人尿胰蛋白酶抑制剂的快速纯化

A rapid purification method of trypsin inhibitor from the concentrated human urine

目的以人尿浓缩物为原料 ,对人尿胰蛋白酶抑制剂的分离纯化技术进行研究。方法利用纯胰蛋白酶作为配体偶联于CNBr Sepharose 4B为亲和载体 ,在不同条件下洗脱亲和蛋白 ,可从粗品溶液中快速分离纯化具有胰蛋白酶抑制活性的两种纯产物。结果层析图谱与SDS PAGE分析表明 ,根据洗脱条件 ,可将有抑制活性的 2 0kD和 40kD蛋白质分离为两个单独组份或混合物。其中 2 0kD组份的抑制活性回收为 35 % ,抑制比活为 3 387U/mg;40kD组份的相应值分别为 5 3 %和 3 116U/mg。混合物的抑制活性回收和抑制比活分别为 89%和 3 0 76U/mg ,再经Su perose 12或Superdex 75凝胶渗透层析 ,分别获得两种组份的纯产物。 结论通过选择性洗脱亲和柱 ,可快速获得高收率。

PurposeA rapid method of purifying human urinary trypsin inhibitor (HUTI) was investigated.MethodsAn affinity column was prepared with trypsin as the ligand coupled to the support, CNBr Sepharose 4B. After loading the concentrated human urine on the affinity column, two pure products both having trypsin inhibitory activity were eluted as the different elution forms.ResultsIt was indicated from chromatographic profiles and SDS PAGE analysis of the samples that the two proteins, 20kD and 40kD, could be separated to be two pure components or a mixture under different elution conditions. For the former, the recovery of the inhibitory activity and the inhibitory specific activity for 20kD component was 35% and 3 387 U/mg, and the relevant values for 40kD were 53% and 3116U/mg, respectively. For the latter, the total recovery of inhibitory activity and the specific activity was 90% and 3076U/mg. If the mixture was loaded on Superose 12 or Superdex 75 for gel permeation chromatography, the two components (20kD and 40kD) could be separated thoroughly.ConclusionThe pure trypsin inhibitor prepared from human urine was effectively performed on an affinity column.