Localization of the two free thiol groups in the porcine pancreatic alpha-amylase I sequence.
Porcine pancreatic alpha-amylase I, a single 496 residue long polypeptide chain, contains 5 disulfide bridges and 2 free -SH groups. The conditions for specific blocking of native amylase either with radioactive N-ethyl maleimide or with labeled iodoacetic acid were determined. Under these conditions 2 moles of blocking reagent are incorporated per mole of amylase. [14C]-S-succinimido amylase was cleaved by CNBr and the resulting peptides were purified. Only one of them the CNBr 2 + 3 peptide (178 residues) was found labeled. Ts1 a 33-residue peptide containing the whole radioactivity was purified from the tryptic digest of this large fragment. After reduction and carboxymethylation Ts1A, (22 residues) was obtained which contains 2 moles of succinyl-Cys and one mole of CM-Cys per mole of peptide. Chymotryptic digestion of Ts1A yielded 2 equally labeled peptides: C1 (16 residues) and C2 (6 residues). Automated sequencing of both peptides and counting of the PTH-amino acids shows that the free cysteines are only 15 residues apart in the sequence.
D000595:Amino Acid Sequence; D000681:Amylases; D000818:Animals; D002918:Chymotrypsin; D003488:Cyanogen Bromide; D010179:Pancreas / Q000201:enzymology*; D010446:Peptide Fragments / Q000032:analysis; D013438:Sulfhydryl Compounds / Q000032:analysis; D013552:Swine; D014357:Trypsin; D000516:alpha-Amylases
D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D000681:Amylases; D000516:alpha-Amylases; D002918:Chymotrypsin; D014357:Trypsin; D003488:Cyanogen Bromide