Quantitative identification of N-terminal amino acids in proteins by radiolabeled reductive methylation and amino acid analysis: Application to human erythrocyte acetylcholinesterase

Robert Haas, Terrone L. Rosenberry

Research output: Contribution to journalArticle

32 Scopus citations

Abstract

A novel method of determining N-terminal amino acids in proteins is introduced. Reductive methylation of a protein with radiolabeled formaldehyde methylates both the α-amino group of the N-terminal amino acid and the ε-amino groups of Lys residues. The radiomethylated amino acids are stable to acid hydrolysis, and each of 16 possible hydrolysis-stable N-terminal amino acids can be identified by the unique elution positions of its Nα-methyl and Nα,Nα-dimethyl derivatives with an appropriate amino acid analyzer elution schedule. The technique is at least as sensitive as other N-terminal amino acid determinations and, in addition, permits a quantitative evaluation of the number of N-terminal groups in a sample. Reductive methylation of bovine serum albumin revealed N-terminal Asp at a stoichiometry of 0.97 amino acid residue per polypeptide, while methylation of prolactin resulted in 0.86 residue of N-terminal Thr per polypeptide. Human erythrocyte acetylcholinesterase contained two N-terminal amino acids with stoichiometries of 0.66 Glu and 0.34 Arg per 70-kDa subunit. Identification of Glu as the principal N-terminus of acetylcholinesterase was confirmed by Edman sequencing.

Original languageEnglish (US)
Pages (from-to)154-162
Number of pages9
JournalAnalytical Biochemistry
Volume148
Issue number1
DOIs
StatePublished - Jul 1985

Keywords

  • acetylcholine
  • amino acid analysis
  • hydrolases
  • membranebound enzymes
  • protein sequencing
  • protein structure

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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