N‐terminal heterogeneity of methylamine dehydrogenase from <i>Thiobacillus versutus</i>

Two enzymatic methods commonly used in N-terminal sequence analysis of blocked proteins are presented in this unit; one uses pyroglutamate aminopeptidase for N(alpha)-pyrrolidone carboxyl-proteins in solution or blotted onto a membrane, and the other uses acylaminoacyl-peptide hydrolase for N(alpha)-acyl-proteins blocked with other acyl groups. A Support Protocol describes a colorimetric assay for pyroglutamate aminopeptidase activity. Sequencing with acylaminoacyl-peptide hydrolase must include fragmentation of the protein before unblocking can be carried out, so procedures are provided for chemically blocking newly generated peptides with either succinic anhydride or phenylisothiocyanate/performic acid. The hydrolase is then applied to the total mixture of peptides, only one of which, the acylated N-terminal peptide, should be a substrate for hydrolase. After incubation, the mixture of peptides is subjected to sequence analysis. Protocols are also provided for unblocking N-terminally blocked proteins using acid-catalyzed hydrolysis or methanolysis, hydrazinolysis, and beta-elimination after acid-catalyzed N-O shift. Alternate protocols describe chemical removal of acetyl and longer-chain alkanoyl groups, as well as formyl groups to open the cyclic imide of pyrrolidone carboxylate.

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Removal of N‐Terminal Blocking Groups from Proteins

Fowler

Moyer

Krishna³

et al. 1996

CP Protein Science

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Removal of N‐Terminal Blocking Groups from Proteins

et al. 2011

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Two enzymatic methods commonly used in N-terminal sequence analysis of blocked proteins are presented: one uses pyroglutamate aminopeptidase for N(α)-pyrrolidone carboxyl-proteins in solution or blotted onto a membrane, and the other uses acylaminoacyl-peptide hydrolase for N(α)-acyl-proteins blocked with other acyl groups. A Support Protocol describes a colorimetric assay for pyroglutamate aminopeptidase activity. Sequencing with acylaminoacyl-peptide hydrolase must include fragmentation of the protein before unblocking, so procedures are provided for chemically blocking newly generated peptides with either succinic anhydride or phenylisothiocyanate/performic acid. The hydrolase is then applied to the total mixture of peptides, only one of which, the acylated N-terminal peptide, should be a substrate for hydrolase. After incubation, the mixture of peptides is subjected to sequence analysis.

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Refined crystal structure of methylamine dehydrogenase from Paracoccus denitrificans at 1.75 Å resolution

Chen

Doi

Durley

et al. 1998

Journal of Molecular Biology

107

119

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N‐terminal heterogeneity of methylamine dehydrogenase from Thiobacillus versutus

Cited by 6 publications

References 8 publications

Removal of N‐Terminal Blocking Groups from Proteins

Removal of N‐Terminal Blocking Groups from Proteins

Removal of N‐Terminal Blocking Groups from Proteins

Refined crystal structure of methylamine dehydrogenase from Paracoccus denitrificans at 1.75 Å resolution

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