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Analytical Biochemistry 280, 286–290 (2000)doi:10.1006/abio.2000.4535, available online at http://www.idealibrary.com on

An Assay for Angiotensin-Converting Enzyme UsingCapillary Zone Electrophoresis

Rong-zhen Zhang,1 Xiao-hua Xu, Tian-bao Chen, Long Li, and Ping-fan Rao2

Institute of Biotechnology, Fuzhou University, Fujian 350002, People’s Republic of China

Received November 19, 1999

A sensitive and rapid method was developed for an-giotensin-converting enzyme (ACE) activity determi-nation by capillary zone electrophoresis. Hippuryl-L-histidyl-L-leucine, a synthetic tripeptide, was used asthe ACE-specific substrate. Capillary zone electro-phoresis was employed to separate the products of theenzymatic reaction and the ACE activity was deter-mined by quantification of hippuric acid, a result ofthe enzymatic reaction on the tripeptide. The capil-lary electrophoresis was performed in a 27 cm 3 75 mmi.d. fused-silica capillary using 200 mM boric acid–borate buffer (pH 9.0) as a run buffer with an appliedvoltage of 8.1 kV at a capillary temperature of 23°C.The electrophoresis was monitored at 228 nm. Eachelectrophoretic run requires only a nanoliter of theenzymatic reactant solution, at only 6 min, rendering apowerful tool for the ACE assay. © 2000 Academic Press

Key Words: angiotensin-converting enzyme; hip-puryl-L-histidyl-L-leucine; hippuric acid; capillaryelectrophoresis.

Angiotensin-converting enzyme (ACE),3 a mem-brane-bound glycoprotein, is a dipeptidyl carboxypep-tidase (EC 3.4.15.1), which has mainly been localizedto the endothelial cells of pulmonary capillaries (1).ACE plays an important role in the regulation of bloodpressure and fluid balance. It does this by convertingthe inactive decapeptide angiotensin I to the pressoroctapeptide angiotensin II, liberating the powerful va-

1 Present address: Department of Pathology & AIDS ResearchProgram, San Francisco General Hospital, Box 0874, University ofCalifornia, San Francisco, CA 94143. E-mail: jzhang@sfaids.ucsf.edu.

2 To whom correspondence should be addressed. Fax: 01186 (591)3732462. E-mail: ibfu@fzu.edu.cn.

3 Abbreviations used: ACE, angiotensin-converting enzyme; His-Leu, L-histidyl-L-leucine; HHL, hippuryl-L-histidyl-L-leucine; HA,

ippuric acid; CE, capillary electrophoresis.

86

soconstricting angiotensin II from the decapetide bymediating the cleavage of the C-terminal dipeptideHis-Leu of the decapeptide angiotensin I (2). It is alsoinvolved in the degradation of the vasodilator brady-kinsin (3, 4).

The ACE inhibitors captopril (5), enalapril, and lis-inopril (6) have made it possible to treat hypertensioneffectively, acting as antihypertensive drugs. Foryears, large quantities of ACE inhibitory componentsderived from functional foods and other natural mate-rials with hypotensive effect have been developed onthe basis of ACE inhibitory activity screening (7–17).They are potentially effective resources for antihyper-tensive drugs as well. On the other hand, increasedACE activity has also been correlated to sarcoidosis(18) and other diseases (19). It is apparent that deter-mination of ACE activity is important clinically as wellas in ACE inhibitor screening.

Various methods for ACE activity determinationhave been reported, including bioassay (20, 21), radio-isotopic (22, 23), spectrophotometric (18, 24), and flu-orometric (15, 25) methods. High-performance liquidchromatography was introduced (26–30), since hip-puric acid generated by the enzymatic reaction cannotbe completely separated from the substrate by solventextraction. Isolation and quantification of hippuric acidcan be obtained in a single step in the HPLC procedure.All the reported methods used reverse-phase chroma-tography, which inevitably involves organic solventssuch as acetonitrile and methanol of HPLC grade. Wehave also developed a method for ACE activity deter-mination by HPLC on a Toyopearl HW-40S column(31). The combined characteristics of reverse-phasechromatography and gel chromatography of the resinare capable of obtaining a simple and satisfactory sep-aration of the substrate and products from the enzy-matic reaction of ACE.

