Isolation of carboxyl-terminal peptides from proteins by diagonal electrophoresis: application to the entomocidal toxin from Bacillus thuringiensis.
A procedure for the selective isolation of the C-terminal peptides from enzymatic digests of proteins is described. The methodology is based on the diagonal electrophoretic procedure described by R. G. Duggleby and H. Kaplan (1975) Anal. Biochem. 65, 346-354). The carboxyl groups in the protein are amidated with [14C]-methylamine followed by enzymatic digestion. Since only the C-terminal peptides lack a free carboxyl group, these peptides will lie on a diagonal line of a two-dimensional electrophoretogram run at pH 2.1 and 4.4. The diagonal line is delineated by autoradiography using [14C]taurine (net charge = 0 at pH 2.1 and 4.4) and [14C]choline (net charge = +1 at pH 2.1 and 4.4). Radioactive C-terminal peptides lie between these markers and can be directly excised for analysis. This procedure permits the detection and selective isolation of C-terminal peptides with minimal losses. The procedure was applied to the test proteins alpha-chymotrypsin and ribonuclease A. It was used to determine the C-terminus of the Bacillus thuringiensis toxin generated by tryptic cleavage of the protoxin.
Bietlot
H P
HP
Department of Chemistry, University of Ottawa, Ontario, Canada.
Carey
P R
PR
Pozsgay
M
M
Kaplan
H
H
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001345:Autoradiography; D001427:Bacterial Toxins; D002918:Chymotrypsin / Q000032:analysis; D004586:Electrophoresis; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification*; D011498:Protein Precursors / Q000032:analysis*; D012259:Ribonuclease, Pancreatic / Q000032:analysis
D000596:Amino Acids; C029103:Bacillus thuringiensis protoxin; D001427:Bacterial Toxins; D010446:Peptide Fragments; D011498:Protein Precursors; D012259:Ribonuclease, Pancreatic; D002918:Chymotrypsin