Extraction And Purification Of The Lung Angiotensin Converting Enzyme And Thermodynamics Primary Explorer Of Binding To Its Inhibitor

Angiotensin converting enzyme (ACE) is membrane anchored dipeptidyl carboxypeptidase that plays a very important role in blood pressure regulation. The first step of carrying out study of ACE biochemical properties, structure, function and ACE inhibitor is preparation of high purity and high activity ACE. In this thesis. This paper about the separation and purification of the lung ACE and study of enzymatic properties of purified ACE, and preliminary study of the thermodynamic parameters of the ACE and its inhibitor binding process.The main results are as follows:Fresh lung as raw material, homogenate and centrifuged in pH8.3of a boric acid buffer containing sucrose.1.6mol·L-1-2.6mol·L-1ammonium sulfate fractional precipitated by follow, the secondary sedimentation crude enzyme solution was obtained to dialysis. Compared to chromatography, ultrafiltration and ion exchange-ultrafiltration was used to purify the dialysis. The greater amount of processing, the purification fold higher of ion exchange-ultrafiltration was selected. Lung ACE was obtained by SDS-PAGE shows ACE-pure product as a band and molecular mass is182KDa. The ACE was purified to357.3-fold with specific activity1.2476U·mg-1and activity recovery12.28%.Part of the enzymatic properties of the study found that lung angiotensin converting enzyme in a neutral or weak alkaline conditions exhibit higher vitality, its maximum enzyme activity at the pH of about8.3. Lung ACE was strong sensitivity to temperature, the optimum reaction temperature is40℃. the reaction rate of ACE catalyse different HHL concentrations were studied, the Lineweaver-Burk equation was obtained from the ACE reaction kinetics curve and calculated Michaelis constant of lung angiotensin converting enzyme was0.849mmol·L-1and the optimum concentration was9.775nmol·min-1.The microcalorimetric study of the banding of inhibitors to ACE, show a positive enthalpy and entropically driven for the banding process at pH8.3buffer. And the dominant driving force for binding is a large positive entropy change. Microcalorimetry technology can get ACE inhibition of the reaction enthalpy of the process, but not the complete thermodynamic parameters. The heat changes of banding of captopril to ACE process can not be monitored in the same enzyme concentration by isothermal titration calorimetry. Enzymes higher concentrations was required in order to monitor the change of the thermal banding process.Although isothermal titration calorimetry method can get a complete thermodynamic parameters, the amount of ACE requires is needed, both methods have their own advantages and disadvantages.