PROTOCOL

A fluorescence-based protocol for quantifying

angiotensin-converting enzyme activity
Miguel Ángel Sentandreu and Fidel Toldrá
Instituto de Agroquı́mica y Tecnologı́a de Alimentos (CSIC), P.O. Box 73, 46100 Burjassot (Valencia), Spain. Correspondence should be addressed to
M.A.S. (ciesen@iata.csic.es) and F.T. (ftoldra@iata.csic.es).

Published online 29 December 2006; doi:10.1038/nprot.2006.349

The determination of angiotensin-converting enzyme (ACE) activity represents a useful tool in the study of different health
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols

pathologies, such as hypertension. This protocol describes a fluorescent assay for measuring ACE activity in vitro with high
precision and sensitivity. The method relies on the ability of ACE to hydrolyse the internally quenched fluorescent substrate
o-aminobenzoylglycyl-p-nitro-L-phenylalanyl-L-proline. The generation of the fluorescent product o-aminobenzoylglycine can be
continuously monitored, preferably using a microtiter-plate fluorometer, though the use of a conventional cuvette fluorometer would
also be possible. The method has important advantages with respect to other assays, because it involves only a one-step reagent, is
easy to carry out and allows the analysis of an elevated number of samples in shorter times. It can be completed in one and a half
hours. In addition, the fact that all reagents are commercially available allows the rapid introduction of the assay into the laboratory.

INTRODUCTION
Angiotensin-converting enzyme (ACE 3.4.15.1) is also named
peptidyl-dipeptidase A because it removes C-terminal dipeptides
from different oligopeptide substrates that have a free C terminus.
In addition to this, an endopeptidase ACE action on several
substrates that have a blocked C-terminus has been reported1.
ACE is a key enzyme of the renin-angiotensin system that regulates
blood pressure in the organism because it cleaves angiotensin I into
the potent vasoconstrictor angiotensin II, which also promotes salt
retention. ACE also inactivates the vasodilator bradykinin by the
sequential removal of two C-terminal dipeptides. Therefore, the
overall effect of ACE activity is an elevation of blood pressure. In
this respect, the discovery of compounds that are capable of
strongly suppressing ACE activity represents a successful strategy
for the treatment of hypertension, which is one of the most
widespread chronic diseases in developed countries2.
Apart from angiotensin I and bradykinin, ACE has been shown
to cleave other bioactive peptides, for example neurotensin, sub-
stance P or the insulin B chain1. For that reason, and considering
the different tissues in which this enzyme has been localized, ACE
has been implicated in different physiological processes, other than
blood-pressure regulation, so that abnormal levels of ACE activity
have been related to conditions such as sarcoidosis3, infertility4,
anemia5 or migraine6. The central role that ACE has in blood
pressure regulation and other physiological processes makes the
measurement of its enzyme activity a valuable tool for medical and
clinical research, so efforts to develop different in vitro ACE assays
are widely described in the literature. In this respect, the main
reference is the method developed by Cushman and Cheung in
1971 (ref. 7), an assay that was decisive in the discovery of the first
ACE inhibitory peptides and in the further design of therapeutic
drugs, such as captopril. This method has been further used by
many other authors, either in the same way as initially described by
Cushman and Cheung7 or with some modifications8–10. It is based
in the hydrolysis of the synthetic peptide hippuryl-L-histidine-L-
leucine (Hip-His-Leu) by the action of ACE and further spectro-
photometric determination of hippuric acid, which is one of the
reaction products. Even though this assay has been useful for
decades, it has some important limitations, such as the required
extraction of the product from the reaction mixture with an
organic solvent, which limits the number of samples that can be
analyzed per day and introduces an additional source of error. This
makes the continuous monitoring of the enzyme reaction unfea-
sible, which is another important limitation.
Here we describe the protocol for an accurate, rapid and sensitive
assay of ACE activity, which has been recently developed in our
laboratory11. The method is based in the intramolecularly
quenched fluorescent tripeptide o-aminobenzoylglycyl-p-nitro-
L-phenylalanyl-L-proline (Abz-Gly-Phe(NO2)-Pro) developed by
Carmel and Yaron12. Hydrolysis of this substrate by the action of
ACE generates the fluorescent product o-aminobenzoylglycine
(Abz-Gly), which can be easily quantified fluorimetrically using
appropriate excitation and emission wavelengths. We present the
optimal conditions for a continuous assay in a fluorescent micro-
plate reader (recommended), or with a standard cuvette fluorom-
eter if a fluorescent microplate reader is not available.
This protocol involves only a one-step reagent, avoiding further
derivatization13, extraction with organic solvents9,10 or chromato-
graphic separations of the reaction products14,15. The simplicity of
the reaction steps also makes the assay easy for non-specialised staff
to do. Fluorescence detection of the reaction products results in
high sensitivity and precision, and the fact that all reagents are
commercially available is a major advantage allowing the easy
introduction of the assay in the laboratory. Another main advan-
tage of this protocol with respect to others currently in use is the
possibility of processing a higher number of samples in a shorter
time, when the use of a microplate fluorometer is feasible.
The protocol described here can be applied to directly quantify
ACE activity in biological samples but also to determine ACE-
inhibitory activity and IC50 values for those compounds that are
potential ACE inhibitors and therefore possible antihypertensive
agents. In this latter case, ACE activity that is obtained in the
presence of the studied compound must always be referred to as the
activity in the absence of any added compound to the reaction
mixture (control for absence of ACE inhibition). The steps and

