Purification and Properties of Angiotensin-Converting Enzyme from Hog Lung

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Abstract
Angiotensin-converting enzyme was purified 1500-fold from a homogenate of hog lungs. The purification procedure included fractionation with ammonium sulfate, inactivation of contaminating enzymes at pH 4.7, batch treatment with CM-cellulose, and chromatography on columns of DEAE-cellulose, hydroxyapatite, and Sephadex G-200. The enzyme was assayed with hippurylglycylglycine and, after pH 4.7 was established, with both hippurylglycylglycine and angiotensin I as substrates. The ratio of activity toward the two substrates remained constant throughout the later stages of purification. The final enzyme preparation, on analytical disc gel electrophoresis, showed one major protein band with the expected ratio of activities toward both substrates. The molecular weight was estimated to be approximately 300,000 by gel filtration. The pH optimums with hippurylglycylglycine and angiotensin I as substrates were 8.4 and 7.5, respectively, and chloride ion was required for optimal activity of the enzyme with both substrates. Hydrolysis of both substrates was inhibited by ethylenediaminetetraacetic acid (EDTA) or the pentapeptide pyroglutamyllysyltryptophanylalanylproline. It would, therefore, appear that a single enzyme, purified in the described manner, hydrolyzes both angiotensin I and hippurylglycylglycine.