Removal of N-terminal blocking groups from proteins.
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.
Fowler
E
E
AutoImmune, Inc., Lexington, Massachusetts, USA.
Moyer
M
M
Krishna
R G
RG
Chin
C C
CC
Wold
F
F
D001671:Biochemistry / Q000379:methods*; D015153:Blotting, Western; D003124:Colorimetry; D005561:Formates / Q000378:metabolism; D006834:Hydrazines / Q000378:metabolism; D006867:Hydrolases / Q000378:metabolism; D006868:Hydrolysis; D017879:Isothiocyanates / Q000378:metabolism; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D011751:Pyroglutamyl-Peptidase I / Q000378:metabolism; D011761:Pyrrolidonecarboxylic Acid / Q000737:chemistry / Q000302:isolation & purification; D012996:Solutions; D013387:Succinic Anhydrides / Q000378:metabolism; D014269:Trifluoroacetic Acid / Q000378:metabolism
D005561:Formates; D006834:Hydrazines; D017879:Isothiocyanates; D011506:Proteins; D012996:Solutions; D013387:Succinic Anhydrides; C005441:phenylisothiocyanate; C008817:peroxyformic acid; C031801:succinic anhydride; D014269:Trifluoroacetic Acid; D006867:Hydrolases; D011751:Pyroglutamyl-Peptidase I; D011761:Pyrrolidonecarboxylic Acid