Coumarin-Ser-Asp-Lys-Pro-OH, a fluorescent substrate for determination of angiotensin-converting enzyme activity via high-performance liquid chromatography
N. Cheviron et al., Coumarin-Ser-Asp-Lys-Pro-OH, a fluorescent substrate for determination of angiotensin-converting enzyme activity via high-performance liquid chromatography, ANALYT BIOC, 280(1), 2000, pp. 58-64
N-Acetyl-Ser-Asp-Lys-Pro-OH (AcSDKP-OH), a negative regulator of hematopoie
tic stem cell proliferation, is shown to be a physiological substrate of an
giotensin I-converting enzyme (ACE), a zinc-dipeptidyl carboxypeptidase, in
volved in cardiovascular homeostasis. Recently, a study carried out on capt
opril-treated volunteers revealed that the kinetics of [H-3]AcSDKP-OH hydro
lysis in vitro in the plasma of donors correlates closely to the plasmatic
ratio angiotensin II/angiotensin I, which characterized the conversion acti
vity of ACE. This prompted us to design a fluorescent substrate, 2-[7-(dime
thylamino)-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, a
llowing an easier and more rapid determination of enzyme activity. We repor
t here the synthesis and the determination of the kinetics constants of thi
s fluorescent derivative compared with those of [H-3]AcSDKP-OH with human p
lasma ACE (133 and 125 mu M, respectively), which are in the same range as
those of the physiological substrate angiotensin I. Furthermore, the hydrol
ysis of the fluorescent substrate shows the same sensitivity toward chlorid
e concentration as the natural substrate, demonstrating its specificity for
N-domain hydrolysis. This fluorescent derivative was used to develop a sen
sitive assay for the determination of ACE activity in human plasma. (C) 200
0 Academic Press.