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quantity of reagents indicated here are given for an individual assay appropriate volume, normally 300–500 ml. If using cuvettes with
in one well of a microplate. The volumes indicated for the different larger volumes, then the proportion of the different reagents should
reagents can be maintained if using a microcuvette with the be maintained while adjusting to a larger total reaction volume.

MATERIALS
REAGENTS
. o-aminobenzoylglycyl-p-nitro-L-phenylalanyl-L-proline
(Abz-Gly-Phe(NO )-Pro) (Bachem, cat. no. M-1100)
2 make a protein concentration of approximately 150 mg ml–1. Make sure that the
. o-aminobenzoylglycine (Abz-Gly) (Bachem, cat. no. E-2920) to make the
calibration curve of product generation
. Angiotensin-converting enzyme (ACE) from different sources (for example,
rabbit-lung ACE from Sigma, cat. no. A-6778) when the protocol is applied
to the study of ACE inhibitors
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols
REAGENT SETUP
ACE stock solution Dilute lyophilized ACE in glycerol at 50% with buffer A to
enzyme is correctly dissolved. This solution can be used either immediately or
stored at –201C as liquid until use. In that state, ACE can be maintained for more
than one year without a significant loss of activity.
ACE working solution Dilute ACE stock solution with buffer B to make an
enzyme concentration of approximately 7.5 mg ml–1. This will give an enzyme

. ZnCl2
. NaCl
. Buffer A: 300 mM Tris-base buffer (pH 8.3) with 2 mM ZnCl2
. Buffer B: 150 mM Tris-base buffer (pH 8.3)
. Buffer C: 150 mM Tris-base buffer (pH 8.3) with 1.125 M NaCl
EQUIPMENT
. Automatic pipette (volume range 25–200 ml)
. Multi-channel pipette (volume range 25–200 ml)
. Reservoirs for holding and pipetting the reagents
. Pipette tips
. Timer (if the fluorometer cannot do kinetic measurements)
. 96-well microplates or micro-cuvettes (300–500 ml) for fluorescence
measurements with reduced background signal. In our assay we used flat-
bottom, black microtiter plates (Thermo Electron, cat. no. 9502867)
. Fluoroskan Ascent microplate fluorometer (Thermo Electron) or microtiter-
plate fluorometer with the following characteristics:
lðexcitationÞ ¼ 355
375 nm
lðemissionÞ ¼ 400
430 nm
activity of B3 mU ml–1 in this working solution using rabbit-lung ACE from
Sigma as the enzyme source and defining one unit of enzyme activity (U) as the
amount of ACE hydrolysing 1 mmol of Abz-Gly-Phe(NO2)-Pro per minute
at 37 1C and pH 8.3. As reference, this enzyme solution will hydrolyse 1.5
nanomoles of hippuryl-His-Leu per min per ml under conditions described
previously7. Mix the solution well. m CRITICALThis should be made fresh on the
day of the experiment.
Substrate stock solution Dissolve approximately 30 mg of Abz-Gly-Phe
(NO2)-Pro in buffer C to make a concentration of approximately 10 mM. Once
completely dissolved, place aliquots of the solution in volumes of approximately
1 ml in Eppendorf tubes. The solution can be used immediately for the present
assay and the remaining aliquots can be stored at –20 1C for further experiments.
m CRITICAL This substrate is in powder form but, on contact with the buffer, it
becomes sticky and difficult to dissolve. To facilitate its complete dissolution, the
use of ultrasounds (10–15 min) and a spatula are recommended.
Substrate working solution Take into consideration the concentration
of the substrate stock solution and dilute it with buffer C to make a final
Abz-Gly-Phe(NO2)-Pro concentration of 0.45 mM. m CRITICAL Avoid exposure
to light until use. This should be made fresh on the day of the experiment.

