Assessment and optimization of kinetic methods for angiotensin-converting enzyme in plasma

Abstract

In the kinetic angiotensin-converting enzyme (ACE) method, a practical and optimal buffer is 80 mmol/L borate buffer at pH 8.2 (37 degrees C). A lag phase is detected in the reaction, and a 5-min incubation of substrate and plasma is suggested before the kinetic measurement. The substrate, N-[3-(2-furyl)acryloyl]-L-phenylalanylglycylglycine (FAPGG), concentration is maximized at 1.0 mmol/L and the measurement wavelength is at 345 nm to ensure linearity of measurement. The proposed procedure uses a 1:9 plasma-to-reagent volume ratio. The linear range of the assay extends to approximately 170 U/L, representing a 25% substrate hydrolysis. The FAPGG absorptivity is determined by measuring the difference in absorbance between 1.0 mmol/L FAPGG and the product solutions. The wavelength fidelity is checked by noting the expected absorbance value of the FAPGG solution, and a 1.0-nm deviation from 345 nm alters the absorbance by 15.5%. The precision of ACE assays at approximately 60 and 100 U/L is 3.5% and 2.4% within batch and 2.9% and 2.6% between batch, respectively. The reference interval (2.5th to 97.5th percentiles) is 41-139 U/L, and there is no difference between values for men and women.

Purification of swine serum angiotensin converting enzyme with high recovery of activity using lisinopril coupled to epoxy‐activated sepharose 6B

“…ACE activity was quantified from the moles of FAP formed. This method is a modification of Buttery and Stuart's procedure [7], which uses a spectrophotometric detection of FAP, showing improved sensitivity and accurate quantitation of ACE activity on samples of less then 1 ~tl. Table 1 Purification of swine serum ACE.…”

“…FAPGG hydrolytic activity. FAPGG hydrolytic activity was measured following the kinetic ACE methods proposed by Buttery and Stuart [7].…”

“…The active fraction was etuted from the affinity column with 50 mM sodium tetraborate buffer, pH 9.5, containing 100 ~tM ZnCI2. The hydrolytic activity was measured following the kinetic ACE methods proposed by Buttery and Stuart[7] using FAPGG as substrate. No activity was found in the loading eluate nor in the washing step.…”

Relationships between left ventricular mass and the renin–angiotensin system, catecholamines, insulin and leptin

“…Plasma renin activity (PRA) was determined as generated Ang I, by radioimmunoassay (RIA) [27]. Serum angiotensin converting enzyme (ACE) was measured using the artificial substrate 3‐(2‐furyl‐akryloyl)‐ l ‐phenylalanyl‐glycyl‐glycin (FAPGG) from which 3‐(2‐furyl‐akryloyl)‐ l ‐phenylalanyl (FAP) is formed and was determined by spectrophotometry [28]. Serum aldosterone was analysed by RIA [29].…”

Presence and comparison of angiotensin converting enzyme in commercial cell culture sera

“…FAPGG hydrolytic activity. FAPGG hydrolytic activity was measured following the kinetic ACE methods [12].…”