Volume 11, Issue 44 (3-2005)
RJMS 2005, 11(44): 979-985 |
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Shafiee S, Keyhani M, Shabani M, Koochaki Shalmani I, Kaviani M. Extraction and Development of Multi-Step Purification of Angiotensin-I Converting Enzyme(ACE) from Rabbit Lung. RJMS 2005; 11 (44) :979-985
URL: http://rjms.iums.ac.ir/article-1-99-en.html
URL: http://rjms.iums.ac.ir/article-1-99-en.html
Abstract: (9215 Views)
Angiotensin-converting enzyme(ACE)(EC: 3.4.15.1) is a peptidyl dipeptide hydrolase that removes the carboxyl terminal (His-Leu) from angiotensin I to produce the octapeptide angiotensin II. Due to the importance of ACE and its active site-directed inhibitors in the pathogenesis and treatment of cardiovascular disorders such as hypertension and heart failure, ACE purification and assay are of clinical and commercial as well as scientific interest. In the present study it was attempted to purify ACE faster and simpler. Purification procedure was finally performed in three steps. After homogenization and centrifugation, ACE was solubilized from pellet using Nonidet-P40, a non-ionic detergent. The supernatant solution brought to 50% and 70% saturation concentration of ammonium sulfate. After ammonium sulfate precipitation, the supernatant solution was used to purify ACE by Q-Sepharose HP as a strong anion exchanger and then by Sephacryl HR S200(gel filtration). After these steps, ACE purification was confirmed by PAGE and SDS-PAGE. The molecular weight found for ACE was 175 KD. Final specific activity was 39.1 units/mg which was achieved through 651 fold purification and the activity recovered was 5.2%. After purification, Km of ACE for Hippuryl-Histidyl-Leucine, a synthetic substrate, was 2.17 mM. Concentrations between 0.001-0.1mM of Captopril, a competitive inhibitor, inhibited ACE almost completely. Optimal pH determined for ACE activity was 8.3. Incubation temperature above 37˚c decreased ACE activity remarkably. ACE purification in three steps(previously often in 5 steps), a decrease in purification steps, procedure time and expenditure and also acceptable activity and yield, were all due to using resins with high resolution and also FPLC (Fast Protein Liquid Chromatography) system which, finally, facilitated the chromatographic procedures.
Type of Study: Research |
Subject:
Biochemistry
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