Proteins pp 169-176 | Cite as

Reversible Dihydroxypropylation of Amino Groups in Proteins: Application in Primary Structural Studies of Streptococcal M-Proteins

  • Belur N. Manjula
  • Vincent A. Fischetti
  • Thomas Fairwell
  • A. Seetharama Acharya


M-protein of group A streptococcus is an immunologically diverse antiphagocytic determinant of the bacteria1. In order to better understand the structure-function relationships of the M-proteins, we undertook the determination of their primary structure. The Pep M5 protein, a biologically active, pepsin-derived N-terminal half of the type 5 M protein, was selected for the initial study2. Pep M5 protein contains 6 arginines, 35 lysines, and 47 glutamates, with no methionines and tryptophans, thus limiting the choice for obtaining large peptides for sequence studies to cleavage at its arginyl peptide bonds2. Though arginine specific clostripain appeared to be satisfactory initially, detailed studies of the peptides generated by clostripain digestion of Pep M5 protein revealed that in addition to the major arginine cleavages, digestion occurred at some of the lysine residues. Furthermore, clostripain failed to cleave some of the arginyl bonds quantitatively3. Thus, a better choice for obtaining arginyl peptides appeared to be to take advantage of the high specificity of tryptic cleavage after chemically modifying the e-amino groups of the Pep M5 protein4. The relatively high lysine content of M-protein makes it essential that the reagent used for the modification be very selective to the amino functions and also be capable of derivatizing the lysine residues of the protein completely.


Lysine Residue Tryptic Peptide Periodate Oxidation Complete Amino Acid Sequence Reductive Alkylation 
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  1. 1.
    R.C. Lancefield, Current Knowledge of Type-Specific M-Antigens of Group A Streptococci, J. Immunol. 89: 307 (1962).PubMedGoogle Scholar
  2. 2.
    B.N. Manjula and V.A. Fischetti, Studies on Group A Streptococcal M Proteins: Purification of Type 5 M Protein and Comparison of its Amino-Terminal Sequence with Two Immunologically Unrelated M Protein Molecules, J. Immunol. 124: 261 (1980).PubMedGoogle Scholar
  3. 3.
    B.N. Manjula, S.M. Mische, and V.A. Fischetti, Primary Structure of Streptococcal Pep M Protein: Absence of Extensive Sequence Repeats, Proc. Natl. Acad. Sci. USA 80: 5475 (1983).PubMedCrossRefGoogle Scholar
  4. 4.
    B.N. Manjula, A.S. Acharya, S.M. Mische, T. Fairwell, and V.A. Fischetti, The Complete Amino Acid Sequence of a Biologically Active 197-Residue Fragment of M Protein Isolated from Type 5 Group A Streptococci, J. Biol. Chem. 259: 3686 (1984).PubMedGoogle Scholar
  5. 5.
    A.S. Acharya and J.M. Manning, Amadori Rearrangement of Glyceraldehyde-Hemoglobin Schiff Base Adducts: A New Procedure for the Determination of Ketoamine Adducts in Proteins, J. Biol. Chem. 255: 7218 (1980).PubMedGoogle Scholar
  6. 6.
    A.S. Acharya, L.G, Sussman, and J.M. Manning, Schiff Base Adducts of Glyceraldehyde with Hemoglobin: Differences in the Amadori Rearrangement at the α-Amino Groups, J. Biol. Chem. 258: 2296 (1983).PubMedGoogle Scholar
  7. 7.
    A.S. Acharya, L.G. Sussman, and B. N. Manjula, Application of Reductive Dihydroxypropylation of Amino Groups of Proteins in Primary Structural Studies: Identification of Phenylthiohydantoin Derivative of ε- Dihydroxypropyllysine Residues by High-Performance Liquid Chromatography, J. Chromatogr. 297: 37 (1984).PubMedCrossRefGoogle Scholar
  8. 8.
    L.R. Lundblad and M.C. Noyes, Chemical Reagents for Protein Modification, Vol 1, CRC Press, Inc., Boca Raton, Florida.Google Scholar
  9. 9.
    K.F, Geoghegan, D.M. Ybarra, and R.E. Feeney, Reversibile Reductive Alkylation of Amino Groups in Protein, Biochemistry 18: 5392 (1979).PubMedCrossRefGoogle Scholar
  10. 10.
    G.E. Means and R.E. Feeney, Reductive Alkylation of Amino Groups in Proteins, Biochemistry 7: 2192 (1968).PubMedCrossRefGoogle Scholar
  11. 11.
    W.S.D. Wong, M.M. Kristjansson, D.T. Osuga, and R.E. Feeney, 1-Deoxyglycitolation of Protein Amino Groups and Their Regeneration by Periodate Oxidation, Int. J. Pen. Prot. Res. 26: 55 (1985).CrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1987

Authors and Affiliations

  • Belur N. Manjula
    • 1
  • Vincent A. Fischetti
    • 1
  • Thomas Fairwell
    • 2
  • A. Seetharama Acharya
    • 1
  1. 1.The Rockefeller UniversityNew YorkUSA
  2. 2.Molecular Disease BranchNational Heart, Lung, and Blood Institute, NIHBethesdaUSA

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