Chromatographic determination of angiotensin-converting enzyme and angiotensinase activity

An analytical method utilizing an automatic amino acid analyzer is described for the separation, identification, and measurement of 5 to 50 nmol of angiotensin I, angiotensin II, [Des-Phe8]angiotensin II, Phe-His-Leu, His-Leu, isoleucine, leucine, tyrosine, and phenylalanine. Aminex A-5 cation-exchange resin (0.9 x 15 cm) is sequentially eluted with three sodium citrate buffers: pH 3.25, 0.2 N; pH 4.85, 0.54 N, and pH 6.5, 0.39 N at 60 and 80 degrees C. Reaction with ninhydrin is used for detection. This chromatographic system was used to determine angiotensin-converting enzyme activity and the angiotensinase activity of rabbit brain endopeptidase B. In each assay, the unhydrolyzed substrate and both products were measured simultaneously in one step without pretreatment of the hydrolysate. Products were recovered in 1:1 molar ratios and the overall recovery of an hydrolyzed substrate of products was quantitative.