Angiotensin Converting Enzyme Extraction And The Study On The Interaction With Inhibitors

Angiotensin Cconverting Enzyme(ACE;EC.3.4.15.1)is a zinc-containing dipeptide carboxyase that plays an important role in blood pressure regulation.In order to study the biochemical properties,structure and function of ACE and the binding mechanism of ACE and inhibitor,it is necessary to prepare high activity and high purity ACE.In this paper,high purity ACE was isolated and purified from pig lungs,and its enzymatic properties were studied.Valine-alanine-proline(Val-Ala-Pro,VAP)and Phenylalanine-valine-alanine-proline(Phe-Val-Ala-Pro,FVAP)binding mechanism with pig ACE was studied by means of isothermal titration calorimetry and UV-visible spectroscopy.The main contents are as follows:(1)The fresh pig lungs were used as raw materials,and then purified by means of homogenization,ammonium sulfate fractionation salting,dialysis and ion exchange chromatography.Then,the enzyme solution was further purified by ultrafiltration membrane bag capable of mass processing of enzyme solution.The optimum conditions were as follows:the flow rate of the ultrafiltration was 5mL·min-1,the volume of the washing liquid was 300mL,and the volume of the retentate was 20mL.After a series of purified enzymes were obtained by electrophoresis grade purity.Compared with the homogenate,the purification ratio of the purified enzyme was 467.6 times,the recovery rate was 37.4%and the specific activity was 2.15U·mg-1.The molecular weight was determined to be 180KD by SDS-PAGE.The optimum temperature for enzyme catalysis was 40?and the optimum pH was 8.3,and the Michaelis-Menten constant was 2.54mmol·L-1.(2)The inhibitory types of VAP and FVAP were determined by isothermal titration calorimetry.The results showed that both of these inhibitory peptides were noncompetitive inhibitory peptides.The IC50 values of VAP and FVAP were 1.36?mol·L-1 and 8.19?mol·L-1,respectively.The thermodynamic parameters of ACE binding to two peptides were determined by isothermal titration calorimetry.The results were as follows:the number of binding sites combined with VAP and ACE increased with the increase of temperature,and the enthalpy of binding is 12.07 KJ·mol-1 at 288.15 K,the molar constant pressure heat capacity ?Cp is-0.9810 KJ·mol-1·K-1,the entropy change is-19.36 k·-mol-1,and the Gibbs free energy change is-31.06 KJ·mol-1,which belongs to the entropy and enthalpy co-driving process.The binding entropy of FVAP and ACE binding process increased with the increase of temperature,the enthalpy of fusion is-2.33 KJ·mol-1 at 288.15 K,the heat capacity Cp is-0.6880 KJ·mol-1·K-1,Gibbs free energy becomes-23.68 KJ·mol-1,tne binding constant is 2.0 X 103 1/M.The entropy is significantly greater than the enthalpy,which is an entropy-driven process.(3)The interaction between peptide VAP and FVAP and ACE was studied by UV-Vis spectrophotometry.The results showed that VAP and FVAP were mixed with ACE enzyme solution,and it was found that redemption and hyperchromicization at 230nm compared with that without inhibition of peptidase spectroscopy showed that VAP and FVAP were combined with ACE.The chain structure has changed.