PROCEDURE
1| Both ACE activity and ACE-inhibitory activity can be determined.
(A) Determining ACE activity
(i) When determining ACE activity in biological samples, it is recommended that controls for determining ACE-specific activity
are included in the sample. Add 50 ml of a specific ACE inhibitor (for example, 0.1 mM captopril) dissolved in buffer B. In
the absence of any interfering enzyme activity, no fluorescence will be generated in these controls. For samples, add 50 ml
of buffer B.
(B) Determining ACE-inhibitory activity
(i) Add 50 ml of the compound for which you wish to evaluate the ACE-inhibitory activity. For reference of no inhibition of
the enzyme reaction, add 50 ml of the solution in which samples are diluted.
m CRITICAL STEP It is important to dispense this volume correctly in the bottom of the wells or cuvettes in order to
avoid errors in the determination. Therefore, using either a single or a multichannel pipette, always pipette using the
reverse technique, touching the bottom slightly with the tip in order to recover all drops, avoiding aspiration of liquid in
the meantime. It is also advisable to make replicates of samples (duplicate at least, triplicate ideal).

2| Add 50 ml of the sample in which you wish to evaluate ACE activity (Step 1A(i)) or, in inhibition studies (Step 1B(i)),
50 ml of ACE working solution. When filling many wells of a microplate, the use of a multi-channel pipette is recommended.
m CRITICAL STEP Dispense the volume of this solution carefully, as described in Step 1.
3| Carefully shake the microplate or cuvette for a few seconds in order to mix the components. When working with microtiter
plates, automatic shaking rather than manual shaking would be preferable in order to avoid cross-contamination between the wells.
4| In order to have a linear response of the enzyme reaction from the start of monitoring, pre-incubate the plate or cuvette at
37 1C for 10 min. Separately, pre-incubate the substrate in the same conditions.

5| Start the enzyme reaction by adding 200 ml of substrate working solution.

m CRITICAL STEP As in Steps 1 and 2 it is important to properly dispense this solution into the well or cuvette, but also to
fill the different wells in a reasonably short time. This time can easily be less than 2 minutes for filling a complete 96-well
microplate with the aid of a multi-channel pipette.

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6| Gently shake the microplate or cuvette as described in 7

Step 3.
6

7| Read the plate or cuvette in the fluorometer using 5

excitation and emission wavelengths indicated above and

nmoles Abz-Gly
4
immediately turn the timer on. This corresponds to the
fluorescence in the reaction mixture before the action of 3
the enzyme (t0). y = 0,1364x
? TROUBLESHOOTING 2 R 2 = 0,9981

1
8| Incubate the plate or cuvette at 37 1C. Normally an
appreciable fluorescence generation due to the release of free 0
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols

0 10 20 30 40
o-aminobenzoylglycine (Abz-Gly) from Abz-Gly-Phe(NO2)-Pro is
Incubation time (minutes)
observed after 5 min of incubation. However, such a short
incubation time is not recommended because a small delay Figure 1 | Generation of Abz-Gly from Abz-Gly-Phe(NO2)-Pro hydrolysis by
at the moment at which the zero value is taken can greatly rabbit-lung angiotensin-converting enzyme (ACE) as a function of time.
influence the obtained results. Under the assay conditions
explained here, the incubation time can be extended up to 40 minutes without a significant loss in the linearity of the reaction
rate (Fig. 1). Therefore, a recommended incubation time for single measurements is 30 min (ti ¼ 30).

