The Amino Acid Sequence of P-Galactosidase

The method of Bruton and Hartley for the isolation of short NH2-terminal sequences of peptides has been applied to /3-galactosidase. Maleyl-P-galactosidase was hydrolyzed with thermolysin and the digest was acidified. After passage through Dowex 50, Thr-Met was obtained from the eluate. This procedure has been extended. For the isolation of a larger NH&erminal peptide, maleyl-protein was treated with trypsin, and the digest was subjected to additional hydrolysis with carboxypeptidase B. The peptide mixture was acidified, centrifuged, and filtered through Dowex 50. From the eluate an octapeptide was purified which corresponded to the NH2-terminal portion of a tridecapeptide isolated previously from another hydrolysate. The sequence was determined to be Thr-Met-Ile-Thr-Asp-Ser-Leu-Ala-Val-ValLeu-Gln-Arg. Another modification of this method is proposed for the isolation of a COOH-terminal peptide. Following hydrolysis of maleyl-protein with trypsin, the peptide mixture may be maleylated before passage through Dowex 50. The COOHterminal peptide is normally not expected to be adsorbed to the resin. In the case of fl-galactosidase a COOH-terminal decapeptide was obtained from such a mixture in the fraction insoluble at pH 4. Its sequence was found to be TyrHis-Ser-Gln-Val-Leu-Tyr-Cys-Gln-Lys. These procedures for the isolation of NH2and COOHterminal peptides may be useful for other proteins.