Coumarin-Ser-Asp-Lys-Pro-OH, a fluorescent substrate for determination of angiotensin-converting enzyme activity via high-performance liquid chromatography.
N-Acetyl-Ser-Asp-Lys-Pro-OH (AcSDKP-OH), a negative regulator of hematopoietic stem cell proliferation, is shown to be a physiological substrate of angiotensin I-converting enzyme (ACE), a zinc-dipeptidyl carboxypeptidase, involved in cardiovascular homeostasis. Recently, a study carried out on captopril-treated volunteers revealed that the kinetics of [3H]AcSDKP-OH hydrolysis in vitro in the plasma of donors correlates closely to the plasmatic ratio angiotensin II/angiotensin I, which characterized the conversion activity of ACE. This prompted us to design a fluorescent substrate, 2-[7-(dimethylamino)-2-oxo-2H-chromen-4-yl]acetyl-SDKP-OH, or coumarin-SDKP-OH, which could be an alternative to the radiolabeled analogue used in that study, allowing an easier and more rapid determination of enzyme activity. We report here the synthesis and the determination of the kinetics constants of this fluorescent derivative compared with those of [3H]AcSDKP-OH with human plasma ACE (133 and 125 microM, respectively), which are in the same range as those of the physiological substrate angiotensin I. Furthermore, the hydrolysis of the fluorescent substrate shows the same sensitivity toward chloride concentration as the natural substrate, demonstrating its specificity for N-domain hydrolysis. This fluorescent derivative was used to develop a sensitive assay for the determination of ACE activity in human plasma.
Cheviron
N
N
Institut de Chimie des Substances Naturelles, Centre National de la Recherche Scientifique, Gif sur Yvette, France.
Rousseau-Plasse
A
A
Lenfant
M
M
Adeline
M T
MT
Potier
P
P
Thierry
J
J
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003374:Coumarins / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D009842:Oligopeptides / Q000737:chemistry*; D007703:Peptidyl-Dipeptidase A / Q000097:blood*; D013050:Spectrometry, Fluorescence
C410033:(7-(dimethylamino)-2-oxobenzopyran-4-yl)acetyl-seryl-aspartyl-lysyl-proline; D003374:Coumarins; D005456:Fluorescent Dyes; D009842:Oligopeptides; D007703:Peptidyl-Dipeptidase A