studies of reversible inhibition, irreversible inhibition, and activation of alkaline phosphatase by...
TRANSCRIPT
Studies of reversible inhibition, irreversible inhibition, andactivation of alkaline phosphatase by capillary electrophoresisq
Angela R. Whisnant and S. Douglass Gilman*
Department of Chemistry, University of Tennessee, Knoxville, TN 37996-1600, USA
Received 14 November 2001
Abstract
Reversible inhibition, irreversible inhibition, and activation of calf intestinal alkaline phosphatase (EC 3.1.3.1) have been studied
by capillary electrophoresis. The capillary electrophoretic enzyme–inhibitor assays were based on electrophoretic mixing of inhibitor
and enzyme zones in a substrate-filled capillary. Enzyme inhibition was indicated by a decrease in product formation detected in the
capillary by laser-induced fluorescence. Reversible enzyme inhibitors could be quantified by Michaelis–Menten treatment of the
electrophoretic data. Reversible, competitive inhibition of alkaline phosphatase by sodium vanadate and sodium arsenate has been
examined, and reversible, noncompetitive inhibition by theophylline has been studied. The Ki values determined for these reversible
inhibitors using capillary electrophoresis are within the range of values reported in the literature for the same enzyme–inhibitor
combinations. Irreversible inhibition of alkaline phosphatase by EDTA at concentrations of 1.0mM and above has been observed.
Activation of alkaline phosphatase has also been observed for EDTA at concentrations from 20 to 400lM. � 2002 Elsevier Science
(USA). All rights reserved.
Keywords: Capillary electrophoresis; Alkaline phosphatase; Enzyme assay; Enzyme inhibition; Enzyme activation
Enzyme assays based on capillary electrophoresis(CE)1 have been developed to take advantage of this highefficiency separation technique, its extremely low samplevolume requirements, and its ability to electrophoreti-cally mix and separate zones of enzymes, substrates, andproducts [1,2]. The first on-column capillary electroph-oretic enzyme assays were described by Bao and Regnier[3] and were performed by injecting a zone of enzyme intoa capillary filled with substrate and the required coen-zyme for the catalyzed reaction. Product was formed asthe enzyme migrated through the capillary, and theproduct was detected at a downstream absorbance de-tector. This type of assay, based on a reaction carried outin a CE column, was later termed electrophoreticallymediated microanalysis (EMMA), and this specific for-mat was defined as ‘‘continuous-engagement’’ EMMA
[4,5]. Capillary electrophoretic enzyme assays have alsobeen carried out by injecting the enzyme and substrate astwo separate zones and allowing them to mix electroph-oretically [6]. Product was formed when the enzyme andsubstrate zones mixed in the capillary, and this productwas detected as a separate electrophoretic zone. Thisformat was defined as ‘‘transient-engagement’’ EMMA[4,5]. Enzyme assays based on EMMA have been used toquantify enzyme and substrate and to determineMichaelis–Menten constants [1–5,7–9]. Enzyme assayshave been developed using microchip devices and elec-trophoretic mixing of reagents (EMMA) [10–16]. Capil-lary electrophoresis and electrophoretic microchipdevices have also been used to study enzyme reactions byincubating reagents together, injecting an aliquot of thereaction mixture into a capillary or microchip, and sep-arating and detecting product formed prior to injectionduring the off-column incubation [1,2,17–21].
Capillary electrophoresis and electrophoretic micro-chip devices have been used to study enzyme inhibition.An electrophoretic microchip device was used to studythe inhibition of b-galactosidase by several inhibitorsusing continuous-engagement EMMA, and the Ki of
Analytical Biochemistry 307 (2002) 226–234
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ANALYTICALBIOCHEMISTRY
qFinancial support was provided by the University of Tennessee.* Corresponding author. Fax: 865-974-3454.
E-mail address: [email protected] (S.D. Gilman).1 Abbreviations used: CE, capillary electrophoresis; EMMA, elec-
trophoretically mediated microanalysis; LIF, laser-induced fluores-
cence; DEA, diethanolamine.
