N-terminal sequence analysis of N alpha-acetylated proteins after unblocking with N-acylaminoacyl-peptide hydrolase.
The enzyme acylaminoacyl-peptide hydrolase represents an attractive reagent for the removal of acetylamino acids from the N-terminus of proteins prior to sequencing. However, the enzyme will not accept intact proteins as substrates, and a blocked protein must consequently be fragmented to generate a relative short blocked peptide, and all the newly generated amino termini must be blocked with an hydrolase-resistant reagent before the enzyme can be used to specifically unblock the N-terminus. When a number of N-acetylated proteins (enolase, alpha-crystallin, ovalbumin, cytochrome c, parvalbumin, superoxide dismutase, and myelin basic protein) were subjected to fragmentation with proteases or cyanogen bromide, treatment with succinic anhydride and exhaustive extraction with ether, and the resulting salt-free, succinylated peptides were incubated with the hydrolase, the N-terminal sequence was specifically unblocked. An aliquot of the entire peptide mixture was applied to the protein sequencer, and a single sequence, corresponding to the known N-terminal sequence starting at residue 2, was obtained. When another aliquot of the same hydrolase-treated peptide mixture was treated with the enzyme acylase I, the liberated acetylamino acid was cleaved, and the N-terminal amino acid (residue 1) could be identified by amino acid analysis. The amount of sequence information obtained from different proteins with different fragmentation methods varied considerably; in the case of parvalbumin a sequence of 12 residues was obtained, while for myelin basic protein, only 3 residues could be identified; the other proteins yielded from 5- to 9-residue sequences.(ABSTRACT TRUNCATED AT 250 WORDS)
Krishna
R G
RG
Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston 77225.
Chin
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CC
Wold
F
F
D000107:Acetylation; D000581:Amidohydrolases; D000595:Amino Acid Sequence; D000818:Animals; D010450:Endopeptidases; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D010446:Peptide Fragments / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D011817:Rabbits; D051381:Rats
D010446:Peptide Fragments; D011506:Proteins; D010450:Endopeptidases; D000581:Amidohydrolases; C019672:aminoacylase I