Capillary electrophoresis (CE) is a powerful new an-

alytical tool with improved resolution, less sample

0003-2697/00 $35.00Copyright © 2000 by Academic Press

All rights of reproduction in any form reserved.

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287ANGIOTENSIN-CONVERTING ENZYME ACTIVITY ASSAY BY ELECTROPHORESIS

load, and shorter operation time than previous meth-ods. In this study, capillary electrophoresis was used todevelop a more efficient method for ACE activity de-termination by exploiting the instrumentation to itsfullest advantages.

MATERIALS AND METHODS

ACE Assay

The enzymatic reaction was based on the methodproposed by Saito et al. (12), using hippuryl-L-histidyl-L-leucine (HHL) as a substrate, which is a modificationof Cushman’s method (24). Angiotensin-converting en-zyme (ACE) from rabbit lung, with a marked activity of2.4 U/mg of protein, HHL, and hippuric acid (HA) werepurchased from Sigma (St. Louis, MO). All other re-agents were analytical grade. The substrate solution of5 mM HHL and ACE solution of 100 mU/mL markedactivity were used as test solutions throughout thismethod. They were prepared by dissolving HHL andACE in 100 mM boric acid–borax buffer containing 0.5N NaCl at pH 8.3. To begin the assay, 5 mL of ACEsolution was added to 10 mL of deionized water andincubated at 37°C for 3 min in a water incubator(Model 501, Shanghai, China). In the case of ACEinhibitory activity determination, a 10-mL inhibitorsample solution was used instead of deionized water,while a corresponding buffer solution was used in thecontrol assay. After 3 min of incubation, 50 mL of thesubstrate solution was added to the mixture and thenincubated at 37°C for 30 min. The enzymatic reactionswere terminated by the addition of 85 mL of 1 N HCl.Then 10 mL of the thoroughly mixed reaction solutionwas applied to the CE system for separation and quan-tification of HA.

CE Instrumentation/Run Conditions

Capillary electrophoresis was performed using aBeckman P/ACE 5510 system equipped with a diodearray detector. All separations were carried out usingan uncoated fused-silica capillary with an internal di-ameter of 75 mm (the total length of the capillary was7 cm and the length from the inlet to the detector was0 cm). The CE system was used in the normal polarityode. Data were collected and peak migration time

nd area were analyzed by Beckman System Gold ver-ion 8.1 software.The CE run buffer consisted of 200 mM boric acid–

orate buffer, pH 9.0. The detection wavelength waset at 228 nm and the electrophoresis was carried outt 8.1 kV at a capillary temperature of 23°C. Newapillaries were conditioned by rinsing with 0.1 NaOH followed by deionized water and 20 volumes of

he run buffer. The capillary was rinsed with the run

uffer solution for 1.5 min before each run. A sample

run was performed in approximately 5.5 min after a 1-spressure injection. At the end of each run, the capillarywas rinsed with 0.1 N NaOH for 1.5 min, followed by arinse with deionized water for 1.5 min. Prior to use, allbuffers and solutions used in the assay were filteredthrough a 0.22-mm filter.

Calibration of HA Concentration

A stock standard solution of HA was prepared bydissolving HA in 100 mM boric acid–borax buffer (pH8.3) containing 0.5 N NaCl to a concentration of 2 mMand stored at 4°C. A series of working standards of HAwas obtained from the stock standard solution by dilu-tion with the same buffer over the range 0.01–0.20 mMfor CE analysis. Injections of standards were per-formed in triplicate in order of increasing concentra-tion. A calibration curve was obtained by plotting thearea of the HA peak on the obtained electrophoreticpattern against its concentration.