0003-2697/02/$ - see front matter � 2002 Elsevier Science (USA). All rights reserved.
PII: S0003 -2697 (02 )00062-3
phenylethyl-b-D-thiogalactoside was determined [10].The inhibition of protein kinase A by the competitiveinhibitor H-89 was studied using an electrophoreticmicrochip device [22]. The substrate, enzyme, and in-hibitor were allowed to mix electrophoretically by con-tinuous-engagement EMMA, and an aliquot of thereaction mixture was injected into another channel forseparation and detection of the substrate and product.The Ki value for H-89 was determined. Acetylcholin-esterase inhibition by several competitive and irrevers-ible inhibitors was studied in an electrophoreticmicrochip device, using continuous-engagement EMMAto electrophoretically mix the substrate, enzyme, andinhibitor [23]. The Ki value for the competitive inhibitor,tarcine, was determined. Capillary electrophoresis wasused to study the inhibition of adenosine deaminase byerythro-9-(2-hydroxy-3-nonyl)adenine using a combina-tion of continuous-engagement EMMA and transient-engagement EMMA [24]. The assays were performed byinjecting zones of enzyme and substrate separately intoan inhibitor-filled capillary and monitoring the resultingproduct peaks. The Ki for erythro-9-(2-hydroxy-3-nonyl)adenine was determined. Noncompetitive inhibitionof calf intestinal alkaline phosphatase by theophyllinewas studied by CE [25]. A combination of continuous-engagement EMMA and transient-engagement EMMAwas used to quantify theophylline and measure the Ki
value for this inhibitor. The CE enzyme–inhibitor assaywas performed by injecting zones of inhibitor and en-zyme separately into a substrate-filled capillary and de-tecting decreased product formation where the inhibitorand enzyme interacted in the capillary [25].
In this report, different classes of alkaline phosph-atase inhibitors have been investigated by CE, using acombination of continuous-engagement EMMA andtransient-engagement EMMA. These inhibitors includea noncompetitive, reversible inhibitor (theophylline),competitive, reversible inhibitors (sodium vanadate andsodium arsenate), and an irreversible inhibitor (EDTA).Theophylline, sodium vanadate, and sodium arsenatewere quantified, and their Ki values were determined.The fractional activity of the enzyme was determined forexperiments with EDTA. The enzyme assays have alsobeen optimized by altering the running buffer compo-sition, reducing the amount of enzyme used for eachassay, thermostating the capillary, and reducing the timerequired for each experiment. The enzyme studied wasalkaline phosphatase (EC 3.1.3.1 from calf intestine).
Materials and methods
Reagents
Alkaline phosphatase (EC 3.1.3.1 from calf intestine)was purchased from ICN Biomedicals (Aurora, OH).
AttoPhos ([2,20-bibenzothiazol]-6-hydroxy-benzathiaz-ole phosphate) was obtained from Promega (Madison,WI). Theophylline (99%), sodium vanadate, and dieth-anolamine (DEA) were supplied by Acros (Pittsburgh,PA). Disodium EDTA and sodium chloride were pur-chased from Fisher Scientific. Sodium arsenate wassupplied by Mallinckrodt (St Louis, MO). All solutionsand buffers were prepared in ultrapure water(>18MXcm, Barnstead E-pure System, Dubuque, IA).
CE-LIF instrumentation and experimental conditions
The CE instrument with laser-induced fluorescence(LIF) detection was constructed in-house and describedin detail previously [25]. The 457.9-nm line of a Coher-ent Innova 90C-5 argon ion laser (Santa Clara, CA) wasused for LIF excitation at 35mW. Fused-silica capil-laries with a 50lm i.d. and 220lm o.d. (SGE, Austin,TX) were used. The detection window was made byremoving a portion of the polyimide coating using a gasflame.
