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The Amino Acid Sequence of ß-Galactosidase

III. THE SEQUENCE OF NH₂- AND COOH-TERMINAL TRYPTIC PEPTIDES

From the ¹ From the Department of Biological Chemistry, UCLA School of Medicine and Molecular Biology Institute, University of California, Los Angeles, California 90024

The method of Bruton and Hartley for the isolation of short NH₂-terminal sequences of peptides has been applied to ß-galactosidase. Maleyl-ß-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 NH₂-terminal 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-Val-Leu-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 COOH-terminal peptide is normally not expected to be adsorbed to the resin. In the case of ß-galactosidase a COOH-terminal decapeptide was obtained from such a mixture in the fraction insoluble at pH 4. Its sequence was found to be Tyr-His-Ser-Gln-Val-Leu-Tyr-Cys-Gln-Lys.

These procedures for the isolation of NH₂- and COOH-terminal peptides may be useful for other proteins.

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