Definition of ACE Activity

One unit (U) of ACE activity was defined as theamount of enzyme required to catalyze formation of 1mmol of HA from HHL per minute at 37°C under thegiven conditions (24). The ACE activity of each sampleapplied to the CE system could be easily calculated byquantifying the amount of the HA formed in the enzy-matic reaction.

Kinetic Studies of ACE

Kinetic study of ACE was carried out at varioussubstrate concentrations ranging from 0 to 8.40 mM,all in the presence of the same amount (500 mU) of theenzyme to verify the above described method for ACEassay. The velocities of the ACE-catalyzed reactionwere calculated from the amounts of HA generated inthe enzymatic reaction. The initial velocities at varioussubstrate concentrations were determined from theinitial slopes of their corresponding time dependentstudies and then plotted as a function of the HHLconcentrations. Kinetic data were analyzed by nonlin-ear regression analysis of Michaelis–Menten plots.

Statistical Analysis

Statistica V4.5 for Windows and the Beckman Sys-tem Gold Software V8.1 were used for the major dataprocessing throughout this work.

RESULTS AND DISCUSSION

Separation of ACE Assay Reaction Mixture

The optimum capillary electrophoretic conditions for

the ACE reaction mixture were established by investi-

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288 ZHANG ET AL.

gating the influences of the concentration and pH ofthe running buffer. In 200 mM boric acid–borax buffer,pH 9.0, satisfactory separation of the reactants andproducts of the reaction, including HHL, HA, and His-Leu (L-histidyl-L-leucine), was achieved as shown in

ig. 1. The HA is the most anionic component, whileess anionic His-Leu and HHL migrated at 3.8 and 3.9

in, respectively. The reproducibility and precision ofhe assay were excellent as demonstrated in Table 1.his implies the successful suppression of electro-os-atic flow under the electrophoretic conditions ap-

lied.In the present study, each CE run was completed in

ess than 10 min, with a satisfactory separation done in.5 min. The current method compares favorably with

FIG. 1. Typical electropherogram of ACE reaction mixture. En-yme reaction conditions: 5 mM HHL and 500 mU of ACE in 100 mM

boric acid–borate buffer (pH 8.3) with 0.5 N NaCl; total volume, 150mL; enzyme reaction time, 30 min at 37°C. CE conditions: Capillary,fused silica [27 cm (20 cm to detector) 3 75 mm i.d.]; run buffer, 200mM, pH 9.0, boric acid–borate buffer; applied voltage, 8.1 kV; detec-tion at 228 nm; temperature of capillary, 23°C. Peaks: A, histidyl-leucine; B, HHL; C, HA.

TABLE 1

Reproducibility, Linearity, and Sensitivityof HA in the CE System

HA

igration time reproducibility (n 5 8) 0.15%eak area reproducibility (n 5 8) 0.71%inearity 0.01–0.20 mMorrelation coefficient 0.9998

imit detection 1 mM

all other reported ACE assays, which usually requiremore than 20 min. A short analysis time and the fullyautomated feature of data collection and analysis of theCE system render this procedure an ideal method forheavy-load ACE assays and sample screening.

Another remarkable advantage of the proposedmethod is that only boric acid–borate buffer was usedin the CE analysis, and no organic solvents were re-quired, which means lower operation cost and greateravailability of the necessary reagents. The totalamount of the sample required for CE analysis is lessthan 10 mL, a tiny fraction of the sample required bytraditional extraction and spectrophotometric meth-ods. That is also significantly less than HPLC detec-tion. The total volume of 150 mL of the enzymaticreaction mixture is enough for the entire CE analysis.It is apparent that samples, enzyme, and the substraterequired can be proportionally and drastically reducedby this new procedure.