The running buffer contained 50mM DEA at pH 9.5and 0.10mM AttoPhos, a fluorogenic alkaline phos-phatase substrate. Previously, chloroform was used toextract the running buffer to reduce the amount ofAttoFluor present [25,26]. It was determined that pre-paring fresh running buffer daily was as effective as thechloroform extraction, and the extraction was not usedfor this work. This eliminated flow irregularities thatwere encountered if any chloroform remained in therunning buffer. The enzyme solution contained 0.18 nMalkaline phosphatase and 50mM DEA buffer at pH 9.5.In addition to the inhibitor, the inhibitor solutionscontained 0.10mM AttoPhos and 50mM DEA at pH9.5. The applied electric field was 310V/cm for all sep-arations and injections. All injections were performedelectrokinetically. The electrode and capillary wererinsed externally with 50mM DEA buffer at pH 9.5before and after each injection to prevent cross-con-tamination of the running buffer, enzyme solution, andinhibitor solutions.
The capillary was thermostated by enclosing thecapillary from the injection end to the detection windowin Teflon tubing (1.6mm i.d.; 3.2mm o.d.) (Nalgene,Rochester, NY) and flowing heated N2 gas (40 �C)through the Teflon tubing around the capillary. To en-sure that the air flowed evenly over the entire thermo-stated section of the capillary, two pieces of Teflontubing of equal length were placed over the capillaryfrom the injection end to the detection window and wereconnected by a T-union Swagelock fitting. The thirdconnection of the T-union was the tubing from the he-ated N2 source. The ends of the tubing at the injectionend of the capillary and at the detection window weresealed with Duco cement. To allow airflow, two smallholes were placed in the Teflon tubing at equal distances
A.R. Whisnant, S.D. Gilman / Analytical Biochemistry 307 (2002) 226–234 227
from the injection end and the detection window. Thisreduced evaporation of solution in the buffer reservoirsby directing N2 flow away from the reservoirs. At theinjection end, 1 cm of the capillary was not enclosed inTeflon tubing.
Microplate experiments
Experiments were performed using a FLUOstar mi-croplate fluorometer (BMG, Durham, NC). The excita-tion filter was centered at 450 nm, and the emission filterwas centered at 555 nm. The instrument was thermo-stated at 25 �C. Microplates used were 96-well platesfrom Nunc (V-96 Polypropylene MicroWell Plate, Ros-kilde, Denmark). For the inhibitor assays, each wellcontained final concentrations of 0.10mM AttoPhos,50mM DEA at pH 9.5, and the inhibitor at variousconcentrations. For the assays used to generate Linewe-aver–Burk plots, each well contained a final concentra-tion of 50mM DEA at pH 9.5 and AttoPhos at variousconcentrations. Each well contained a final volume of200lL. For all enzyme assays, 10lL of alkaline phos-phatase was injected into each well separately by the in-strument to give a final concentration of 0.18 nM. Theproduct formation was measured at 0.1-s intervals.
Results and discussion
Optimization of capillary electrophoretic enzyme assays
Several improvements have been made to the capil-lary electrophoretic enzyme–inhibitor assays since ourinitial report [25]. Originally a 100 mM borate buffer atpH 9.5 was used [25]. However, borate has been re-ported to be a weak inhibitor of alkaline phosphatase[27–29], and DEA (50mM, pH 9.5) has been used in thiswork, since DEA has been reported to produce relativityhigh alkaline phosphatase activity [28].
The concentrations of enzyme and substrate havebeen optimized for the new buffer system. Using DEA,the alkaline phosphatase concentration can be reducedfrom 1.7 (used in our previous work [25]) to 0.18 nM.Only 1.9 amol enzyme is injected for a 3.0-s injection.The concentration of the substrate, AttoPhos, has beenoptimized at the new alkaline phosphatase concentra-tion by determining the minimum substrate concentra-tion that will give a reaction velocity near the maximumvelocity, Vmax. The concentration of AttoPhos has beenreduced from 1.0mM in our previous report [25] to0.10mM in this work. The applied separation potentialhas been increased from 210 to 310V/cm. This change,along with the change in buffer composition, reduces thetotal analysis time from 13 to 5min.
