| heal.abstract |
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 P1) for trypsin biorecognition, a Pro or Ala in site P2, and a Thr or Val in site P3. 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 μM), whereas the sequence H-TAR-OH displayed the lowest (K(D) 38 μM). 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.Design and optimization of peptide-ligand adsorbents for affinity chromatography was carried out, in order to purify trypsin from bovine pancreas. Four tripeptide ligands were synthesized, and each of them was immobilized via its N-terminal amino group on Ultrogel A6R agarose gel, which was previously activated with low concentrations of cyanuric chloride. Distribution constants for immobilized tripeptide-trypsin complexes were determined. Dipeptide ligands have failed to bind trypsin. The most effective affinity chromatography adsorbent was obtained by immobilizing the H-TPR-OH ligand on agarose at a concentration of 14 μmol per g. This adsorbent allowed purification of trypsin from pancreatic crude extract to a specific activity of 15,200 BAEE units per mg (tenfold purification). |
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