Purification Of Porcine Pancreas Kallikrein By Ion-Exchange And Affinity Chromatography

Kallikrein is a serine proteolytic enzyme, which is classified into two groups: the plasma kallikrein and the tissue kallikrein. Now tissue kallikrein are applied to medicines which are used to cure hypertension, angina, atherosis, minu-circulation obstacle and so on. Much attention has been paid to kallikrein in pharmaceutics.Porcine pancreas is usually used as raw material to produce kallikrein in commercial production. Methods utilized to separate and purify kallikrein mostly include organic solvent precipitation, salt classification precipitation, gel filtration, ion-exchange chromatogTam, affinity chromatogram. Separation of the enzymes with ion-exchange is mostly according to the electric charge of protein in solution. Affinity chromatogram is a special chromatogram technology that is dependent to the special affinity of target substance with differential ligand.In the present study, an improved method for extracting kallikrein from porcine pancreas was developed. The optimal conditions were that 0.05M NaAc-HAc buffer (pH5.0) was used as extract solution in the volume of three times to the mass of porcine pancreas, and the concentration of Ca²⁺ was 10%. The crude enzyme was purified by DEAE-Sepharose4B ion-exchange chromatogram and the affinity chromatogram of p-Aminobenzamidine-Sepharose4B. The optimal conditions of DEAE-Sepharose4B were that: starting buffer is 0.02M NaAc-HAc buffer (pH5.5), eluting buffer is 0.02M NaAc-HAc-0.36NaCl solution (pH5.5). The conditions of the affinity chromatogram of p-Aminobenzamidine-Sepharose4B were that: starting buffer is 0.1M PB buffer (pH7.0), eluting buffer is 0.1M PB-0.5M NaCl solution (pH7.0).For the preparation in the lab scale, the specific activity of the kallikrein was about 300.00KU/mg.p after DEAE-Sepharose4B ion-exchange chromatogram and affinity chromatogram of p-Aminobenzamidine-Sepharose4B. The purity of the kallikrein prepared by DEAE-Sepharose4B ion-exchange chromatogram and affinity chromatogram of p-Aminobenzamidine-Sepharose4B was increased by 6 times and...