Fig. 1A shows an electropherogram from a CE en-zyme assay using our previous experimental conditions
[25]. Fluorescent product is produced as a plug of en-zyme migrates through a capillary filled with the fluo-rogenic substrate, and the product is detected later byLIF. The fluorescence signal indicates the reaction rateat the time the detected product was formed in thecapillary. It has been determined that the fluctuations inthe detected product plateau are due to temperaturechanges down the length of the capillary and corres-ponding changes in the reaction rate. Therefore, ther-mostating of the capillary was required in order tomaintain a constant temperature and reaction rate. Thecapillary has been enclosed in a Teflon tube and heatedN2 is circulated over the capillary. The temperature ofthe N2 has been optimized for maximum enzyme activity(40 �C). Fig. 1B shows an electropherogram for the newoptimized enzyme assay conditions with thermostating.The assay in Fig. 1B is performed by injecting a 3.0 s
Fig. 1. (A) Electropherogram of an alkaline phosphatase enzyme assay
without thermostating. A zone of 1.7 nM alkaline phosphatase was
injected for 3.0 s at 15.0 kV (210V/cm) into the capillary filled with
1.0mM AttoPhos and 100mM borate at pH 9.5. The separation po-
tential was 15.0 kV. The current was 16lA. (B) Electropherogram of
an alkaline phosphatase enzyme assay with thermostating. A zone of
0.18 nM alkaline phosphatase was injected for 3.0 s at 17.8 kV (310V/
cm) into the capillary filled with 0.10mM AttoPhos and 50mM DEA
at pH 9.5. The separation potential was 17.8 kV. The current was
10lA.
228 A.R. Whisnant, S.D. Gilman / Analytical Biochemistry 307 (2002) 226–234
zone of 0.18 nM alkaline phosphatase at a potential of17.8 kV (310V/cm) into a capillary containing 0.1mMAttoPhos. Then a constant separation potential of17.8 kV (310V/cm) is applied, and the fluorescentproduct forms continuously as the alkaline phosph-atase–AttoPhos complex migrates through the capillary.The fluctuations in the product plateau (Fig. 1A) havebeen greatly reduced by thermostating, indicating aconstant reaction rate. The small peak at 4.4min (Fig.1B) is due to product formed at the beginning of theexperiment when the enzyme is injected into the capil-lary before the separation potential is applied. This peakis observed at the end of the fluorescence plateau op-posite of where one might expect it. Because the en-zyme–substrate complex migrates to the LIF detectorfaster than the fluorescent product, the first productdetected is product produced near the end of the ex-periment just as the enzyme–substrate complex migratespast the detector. The product produced when the en-zyme is first injected into the capillary is actually de-tected last. In effect, all the electropherograms shown inthis paper are reversed in time from what one wouldexpect [3,25].
