Extraction And Purification Of The Lung Angiotensin Converting Enzyme And Enzymolysis And The Study Of Inhibition Mechanism

Angiotensin converting enzyme (ACE) is membrane anchored dipeptidyl carboxypeptidase that plays a very important role in cardiovascular homeostasis. At the beginning to accomplish the study of ACE physicochemical, enzymatic properties and ACE inhibitor is preparation of high purity and high activity ACE. This paper is mainly study the separation and purification of the lung ACE and to research the enzymatic properties of purified ACE. To further study the inhibitor binding process of ACE and its inhibitor has acquired the thermodynamic parameters. The main results are showed below:The raw material was hog lung and the tissue was cut into small pieces. After homogenate in boric acid buffer, using trypsin in the homogenate at 37℃ and extracted ACE significantly by ultrasonic then centrifuged. And the optimal ultrasonic conditions:ultrasonic power was 360W, total time was 3min,lung/buffer was 1:5. The follow step is 1.6mol·L-1～2.6mol·L-1 ammonium sulfate fractional precipitated and to dialysis the secondary sedimentation crude enzyme solution. ACE was subjected to sepharose DEAE-Sepharose FF column chromatography while optimizing the concentration. Then we could have a great deal of hight activity of enzyme.By using SDS-PAGE, purified lung ACE shows as a band and molecular mass is 69KDa. The purification of ACE is up to 29.48-fold with specific activity 0.1061U·mg-1 and activity recovery 12.33%.Result of the enzymatic properties research show that lung angiotensin converting enzyme has a higher vitality under a neutral or weak alkaline conditions.In the circumstance of the pH about 8.3, the enzyme activity of ACE reach maximum value. The optimum reaction temperature of lung ACE is 40℃ which was very sensitivity to temperature change. To study the reaction rate of ACE catalyse different HHL concentrations, the Lineweaver-Burk equation was obtained from the ACE reaction kinetics curve. The Michaelis constant of the lung angiotensin converting enzyme is 2.7mmol·L-1 and the optimum concentration was 8.1nmol·min-1.The results of the isothermal titration calorimetry experiment which binding process of inhibitors to ACE, show a tight binding Entropic ally driven of Lisinopril and ACEI(VSLPEW) to pig lung ACE and positive enthalpy at pH8.3 buffer. ITC technology can get ACE inhibition of the reaction enthalpy of the process and the complete thermodynamic parameters. ΔH decreases linearly with increasing temperature, and the values of heat capacity changes Δ Cp were determined by the slope of the graphical representation of ΔH versus temperature. The dominant driving force for binding is a large positive entropy change that the stoichiometry value for the Lisinopril complete inhibition of the enzyme is about 1:1 and for the ACEI(VSLPEW) complete inhibition of the enzyme is about 2:1.