A continuous fluorescent assay for the determination of plasma and tissue angiotensin I-converting enzyme activity

Abstract

A continuous assay using internally quenched fluorescent peptides with the general sequence Abz-peptidyl-(Dnp)P-OH (Abz = ortho-aminobenzoic acid; Dnp = 2,4-dinitrophenyl) was optimized for the measurement of angiotensin I-converting enzyme (ACE) in human plasma and rat tissues. Abz-FRK(Dnp)P-OH, which was cleaved at the Arg-Lys bond by ACE, was used for the enzyme evaluation in human plasma. Enzymatic activity was monitored by continuous recording of the fluorescence (lambda ex = 320 nm and lambda em = 420 nm) at 37 degrees C, in 0.1 M Tris-HCl buffer, pH 7.0, with 50 mM NaCl and 10 microM ZnCl2. The assays can be performed directly in the cuvette of the fluorimeter and the hydrolysis followed for 5 to 10 min. ACE measurements in the plasma of 80 healthy patients with Hip-His-Leu and with Abz-FRK(Dnp)P-OH correlated closely (r = 0.90, P < 0.001). The specificity of the assay was demonstrated by the complete inhibition of hydrolysis by 0.5 microM lisinopril or captopril. Abz-FRK(Dnp)P-OH cleavage by ACE was monitored in rat lung, kidney, heart, and liver homogenates in the presence of a cocktail of inhibitors containing trans-epoxy-succinyl-L-leucylamido-(4-guanido)-butene, pepstatin, phenyl-methylsulfonyl fluoride, N-tosyl-L-phenylalanyl-chloromethyl ketone, and N-tosyl-lysyl-chloromethyl ketone to prevent undesirable hydrolysis. ACE activity in lung, heart and kidney homogenates, but not in liver homogenates, was completely abolished by 0.5 microM lisinopril or captopril. The advantages of the method are the procedural simplicity and the high sensitivity providing a rapid assay for ACE determinations.

Characterization of angiotensin-converting enzymes 1 and 2 in the soleus and plantaris muscles of rats

“…The activity of skeletal muscle ACE1 was determined by a fluorometric assay (14) of samples from the soleus and plantaris tissues obtained from Wistar rats, frozen in liquid nitrogen, and stored at -80°C. Briefly, the homogenate supernatant was incubated for 60 min at 37°C with a fluorogenic substrate containing 10 μM Abz-FRK(Dnp) P-OH (Abz = o-aminobenzoic acid; Dnp = dinitrophenyl) in 0.1 M Tris-HCl buffer, 50 mM NaCl, and 10 mM ZnCl 2 , pH 7.0.…”

Enzima conversora de angiotensina no líquido pericárdico: estudo comparativo com a atividade sérica

“…The substrates with internal fluorescence suppression (FRET peptides), Abz-YRK(Dnp)P-OH, Abz-LFK(Dnp)-OH and Abz-SDK(Dnp)P-OH (Abz = o-aminobenzoic acid; Dnp = 2,4-dinitrophenol) were synthesized at the Department of Biophysics of the Universidade Federal de São Paulo, São Paulo, SP, Brazil [22][23][24] . Other reagents were obtained from Merck.…”

“…The assays with substrates that had internal fluorescence suppression were carried out as previously described [22][23][24] . The optimal pH was determined using aliquots (4 ng) of the purified enzyme and a buffer solution containing: 25 mM of glycine, 25 mM of acetic acid, 25 mM of Mes and 75 mM of Tris in the presence of 100 mM of NaCl and 10 µM of ZnCl 2 in a pH range of 5.0 to 9.5, for a final volume of 1 ml, at 37 o C. The kinetic parameters were determined at 37 o C, in 0.1 M Tris-HCl buffer (pH 7.0) containing NaCl (0.05 M), ZnCl 2 (10 µM) and the substrates Abz-YRK(Dnp)P-OH, Abz-LFK(Dnp)-OH and Abz-SDK(Dnp)P-OH.…”

“…One hundred µL of the pericardial fluid and serum samples were added to the substrate solution for a final volume of 2.0 ml and the enzymatic activity was monitored fluorimetrically as previously described 22 . A fluorescence curve was obtained using the peptide standards before and after total hydrolysis.…”

“…However, the natural tetrapeptide N-Acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) 20 and angiotensin 1-7 (Asp-Arg-Val-Tyr-Ile-His-Pro) 21 are more specific for the catalytic N-domain. Peptides with internal fluorescence suppression [FRET (Fluorescence resonance energy transfer) peptides)], which contain sequences that are susceptible to hydrolysis by ACE have been used for the characterization of N-and C-domains of the enzyme [22][23][24] .…”