Modification of Amino Groups
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- Abstract
- Table of Contents
- Materials
- Figures
- Literature Cited
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.
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- 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
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Basic Protocol 1: Amidation using a Succinimidyl Ester
Materials
Basic Protocol 2: Addition of Fluorescein Isothiocyanate
Materials
Basic Protocol 3: Succinylation
Materials
Basic Protocol 4: Reductive Methylation
Materials
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Figure 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. View Image -
Figure 15.2.2 Modification of a protein amino group with fluorescein isothiocyanate. View Image -
Figure 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). View Image -
Figure 15.2.4 Reductive methylation of a protein amino group. View Image -
Figure 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. View Image
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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. |
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