Quantitative identification of N-terminal amino acids in proteins by radiolabeled reductive methylation and amino acid analysis: application to human erythrocyte acetylcholinesterase.
A novel method of determining N-terminal amino acids in proteins is introduced. Reductive methylation of a protein with radiolabeled formaldehyde methylates both the alpha-amino group of the N-terminal amino acid and the epsilon-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 N alpha-methyl and N alpha,N alpha-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.
D000110:Acetylcholinesterase / Q000097:blood*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis*; D001894:Borohydrides; D005557:Formaldehyde; D006801:Humans; D008745:Methylation; D010084:Oxidation-Reduction
D000596:Amino Acids; D001894:Borohydrides; D005557:Formaldehyde; C009282:sodium cyanoborohydride; D000110:Acetylcholinesterase