9| After incubation, read the plate again as in Step 7. For kinetic measurements, read the plate at the desired time intervals.
ACE activity in the different wells is linearly related to the fluorescence generated during the incubation time (ti – t0). This
relation can be easily obtained by making a calibration curve with free Abz-Gly (fluorescence reaction product), plotting Abz-Gly
concentration versus obtained fluorescence. Recommended Abz-Gly concentrations for this curve range between 5 and 30 mM.
? TROUBLESHOOTING

 TIMING
Preparation of the enzyme solutions
10 min

Preparation of substrate solutions

30 min

Assay of ACE activity using a microplate

60 min for 96 samples

Assay of ACE activity using cuvettes

32 min per sample, when using a single microcuvette (this time can be notably reduced if several samples are incubated at
the same time in different fluorescence cuvettes)

? TROUBLESHOOTING
Troubleshooting advice can be found in Table 1.

TABLE 1 | Troubleshooting table.

Step Problem Possible reason Solution
7 High fluorescence values obtained High intrinsic fluorescence of the biolo- Try to dilute the sample until the background
immediately after the addition of the gical sample, in which ACE activity is to fluorescence is reduced but appreciable ACE
enzyme (t0). be determined. activity is retained. If necessary, work with
longer incubation times.
Background fluorescence caused by any Include blank samples containing all
spoiled assay component. reagents of the assay except ACE. If fluor-
escence of blanks is high, then replace
buffers and reagents by new ones.

9 No generated fluorescence is observed Excitation and/or emission wavelengths Make sure that excitation and emission
after incubation. of the fluorometer are not correctly wavelengths are correctly chosen according
established. to requirements described in the equipment
section.

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TABLE 1 | Troubleshooting table (continued).

Step Problem Possible reason
The final assay mixture does not have an
appropriate pH for ACE activity. This can
happen if samples are dissolved in very
strong acid buffers or if buffers A, B or C
are spoiled.
ACE has no enzyme activity.
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols
Solution
Reproduce the proportions of the assay
mixture in a sufficient volume for measuring
pH. Be sure that pH is approximately 8.3. If
this is not the case, dissolve samples in an
adequate buffer yielding an adequate final
pH of the reaction mixture. Be sure that
buffers A, B and C are not spoiled.
Prepare new samples taking care to preserve
the enzyme activity.
In inhibition studies, assay ACE activity in

the absence of any compound or buffer other

than those belonging to the assay. If no
fluorescence is generated, prepare a new ACE
stock solution.

9 Large variability in the fluorescence
obtained for replicates of the same
sample.
Incorrect addition of the reagents Dispense correctly the different reagents
into wells. into wells according to what is stated in
point 1 of the procedure. Normally a coeffi-
cient of variation below 10% should be
obtained for different replicates of the same
sample.

