Affinity chromatography on immobilized anhydrotrypsin: general utility for selective isolation of C-terminal peptides from protease digests of proteins.
Recently we have succeeded in the efficient isolation of the C-terminal peptides from tryptic digests of the tail sheath protein (with C-terminal Gly) and the tube protein (with C-terminal Glu) of bacteriophage T4, by taking advantage of a unique property of immobilized anhydrotrypsin, that is, a strong specific affinity for peptides containing Arg or Lys residues at their C-termini. In this study, the utility of affinity chromatography on immobilized anhydrotrypsin was further demonstrated in the cases of Streptomyces subtilisin inhibitor (as a reduced and S-carboxymethylated form, with C-terminal Phe) and alpha 1-antitrypsin (with C-terminal Lys). By subjecting a tryptic digest of the former protein and a chymotryptic digest of the latter protein to the affinity chromatography, the C-terminal peptides were specifically recovered in the breakthrough fraction and in the adsorbed fraction, respectively. It was further shown that immobilized anhydrotrypsin can also adsorb peptides with C-terminal S-aminoethyl-Cys residues and exerts adsorptive ability even toward the peptides in solution containing urea at a high concentration if appropriate precautions are taken. These findings suggest the general utility of this simple method for C-terminal peptide isolation, which is extremely helpful for studies to confirm amino acid sequences deduced from nucleotide sequences of the cDNA (or genomic DNA) of proteins.
Kumazaki
T
T
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Hokkaido University.
Terasawa
K
K
Ishii
S
S
D000327:Adsorption; D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins / Q000032:analysis / Q000378:metabolism; D055598:Chemical Phenomena; D002621:Chemistry; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000378:metabolism; D004800:Enzymes, Immobilized; D010446:Peptide Fragments / Q000302:isolation & purification*; D010447:Peptide Hydrolases / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism; D014508:Urea / Q000494:pharmacology; D000515:alpha 1-Antitrypsin / Q000032:analysis / Q000378:metabolism
D000596:Amino Acids; D001426:Bacterial Proteins; D004800:Enzymes, Immobilized; D010446:Peptide Fragments; D000515:alpha 1-Antitrypsin; C011661:anhydrotrypsin; C018150:subtilisin inhibitor protein, Streptomyces; D014508:Urea; D010447:Peptide Hydrolases; D002918:Chymotrypsin; D014357:Trypsin