A Sensitive Fluorimetric Assay for Serum Angiotensin-con venrting Enzyme

Abstract

A simple, rapid, highly sensitive and reproducible assay for angiotensin-converting enzyme in untreated serum is described. It is based on the conversion of the substrate analog, hippuryl-L-histidyl-L-leucine (5 mM in 0.1 M K phosphate, pH 8.3-0.3 M NaCl) to hippurate and L-histidyl-L-leucine, which is quantified spectrofluorimetrically (lamdba excitation = 360 nm; lamdba fluorescence = 500 nm) by formation of a fluorescent adduct with ophthaldialdehyde. The chloride requirement and inhibition and activation patterns correspond to those for angiotensin-converting enzyme. The Km for hippuryl-L-histidyl-L-leucine was 1.33 mM. The mean value of serum angiotensin-converting enzyme for 58 normal human subjects (mean age, 32 years; range 19-57) was 32.2 +/- 1.30 (SE), with a standard deviation of 9.87 nmol/min/ml serum. The assay is useful for the diagnosis and possible management of sarcoidosis and may have other applications in the future.

Development and characterization of novel potent and stable inhibitors of endopeptidase EC 3.4.24.15

“…The K m had been determined as 19.8 µM by a Lineweaver-Burk analysis of initial velocities measured in triplicate at a range of QFS concentrations (1.1-140 mM) [13]. ACE (human plasma) was assayed by using the fluorimetric ACE method, as described by Friedland and Silverstein [14], which measures the degradation of hippuryl-His-Leu. JA-2 was added to a final concentration of 0.5 mM.…”

Development of an at-line method for the identification of angiotensin-I inhibiting peptides in protein hydrolysates

“…In our view the HHL substrate is attractive because it shows the closest resemblance to AI, the physiological substrate. Techniques to monitor the conversion rate of HHL include chromatographic or electrophoretic quantification of HHL or its products H and HL [10,11], or colorimetric or fluorimetric detection after OPA [12] or TNBS derivatisation [13]. Chromatographic quantification minimises errors due to background peptides but clearly is much slower than colorimetric methods.…”

Constancy of serum angiotensin-converting enzyme activity in normal and complicated pregnancy

“…All specimens were assayed within 2 weeks of collection and storage. Enzyme activity was determined by the fluorometric assay technique of FRIEDLAND and SIL-VERSTEIN [1]. This procedure measures the dipeptide hydrolysis product histidyl-leucine generated by ACE activity on the substrate hippuryl-L-histidyl-L-leucine.…”

“…Although serum levels of angiotension II appear to remain relatively constant during pregnancy, the pregnant woman's ability to respond to infusion of the pressor substance is markedly diminished [1]. It is hypothesized that angiotensin converting enzyme (ACE), either through it's direct actions or it's inhibitory effect upon bracykinin, could play an important role in the development of the hypertensive disorders of pregnancy.…”