We have reexamined the CE enzyme–inhibitor assayfor theophylline, a reversible, noncompetitive inhibitorof alkaline phosphatase [25]. Fig. 2 presents the data forthe reversible inhibition of alkaline phosphatase bytheophylline. Fig. 2 also includes sketches of the relativepositions of the enzyme and inhibitor zones in the cap-illary as the experiment progresses. As described above,
the time scale for detection of fluorescent product isreversed relative to the sequence of events taking placein the capillary. First, 100lM theophylline is injected for4.0 s at 17.8 kV (310V/cm). Theophylline is injected firstbecause the enzyme–substrate complex has a greatermobility than the inhibitor. Then a constant potential of17.8 kV is applied for 20.0 s. Next, 0.18 nM alkalinephosphatase is injected for 3.0 s at 17.8 kV, and finallythe 17.8-kV separation potential is applied. The relativeposition of the enzyme–substrate (E) and inhibitor (I)zones when the separation potential is first applied isshown in Fig. 2(a). As the two zones migrate throughthe capillary, product is formed continuously. When thezones of inhibitor and enzyme–substrate complex mixelectrophoretically, a decrease in product formation isobserved due to inhibition of the enzyme (Fig. 2(b)).Once the zones of inhibitor and enzyme–substratecomplex separate again, the enzyme returns to its orig-inal activity, indicating that theophylline is a reversibleinhibitor (Fig. 2(c)). The reversible inhibition of alkalinephosphatase by theophylline is readily observed in theproduct plateau with the capillary thermostated andfluctuations in the product plateau minimized. The Ki
for theophylline, the equilibrium constant of the inhi-bitor binding to the enzyme,was determined by repeatingthe CE enzyme–inhibitor assay at six concentrations oftheophylline (5–250lM; N ¼ 3) and plotting and ana-lyzing the results as in our previous work [25]. The Ki
value determined is 102� 4lM. The detection limit fortheophylline is 3lM. The detection limit is primarilydetermined by the Ki value for the enzyme and inhibitor.In our previous work, the Ki value determined for the-ophylline was 90� 34lM, and the detection limit was3lM [25].
Alkaline phosphatase inhibition by competitive reversibleinhibitors
Sodium vanadate is a competitive, reversible inhibitorof alkaline phosphatase [28–31]. Reported Ki values forsodium vanadate range from 4lM for rat intestinal al-kaline phosphatase [31] to 12lM for Escherichia colialkaline phosphatase [30]. Vanadate is used in thetreatment of diabetes since it mimics the effects of insulinand reduces glucose levels [32–34]. In the body, vana-date affects the activity of several enzymes includingadenylate cyclase, which regulates the levels of cyclicadenosine monophosphate, thereby affecting insulin se-cretion [34].
Fig. 3 presents the data for a CE enzyme–inhibitorassay for sodium vanadate. Reversible inhibition of al-kaline phosphatase by sodium vanadate is evident fromthese data. Due to the greater mobility of the enzyme–substrate complex, sodium vanadate (75lM) is injectedfirst for 5.0 s at 17.8 kV (310V/cm). Next, a constantpotential of 17.8 kV is applied for 50.0 s. Then 0.18 nM
Fig. 2. Electropherogram of a theophylline–alkaline phosphatase en-
zyme–inhibitor assay. A zone of 100lM theophylline was injected for
4.0 s at 17.8 kV into a capillary filled with 0.10mM AttoPhos and
50mM DEA at pH 9.5. Next, a potential of 17.8 kV was applied for
20 s. Then, a zone of 0.18 nM alkaline phosphatase was injected into
the capillary for 3.0 s at 17.8 kV. Finally, the separation potential
(17.8 kV) was applied. The current was 10lA. (a)–(c) The relative
positions of the enzyme and inhibitor zones in the capillary at the
corresponding positions in the resulting electropherogram.
A.R. Whisnant, S.D. Gilman / Analytical Biochemistry 307 (2002) 226–234 229
alkaline phosphatase is injected for 3.0 s at 17.8 kV.Since sodium vanadate is a reversible inhibitor, the re-sulting product plateau (Fig. 3) closely resembles theproduct plateau for theophylline inhibition (Fig. 2).
According to the Michaelis–Menten treatment of areversible, competitive inhibitor, Vmax is unaffected by theinhibitor, but the apparent Km, the Michaelis constant,will vary with inhibitor concentration. A Dixon plot canbe used to determine the Ki value of a competitive in-hibitor [35]. The equation used for the Dixon plot is
1=v ¼ ðKm=Vmax½S�KiÞ½I � þ 1=Vmaxð1þ Km=½S�Þ; ð1Þwhere v is the velocity of the reaction at inhibitor con-centration, ½I �, Vmax is the maximum velocity of the en-zyme at the concentration of substrate in absence ofinhibitor, ½S� is the concentration of substrate, and Ki isthe equilibrium constant of the inhibitor binding to theenzyme. A plot of 1=v versus ½I � should be linear, and Ki
can be determined from the slope and intercept of theline and Vmax.
