Isolation And Purification Of Pancreatic Kallikrein

Kallikrein is a kind of serine proteinase, which is classified into two types——tissue kallikrein and plasma kallikrein. Pancreatic kallikrein, which is widely used in medical field, is very important in clinical medicine because of its function of regulating blood pressure and improving blood circulation. It has been used to cure diseases such as high blood pressure and atherosis. And more and more medical researchers focus their research on it.Because domestic viscera resources are abundant and cheap, it is economically helpful in medical domain to purify kallikrein from pancreas, which is full of kallikrein. Although many researches have been made several decades before, the lack of a purifying way which has simple operation and high yield in short time is still a problem.In this research, parameters in processes of salting out, ultrafiltration and ion exchange chromatography were studied. What’s more, a series of stable and reliable testing methods were built. The results are as followed:1.Whether trypsin inhibitor was important in enzyme activity tests was studied. It was very effective to inhibit trypsin’s reaction with N-benzoyl-L-arginine ethyl ester (BAEE). So the addition of trypsin inhibitor can ensure results much more accurate and reliable.2.Whether concentration of ammonium sulfate made a difference in enzyme activity tests was studied. The experiment was designed to add 0-0.5M ammonium sulfate in testing as a gradient. The results showed that even if the concentration of ammonium sulfate was 0.5M, it would just made a little difference in enzyme activity testing. The conclusion was that the effection of ammonium sulfate could be ignored.3.The precipitation time of salting out was studied.Among 4 different parameters (30,60,90, and 120 min), kallikrein was precipitation completely after 90 minutes. The optimized precipitation time was 90 minutes.4.The precipitation range of salting out was also studied.6 different ranges were designed——20%-30%,30%-40%,40%-50%,50%-60%,60%-70%, 70%-80%——to seek the optimized one. The results showed that kallikrein could be precipitated from 20% to 70%.5.Then the ultrafiltration process was studied. Different concentration multiples were tested in ultrafiltration.2 × could achieve the best effection among 2 ×,4 × and 8 X. The yield was high and the loss of enzyme activity was low when ultrafiltration was under 2 ×.6.Using single factor experiments, some parameters of ion exchange chromatography, including pH of adsorption, ion concentration of adsorption and concentration of eluent were studied. The optimized conditions were:0.02M pH 5.5 NaAc-HAc buffer solution as equilibrium liquid,0.02M pH 5.5 NaAc-HAc buffer solution with 0.36M NaCl as eluent.7.Using DEAE-Sepharose FF to purify kallikrein with optimized parameters, one eluting peak was obtained. The result was that the purity of solution collected was high. The specific activity was 403.62 U/mg and the yield reached 73.19%. The purification step led to an increase in purity of 29.77 fold.