An Improved Fluorometric Assay of Rat Serum and Plasma Converting Enzyme

SUMMARY The most sensitive nonradiometric routine assay for angiotensin-converting enzyme (ACE) activity uses fluorometry to detect His-Leu released from Hip-His-Leu. Our results indicate that, in contrast to human serum, rat serum and plasma contain large and variable amounts of dipeptidase activity that lead to a subestimation of the ACE activity measured in 0.1 M potassium phosphate buffer, pH 8.3, containing 0.3 M NaCI, the most commonly used assay for human serum and tissue ACE. We describe and validate an assay for 1 to 10 /xL rat and human serum or plasma using 5 mM Hip-His-Leu in 500 fiL of 0.4 M sodium borate buffer, pH 8.3, containing 0.9 M NaCI at 37°C that reduced the subestimation error to ^ 3 % (rat serum) and ^ 0.1 % (human serum) and increased the ACE activity twofold to threefold. The Km and V, ^ are reported for rat serum ACE (Hip-His-Leu) and dipeptidase (His-Leu) in borate buffer and phosphate buffer. Rat serum ACE hydrolysis of Hip-His-Leu measured by fluorometry correlated (r = 0.99, p < 0.05) with the hydroly-sis of angiotensin I measured by high-performance liquid chromatography. A direct method based on ami no acid analysis is described for evaluating the dipeptidase error of complex mixtures such as tissue extracts and other physiological fluids. We have found that the assay can be used to measure ACE activity in 25 samples (in duplicate) in 2 hours with small intraassay (2.2%) and interassay (3.9%) coefficients of variation. It appears to be suitable for serial measurements in small animals in studies of the renin-angiotensin system in experimental hypertension and other physiopathological conditions. (Hypertension 7: 244-252, 1985) KEY WORDS • rat angiotensin-converting enzyme • rat serum conversion of angiotensin I rat dipeptidase activity • fluorometric converting-enzyme assay • renin-angiotensin system RENIN and angiotensin-converting enzyme(ACE) release the vasoconstrictor peptide an-giotensin II (ANG II) from the precursor pro-tein angiotensinogen by sequential, specific limited proteolysis reactions. Renin and ACE have a diffus