To generate a Dixon plot for sodium vanadate, aninhibitor response factor, RIce, has been defined for theCE experiments as follows:
RIce ¼ ðF1 F0Þ=ðF2 F0Þ; ð2Þwhere F1 (Fig. 3) is proportional to the rate of productformation without inhibitor and is used to normalize thedata due to variability in the height of the plateau fromexperiment to experiment. F2 (Fig. 3) is the minimumproduct formation observed in the plateau. F0 is thebaseline fluorescence (Fig. 3). RIce is proportional to 1=vin Eq. (2). A similar approach has been used to analyzetheophylline in this report and in our previous work[25]. In Fig. 4A, RIce values are plotted at differentconcentrations of sodium vanadate. To determine Ki forsodium vanadate, Vmax is required. Vmax is determined
from the RIce value of the CE assay without inhibitor(Fig. 1B). The Ki determined for sodium vanadate fromthe Dixon plot in Fig. 4A is 2:1� 1:5lM (R2 ¼ 0:997).The detection limit for sodium vanadate is 3lM.
Microplate fluorometer experiments analogous to theCE enzyme–inhibitor assays have been performed. Aresponse factor, RImp, for the microplate fluorometer isdefined as 1=v, where 1=v is the inverse of the initialvelocity of the enzyme-catalyzed reaction. The slope ofthe plot of fluorescence versus time for the microplateexperiments is used to determine v. Fig. 4B shows theDixon plot, RImp versus concentration of sodiumvanadate, for the microplate experiments. Vmax is de-termined by carrying out experiments to generate a Li-neweaver–Burk plot as defined by [35]
1=v ¼ ðKm=VmaxÞð1=½S�Þ þ 1=Vmax: ð3ÞA plot of 1=v versus 1=½S� should be linear, and Vmax
can be determined from the inverse of the intercept.From the slope and intercept of the line and the Vmax
value, the Ki determined for sodium vanadate is 3:5�0:2lM. The analysis of the sodium vanadate dataassumes steady-state kinetics, and a comparison of theresults from the CE enzyme–inhibitor assays to the
Fig. 4. (A) A plot of RIce versus sodium vanadate concentration. RIceis proportional to v1, where v is the velocity of the reaction in the
presence of the inhibitor. (B) A plot of RImp versus the concentration
of sodium vanadate concentration where RImp is analogous to RIce for
microplate experiments.
Fig. 3. Electropherogram of a sodium vanadate–alkaline phosphatase
enzyme–inhibitor assay. A zone of 75lM sodium vanadate was injected
for 5.0 s at 17.8 kV into a capillary filled with 0.10mM AttoPhos and
50mMDEA at pH 9.5. Next, a potential of 17.8 kVwas applied for 60 s.
Then, a zone of 0.18 nM alkaline phosphatase was injected for 3.0 s at
17.8 kV (310V/cm). Finally, the separation potential (17.8 kV) was ap-
plied. The current was 10lA. F0, F1, and F2 are defined in the text.
230 A.R. Whisnant, S.D. Gilman / Analytical Biochemistry 307 (2002) 226–234
microplate assays indicates that the CE enzyme–inhib-itor assays exhibit steady-state behavior. The Ki valuesfor the CE and microplate enzyme–inhibitor assays arein agreement with reported values [30,31].
Sodium arsenate, also a reversible, competitive inhib-itor of alkaline phosphatase, has been studied[28,29,31,36]. Reported Ki values for sodium arsenaterange from 2lM for rat intestinal alkaline phosphatase[31] to 33:8lM for membrane-bound alkaline phospha-tase frombone cartilage [29].An electropherogram fromaCE enzyme–inhibitor assay of alkaline phosphatase andsodium arsenate is presented in Fig. 5. In this experiment,125lMsodiumarsenate is injected first for 8.0 s at 17.8 kV(310V/cm) since the enzyme–substrate complex has agreater mobility than arsenate. Then a constant potentialof 17.8 kV is applied for 150.0 s. Alkaline phosphatase(0.18 nM) is injected for 3.0 s at 17.8 kV before the ap-plication of the separation potential (17.8 kV). Since so-dium arsenate is a reversible, competitive inhibitor, itsresponse is similar to that for sodium vanadate.
