Application of reductive dihydroxypropylation of amino groups of proteins in primary structural studies: identification of phenylthiohydantoin derivative of ε-dihydroxypropyl-lysine residues by high-performance liquid chromatography

A. Seetharama Acharya, Leslie G. Sussman, Belur N. Manjula

Research output: Contribution to journalArticle

9 Scopus citations

Abstract

The general utility of reductive alkylation of amino groups of proteins with glyceraldehyde (2,3-dihydroxypropionaldehyde) in the presence of sodium cyano-borohydride, i.e. dihydroxypropylation, as an aid in generating arginine peptides of proteins by tryptic digestion has been investigated. The dihydroxypropylation of the amino groups of ribonuclease A and the streptococcal Pep M5 protein proceeds predominantly to the stage of monoalkylation. The derivatized lysine namely, ε-dihydroxypropyl-lysine is stable to acid hydrolysis, and is eluted slightly ahead of histidine in the amino acid analyzer. The peptide bonds of ε-dihydroxypropyl-lysine residues are resistant to tryptic digestion. The arginine peptides of dihydroxypropylated ribonuclease A, and dihydroxypropylated streptococcal Pep M5 protein have been isolated by reversed-phase high-performance liquid chromatography (HPLC) of the tryptic digest of the derivatized proteins. The phenylthiohydantoin (PTH) derivative of ε-dihydroxypropyl-lysine has been prepared. It is eluted at a position intermediate to that of the PTH derivatives of proline and tryptophan in reversed-phase HPLC on DuPont Zorbax ODS columns. Thus the PTH-ε-dihydroxypropyl-lysine could be identified during the sequence studies of the dihydroxypropylated peptides. The presence of dihydroxypropyl groups on the ε-amino groups of lysine residues in the dihydroxypropylated peptides does not interfere with the Edman degradation studies. The ease of the dihydroxypropylation reaction, the resistance of the peptide bonds of ε-dihydroxypropyl-lysine residues to trypsin, and the identification of the PTH derivative of ε-dihydroxypropyl-lysine residues by reversed-phase HPLC makes the dihydroxypropylation procedure a valuable addition to the arsenal of procedures for limiting the tryptic digestion to the arginine residues of proteins and peptides.

Original languageEnglish (US)
Pages (from-to)37-48
Number of pages12
JournalJournal of Chromatography A
Volume297
Issue numberC
DOIs
StatePublished - Aug 2 1984

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Organic Chemistry

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