ANTICIPATED RESULTS
In order to show the feasibility of the protocol presented here, the assay was carried out using fluorescence microtiter plates
and rabbit-lung ACE as the enzyme source in two different cases: to show the linearity and sensitivity of the assay by kinetic
measurement of ACE activity (A), and to evaluate the ability of several dipeptides in suppressing ACE activity (B). These
dipeptides were previously assayed in vitro using hippuryl-His-Leu as the substrate16, as described by Cushman and Cheung7.
Fluorescence measurements were carried out in a Fluoroskan Ascent microplate fluorometer using excitation and emission
wavelengths of 355 and 405 nm, respectively.
The generation of the fluorescent group Abz-Gly by the action of angiotensin-I converting enzyme hydrolyzing
Abz-Gly-Phe(NO2)-Pro along the time course is illustrated in Figure 1. The continuous fluorometric detection of Abz-Gly makes
the assay highly sensitive and precise, allowing it to detect product that is generated after 5 minutes of incubation time, which
would correspond to the formation of 0.8 nmoles of Abz-Gly. The generation of the product remained linear for incubation times
of up to 40 minutes under the assay conditions described in the protocol. At longer incubation times and/or higher enzyme
concentrations, the linearity can turn towards slightly slower reaction rates11. For that reason, when determining ACE activity
in biological samples, those yielding high activity values should be appropriately diluted. Taking these findings together,
we recommend the assay conditions described in the protocol as a good compromise between low enzyme consumption
(0.15 mU of ACE per assay), appreciable fluorescence generation and a reasonable incubation time (30 min).
IC50 values of ACE activity obtained for several dipeptides using this protocol with a microplate fluorometer are shown in
Table 2. These values were obtained by assaying ACE activity (using the working solution at 3 mU ml–1) in the presence of
each dipeptide at seven different concentrations, ranging from 0.5 to 200 mM in the final assay mixture. The inverse of relative

TABLE 2 | Effect of several dipeptides on ACE activity using the present protocol compared with results
obtained by Cheung et al.7 using Hippuryl-His-Leu as the substrate.
Peptide IC50 (lM)a Abz-Gly-Phe(NO2)-Pro IC50 (lM)b Hippuryl-His-Leu
Val-Tyr 4.6 22
Arg-Pro 15 180
Gly-Pro 66 450
Gly-Phe 117 630
Phe-Gly 516 3,700
Ala-Gly 1,020 2,500
Gly-Gly 3,450 7,200
aPeptide concentration inhibiting 50% of ACE activity, determined by continuous fluorimetric assay at pH 8.3, using 0.3 mM Abz-Gly-Phe(NO )-Pro as substrate and
2
0.75 M NaCl in the reaction mixture. Data adapted from M.A.S and F.T.22,23.
bPeptide concentration inhibiting 50% of ACE activity, determined by end-point spectrophotometric assay with 5 mM hippuryl-His-Leu as substrate and 0.3 M NaCl

in the reaction mixture16.

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 PROTOCOL

ACE-activity values were plotted as a function of peptide concentration and the equation obtained for the linear part of the
curve was used to calculate IC50 as the concentration value obtained for half of the initial activity.
The obtained results are compared with those reported for the same dipeptides by Cheung et al.16. As can be observed, results
are comparable between the two assays with respect to the inhibition degree generated by the different peptides. In both
experiments Val-Tyr was the dipeptide showing the strongest ACE inhibition, followed by Arg-Pro, Gly-Pro and Gly-Phe. This is
in accordance with the inhibition pattern observed for ACE where many peptides containing an aromatic amino acid (Tyr, Phe
or Trp) together with Pro in a C-terminal position, are between the most potent ACE inhibitors17–19. This feature is clearly
appreciated by comparing IC50 values obtained for Gly-Phe and Phe-Gly, the inhibition exerted by Gly-Phe being notably higher.
Using the protocol described here, IC50 values obtained for the different peptides were lower than those obtained in the
previous work16. This can be explained by considering the fact that the units of ACE, the type of assay and the substrate used
for the assay of ACE activity are different. In such cases the obtained IC50 values are difficult to compare20,21 even though,
as pointed out by Fuglsang et al.20, the order in the inhibition rate should be normally maintained. In our case, the order of
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols

the inhibition rate between the two assays was maintained for the assayed dipeptides, except for Phe-Gly and Ala-Gly, which
showed little inhibition of ACE activity.

ACKNOWLEDGMENTS This work was supported by an I3P contract from the
European Social Fund (M.A.S.) and by a Marie Curie ERG grant from the European
Commission (M.A.S.).
COMPETING INTERESTS STATEMENT The authors declare that they have no
competing financial interests.
Published online at http://www.natureprotocols.com
Rights and permissions information is available online at http://npg.nature.com/
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