Affinity Chromatography of Several Proteolytic Enzymes on Carbobenzoxy‐d‐phenylalanyl‐triethylenetetramine‐sepharose*

Carbobenzoxy‐D‐phenylalanyl‐triethylenetetramine‐Sepharose (Z‐D‐Phe‐T‐Sepharose) was found to be an effective affinity adsorbent for several proteolytic enzymes which preferentially catalyze the hydrolytic cleavage of peptide bonds involving amino or carboxyl groups of hydrophobic and bulky amino acid residues. α‐Chymotrypsin, subtilisin and neutral metalloendopeptidase were adsorbed at pH 5–7, while pepsin and Rhizopus niveus acid protease showed affinity to the adsorbent at pH 3.5–5.0. α‐Chymotrypsin was recovered by elution with 0.1 M acetic acid and neutral subtilopeptidase (B. subtilis neutral protease) was eluted with 0.5 M NaCl at pH 9.0. Subtilisin and thermolysin were found in the eluates with 1.5 M guanidine‐HCl, 1 M p‐toluenesulfonate or 30% ethylene glycol at pH 7. Rhizopus acid protease was eluted with 0.5 M acetic acid and pepsin with 1 M guanidine‐HCl or p‐toluenesulfonate at pH 3.5. The chromatographic purifications by this method of highly purified or crystallized enzyme preparations commercially available resulted in 1.3–1.5‐fold increase in their specific activities. Inactivation of these proteolytic enzymes by chemical modification, and formation of enzyme‐inhibitor complex led to loss of the binding ability to the adsorbent. On the other hand, the zinc‐free apoenzymes of neutral subtilopeptidase and thermolysin were adsorbed onto and eluted from the adsorbent column under the same conditions as those for the native enzymes, suggesting that zinc is not essential for the binding of the substrate to these metalloproteases.