The purification of Aeromonas aminopeptidase by affinity chromatography

A competitive inhibitor for Aeromonas aminopeptidase has been prepared from the bromomethyl ketone derived from t-butyloxycarbonyl-l-leucine and successfully coupled to aminomethyl cellulose to form an adsorbent for affinity chromatography. The blocked form of the inhibitor was coupled to aminomethyl cellulose and then deblocked in aqueous trifluoroacetic acid to yield an insolubilized analog of an NH₂-terminal l-leucyl residue. This material was effective in binding the aminopeptidase and separating it from a contaminating endopeptidase, which has a similar isoelectric point and size. Separation of the aminopeptidase and endopeptidase was shown to be due to the specificity of the affinity adsorbent for the aminopeptidase, inasmuch as separation of the enzymes did not occur on a typical anion exchange column or on a hydrophobic column lacking a free amino group.