation
  • Support Protocol 1: Rapid Desalting of Protein Samples for Electrospray Mass Spectrometry
  • Commentary
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Amidation using a Succinimidyl Ester

  Materials
  • 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

  Materials
  • 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; Pharmacia Biotech), equilibrated in PBS ( appendix 2E)

Basic Protocol 3: Succinylation

  Materials
  • 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

  Materials
  • 1 to 10 mg protein/ml in 0.1 M sodium citrate, pH 6.0
  • 37% formaldehyde (Aldrich)
  • Sodium cyanoborohydride (NaBH 3CN, Aldrich; see CAUTION below)
  • 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)
CAUTION: Sodium cyanoborohydride is a highly toxic compound. Follow all precautions recommended in the Material Safety Data Sheet. Do not attempt to sniff the compound's odor, and be aware that exposing it to strong acid will result in the release of cyanide gas.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

  •   FigureFigure 15.2.1 General representation of the modification of a protein amino group by (A) a succinimidyl ester and (B) a sulfosuccinimidyl ester. Sulfosuccinimidyl esters are more water‐soluble than the succinimidyl esters of the same acids. The “tag” denoted in the figure represents the group added to the protein and may take many forms.
  •   FigureFigure 15.2.2 Modification of a protein amino group with fluorescein isothiocyanate.
  •   FigureFigure 15.2.3 The principal reactions of succinic anhydride with proteins. (A) Succinylation of an amino group, which is normally the major result of treating a protein with succinic anhydride. (B) Succinylation of tyrosine, the minor reaction with a phenolic group of tyrosine; this reaction may be reversed by treating the protein with hydroxylamine·HCl at pH 7. Ionizable groups are drawn in their neutral state (amino as NH2 and carboxyl as COOH).
  •   FigureFigure 15.2.4 Reductive methylation of a protein amino group.
  •   FigureFigure 15.2.5 Rapid reversed‐phase HPLC of a 61‐kDa human recombinant intracellular enzyme (purified to homogeneity before this fractionation). The protein peak indicated by the arrow was collected, dried, and successfully analyzed by electrospray mass spectrometry.

Videos

Literature Cited

Literature Cited
   Alouani, S., Gaertner, H.F., Mermod, J.J., Power, C.A., Bacon, K.B., Wells, T.N., and Proudfoot, A.E. 1995. A fluorescent interleukin‐8 receptor probe produced by targetted labelling at the amino terminus. Eur. J. Biochem. 227:328‐334.
   Atassi, M.Z. and Habeeb, A.F.S.A. 1972. Reaction of proteins with citraconic anhydride. Methods Enzymol. 25:546‐553.
   Banks, P.R. and Paquette, D.M. 1995. Comparison of three common amine‐reactive fluorescent probes used for conjugation to biomolecules by capillary zone electrophoresis. Bioconjugate Chem. 6:447‐458.
   Brinkley, M. 1993. A brief survey of methods for preparing protein conjugates with dyes, haptens, and cross‐linking reagents. In Perspectives in Bioconjugate Chemistry (C.F. Meares, ed.) pp. 59‐70. American Chemical Society, Washington, DC.
   Butler, P.J.G. and Hartley, B.S. 1972. Maleylation of amino groups. Methods Enzymol. 25:191‐199.
   Dixon, H.B.F. and Fields, R. 1972. Specific modification of NH2‐terminal residues by transamination. Methods Enzymol. 25:409‐419.
   Elzinga, M. and Alonzo, N. 1983. Analysis for methylated amino acids in proteins. Methods Enzymol. 91:8‐13.
   Feeney, R.E. 1987. Chemical modification of proteins: Comments and perspectives. Int. J. Pept. Protein Res. 29:145‐161.
   Fields, R. 1972. The rapid determination of amino groups with TNBS. Methods Enzymol. 25:464‐468.
   Geoghegan, K.F. and Stroh, J.G. 1992. Site‐directed conjugation of nonpeptide groups to peptides and proteins via periodate oxidation of a 2‐amino alcohol. Application to modification at N‐terminal serine. Bioconjugate Chem. 3:138‐146.
   Gaertner, H.F. and Offord, R.E. 1996. Site‐specific attachment of functionalized poly(ethylene glycol) to the amino terminus of proteins. Bioconjugate Chem. 7:38‐44.
   Geoghegan, K.F., Ybarra, D.M., and Feeney, R.E. 1979. Reversible reductive alkylation of amino groups in proteins. Biochemistry 18:5392‐5399.
   Geoghegan, K.F., Cabacungan, J.C., Dixon, H.B.F., and Feeney, R.E. 1981. Alternative reducing agents for reductive alkylation of amino groups in proteins. Int. J. Pept. Protein Res. 17:345‐352.
   Hirs, C.H.W. 1967. Reactions with reactive aryl halides. Methods Enzymol. 11:548‐555.
   Inman, J.K., Perham, R.N., DuBois, G.C., and Appella, E. 1983. Amidination. Methods Enzymol. 91:559‐569.
   Jentoft, N. and Dearborn, D.G. 1979. Labeling of proteins by reductive methylation using sodium cyanoborohydride. J. Biol. Chem. 254:4359‐4365.
   Jentoft, N. and Dearborn, D.G. 1983. Protein labeling by reductive alkylation. Methods Enzymol. 91:570‐579.
   Ji, T.H. 1983. Bifunctional reagents. Methods Enzymol. 91:580‐609.
   Klotz, I.M. 1967. Succinylation. Methods Enzymol. 11:576‐580.
   Means, G.E. and Feeney, R.E. 1971. Chemical Modification of Proteins. Holden‐Day, San Francisco.
   Scopes, R.K. 1994. Control of pH: Buffers. In Protein Purification: Principles and Practice, pp. 324‐333. Springer‐Verlag, New York.
   Stark, G.R. 1967. Modification of proteins with cyanate. Methods Enzymol. 11:590‐594.
   Tae, H.J. 1983. Bifunctional reagents. Methods Enzymol. 91:580‐609.
   Wetzel, R., Halualani, R., Stults, J.T., and Quan, C. 1990. A general method for highly selective cross‐linking of unprotected polypeptides via pH‐controlled modification of N‐terminal α‐amino groups. Bioconjugate Chem. 1:114‐122.
   Zhang, L. and Tam, J.P. 1996. Thiazolidine formation as a general and site‐specific conjugation method for synthetic peptides and proteins. Anal. Biochem. 233:87‐93.
Key References
   Brinkley, M. 1993. See above.
  Surveys current techniques for preparing protein conjugates.
   Jentoft, N. and Dearborn, D.G. 1983. See above.
  A comprehensive guide to reductive alkylation.
   Means, G.E. and Feeney, R.E. 1971. See above.
  A classic book that remains invaluable for direct advice and fundamental information on chemical modification of proteins.
   Tae, H.J. 1983. See above.
  Comprehensive coverage of modifying agents.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library