Calibration of HA Standards

An electrophoretogram of the HA standard is shownin Fig. 2, with the migration time of approximately 4.8min. As shown in Fig. 3, a strict linear correlationbetween HA concentration and its peak area was ob-tained. The regression equation was calculated to beEq. [1], with a correlation coefficient of 0.9998 (n 5 3)within the HA concentration range from 0.01 to 0.20mM:

FIG. 2. CE separation of standard HA. CE conditions as in Fig. 1.

AHA 5 3.681@HA# 1 0.002~mM!, [1]

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289ANGIOTENSIN-CONVERTING ENZYME ACTIVITY ASSAY BY ELECTROPHORESIS

where [HA] is HA concentration and AHA is HA peakarea. Perfect linearity in the HA concentration rangeindicated no interaction between HA and the electro-phoretic capillary under the proposed conditions. Byexploiting the ultraviolet absorbancy maximum of HAat 228 nm, the detection limits were in the range of 1.0mM HA.

Measurement of ACE Activity

After each capillary electrophoretic run, HA peakarea could be easily obtained from the electrophoreto-gram by the CE system software; then the concentra-tion of HA could be calculated by Eq. [1]. Consequently,the ACE activity (U) was determined according to Eq.[2]:

U 5 150@HA#/30 5 ~1.358AHA 2 0.003!

3 10 26~U!. [2]

Here 150 and 30 represent the total volume (mL) ofACE reaction solution and enzymatic reaction time(min), respectively.

Enzyme Kinetic Analysis

ACE kinetics was studied by changing the amount ofsubstrate, HHL, in the presence of a fixed amount (500mU) of enzyme. The initial velocity for different concen-trations of HHL was determined by plotting the enzy-matic reaction time as a function of HA peak area andthen was plotted as a function of each substrate con-centration to obtain information on the substrate sat-uration of enzyme. The Michaelis–Menten equation for

FIG. 3. Standard curve for HA from CE electropherograms. CEconditions as in Fig. 1.

ACE derived from rabbit lung was expressed as Eq. [3],

with a Km of 1.454 mM for the substrate HHL and aVmax of 5.069 nM z min21 HA:

V 5 5.069S/~1.454 1 S! ~nMzmin21!, [3]

here V is the velocity and S is the concentration ofubstrate HHL.The value of Km obtained in this study is lower than

those reported previously for rabbit lung ACE, whichwere 2.6 mM by Cushman et al. (24) and 2.3 mM by

as et al. (32), but similar to the value of 1.2 mMeported by Friedland et al. by fluorometric determina-ion of the amount of His-Leu (33). It is likely that theigher values of Km obtained by Cushman and Das

arise from the incomplete separation of HA from thesubstrate by solvent extraction and the difficulties en-countered when using the less sensitive spectrophoto-metric method to determine the enzyme activity at lowsubstrate concentrations (24, 32). The differences inKm may also be attributed to the different buffer sys-ems for the enzyme assay, with boric acid–borateuffer in our method instead of phosphate in the re-orted works.

CONCLUSIONS

In comparison with spectrophotometric and otherassays, the capillary electrophoretic method describedabove is more rapid, sensitive, and automatic, requiresmuch less sample, substrates, and other reagents, andhas high reproducibility. This approach should prove tobe valuable in developing new diagnostic methods ofACE activity assay and screening ACE inhibitors.

Using the procedure described above, we are currentlyinvestigating new inhibitors for ACE from snake venomand traditional Chinese food. The huge numbers of frac-tionation samples and the minor contents of the inhibi-tors in those natural products have made ACE activityassay a major bottleneck. Thanks to its high sensitivityand rapidity, the proposed method has proved to be aright approach to the problem in our work.

ACKNOWLEDGMENTS

This work is sponsored by the Fujian Provincial Training Founda-tion for Bai-Qian-Wan Talents Engineering, China. The authors aregrateful to the 211 Project Foundation for an equipment grant.

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