Reversible Dihydroxypropylation of Amino Groups in Proteins: Application in Primary Structural Studies of Streptococcal M-Proteins
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.
KeywordsAdduct Lysine Ketone Arginine Trypsin
Unable to display preview. Download preview PDF.
- 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.L.R. Lundblad and M.C. Noyes, Chemical Reagents for Protein Modification, Vol 1, CRC Press, Inc., Boca Raton, Florida.Google Scholar