Design and study of peptide‐ligand affinity chromatography adsorbents: Application to the case of trypsin purification from bovine pancreas

The purification of trypsin from bovine pancreas was employed in a case study concerning the design and optimization of peptide‐ligand adsorbents for affinity chromatography. Four purpose‐designed tripeptide‐ligands were chemically synthesized (>95% pure), exhibiting an Arg residue as their C‐terminal (site P 1 ) for trypsin bio‐recognition, a Pro or Ala in site P 2 , and a Thr or Val in site P 3 . Each tripeptide‐ligand was immobilized via its N‐terminal amino group on Ultrogel A6R agarose gel, which was previously activated with low concentrations of cyanuric chloride (10.5 to 42.5 μmol/g gel). Well over 90% of the peptide used was immobilized. Three different concentrations were investigated for every immobilized tripeptide‐ligand, 3.5, 7.0, and 14 μmol/g gel. The K D values of immobilized tripeptide‐trypsin complexes were determined as well as the purifying performance and the trypsin‐binding capacity of the affinity adsorbents. The K D values determined were in good agreement with the trypsin purification performance of the respective affinity adsorbents. The tripeptide sequence H‐TPR‐OH displayed the highest affinity for trypsin ( K D 8.7 μ M ), whereas the sequence H‐TAR‐OH displayed the lowest ( K D 38 μ M ). Dipeptide‐ligands have failed to bind trypsin. When the ligand H‐TPR‐OH was immobilized via its N‐terminal on agarose, at a concentration of 14 μmol/g gel, it produced the most effective affinity chromatography adsorbent. This adsorbent exhibited high trypsin‐binding capacity (approximately 310,000 BAEE units/mL of adsorbent); furthermore, it purified trypsin from pancreatic crude extract to a specific activity of 15,200 BAEE units/mg (tenfold purification), and 82% yield. © 1997 John Wiley & Sons, Inc.