Separation And Purification Of Angiotensin Converting Enzyme From PIG And Rabbit Lung

Angiotensin converting enzyme (ACE) is membrane anchored dipeptidyl carboxypeptidase that plays a very influential role in blood pressure regulation. For further in-depth study the structure and function, biochemical properties, and the binding process between ACE and its inhibitors, we must preparate a large amount of high activity enzyme. In this paper, ACE was separated and purificated from pig lung and rabbit lung. Then we measured the product activity in every step, compared to the primary method in the report before, and present a new optimized process.lt made possibility that obtaining a large number of high recovery rate of ACE. The main results are as follows:The material was fresh pig lung from nearby market.After homogenarating with boric acid buffer, the homogenate was prepared in water bath for1h at37℃containing trypsin then centrifugated.1.6mo·L-1-2.6mol·L-1ammonium sulfate fractional precipitated the supernatant. Then the protein was dialyzed. ACE was subjected to sepharose DEAE-Sepharose FF column chromatography while optimizing the loading quantity of sample, the velocity of flow, the concentration and pH of eluent. Then we could get a large amount of high recovery rate of enzyme. The ACE was purified to39.48-fold with specific activity0.1303U·mg-1and activity recovery59.14%, and ACE molecular weight was69kDa, which provided plentiful raw materials for the following purification and other work, and gave a good research foundation.Refering to the separation and purification of pig lung ACE, rabbit lung ACE was purificated in the same way. Rabbit lung ACE was purified to31.90-fold with specific activity0.1303U·mg-1and activity recovery45.20%.Through the study about properties of two enzymes from lung, we can learned the relationship between the activity and pH and temperature, respectively:both angiotensin converting enzymes showed the higher activity in partial neutral or weak alkaline conditions. Their maximum enzyme activity was at the pH8.3. Both angiotensin converting enzymes were more sensitive to the temperature.42℃is the optimum reaction temperature. When the temperature was higher or be lower than42℃, the activity declined obviously. The reaction kinetics curve between ACE and HHL with different concentrations was studied, and Lineweaver-Burk equation was obtained. We could calculate and gain that Michaelis constant of pig lung ACE was1.521mmol-L-1and the optimum concentration was17.301nmol·min-1, Michaelis constant of rabbit lung ACE was1.058mmol·L-1and the maximum reaction rate was12.107nmol·min-1respectively.