RESUMEN
A low-molecular-weight biomimetic affinity ligand selective for binding elastase has been designed and synthesized. The ligand was based on mimicking part of the interaction between a natural inhibitor, turkey ovomucoid inhibitor and elastase, and modelled from the X-ray crystallographic structure of the enzyme-inhibitor complex. Limited solid-phase combinatorial chemistry was used to synthesize 12 variants of the lead ligand using the triazine moiety as the scaffold for assembly. The ligand library was screened for its ability to bind elastase and trypsin, and two ligands were studied further. Ligand C4/6 [2-alanyl-alanyl-4-tryptamino-6-(alpha-lysyl)-s-triazine] was found to bind porcine pancreatic elastase, but not trypsin, with a dissociation constant of 6 x 10(-5) M and a binding capacity of 21 mg elastase per ml gel. The adsorbent was used to purify elastase from a crude extract of porcine pancreas. Immobilized ligand C4/5 6 [2-alanyl-alanyl-4-tyramino-6-(alpha-lysyl)-s-triazine] was similarly chosen for optimal binding of elastase from cod and used to purify the enzyme from a crude extract of cod pyloric caeca. Ligand C4/6 was subsequently synthesized in solution and its structure verified by 1H-NMR.
Asunto(s)
Diseño de Fármacos , Elastasa Pancreática/metabolismo , Animales , Cromatografía de Afinidad , Peces , Ligandos , Modelos Moleculares , Imitación Molecular , Oligopéptidos/química , Oligopéptidos/metabolismo , Elastasa Pancreática/química , Elastasa Pancreática/aislamiento & purificación , Soluciones , PorcinosRESUMEN
Elastase was isolated from ovine pancreas and purified to homogeneity by two different procedures. One involved precipitation with ammonium sulphate, p-aminobenzamidine-Sepharose chromatography, CM-Sepharose ion exchange chromatography and S-300 Sephacryl chromatography. The other involved the direct adsorption of elastase by tri-L-alanyl-Sepharose chromatography and a CM-Sepharose step. The enzyme, which was produced in an inactive form in the pancreas, was activated with a trace of trypsin prior to chromatography. Ovine pancreatic elastase has an isoelectric point above pI 9.3 and its molecular mass is estimated at approximately 25 kDa. The optimal pH range for activity is between 8.0 and 8.4 and the enzyme is unstable at pH below 4.0 and above 10.0 and at temperatures above 65 degrees C. The kinetic properties of the enzyme were determined with succinyl-Ala-Ala-Ala-p-nitroanilide as the substrate. Km and kcat Km-1 proved to be similar to the kinetic parameters of porcine elastase determined simultaneously.