The kinetic treatment of the CE enzyme–inhibitorassay data for sodium arsenate was the same as forsodium vanadate. The plot of RIce at different concen-trations of sodium arsenate (25–250lM; N ¼ 3) is linear(R2 ¼ 0:999), indicating that the arsenate assays exhib-ited steady-state behavior. The Ki determined for so-dium arsenate is 21� 4lM, and the Ki value determinedby microplate fluorometer experiments is 8:6� 1:4lM.These Ki values are in agreement with previously pub-lished values for sodium arsenate. It is unclear why theKi value determined for sodium arsenate by CE is afactor of 2 higher than the Ki determined by microplateexperiments. This could be a kinetic effect since theelectrophoretic experiment takes place on a faster timescale than the microplate experiments. However, the CE
data for sodium arsenate give an excellent fit to theequation for the Dixon plot, and the Ki values obtainedfor other reversible inhibitors (vanadate and theophyl-line) are similar using both techniques.
Alkaline phosphatase inhibition by an irreversible inhib-itor
Alkaline phosphatase is a zinc metalloprotein andcontains four equivalents of zinc per mole of alkalinephosphatase [28,30,37]. Zinc is required for activity of theenzyme [38]. Chelating compounds such as EDTA canirreversibly inhibit alkaline phosphatase by removing thezinc from the enzyme [39–41]. A CE enzyme–inhibitorassay indicating irreversible inhibition of alkaline phos-phatase by EDTA is shown in Fig. 6. In this experiment,1.0mM EDTA is injected first for 55.0 s at 17.8 kV(310V/cm). Next, a constant potential of 17.8 kV is ap-plied for 180.0 s. Then 0.18 nM alkaline phosphatase isinjected for 3.0 s at 17.8 kV. As presented in Fig. 6,product formation decreases when the zones of inhibitorand enzyme–substrate complex mix electrophoretically(4.2min, Fig. 6). After the zones of inhibitor and en-zyme–substrate complex separate, the enzyme does notreturn to its original activity (4.0min, Fig. 6), indicatingthat the enzyme is irreversibly inhibited. The same ex-periment using 0.030mM EDTA is presented in Fig. 7.At this EDTA concentration, when the enzyme–substratecomplex and inhibitor separate, the enzyme is activatedrather than irreversibly inhibited.
The ratio of enzyme activity before and after the en-zyme interacted with EDTA has been measured over arange of EDTA concentrations in order to quantify ir-reversible inhibition or activation of alkaline phospha-tase by EDTA. The fractional activity is calculated as
Fig. 5. Electropherogram of a sodium arsenate–alkaline phosphatase
enzyme–inhibitor assay. A zone of 125lM sodium arsenate was in-
jected for 5.0 s at 17.8 kV into a capillary filled with 0.10mM AttoPhos
and 50mM DEA pH 9.5. Next, a potential of 17.8 kV was applied for
60 s. Then, a zone of 0.18 nM alkaline phosphatase was injected for
3.0 s at 17.8 kV into the capillary. Finally the separation potential was
applied (17.8 kV). The current was 10lA.
Fig. 6. Electropherogram of an EDTA–alkaline phosphatase enzyme–
inhibitor assay. A zone of 1.0mM EDTA was injected for 55.0 s at
17.8 kV into a capillary filled with 0.10mMAttoPhos and 50mMDEA.
Next, a voltage of 17.8 kVwas applied for 180 s. Then, a zone of 0.18 nM
alkalinephosphatasewas injected for 3.0 s at 17.8 kV intoa capillary, and
the separation potential was applied (17.8 kV). The current was 10lA.
A.R. Whisnant, S.D. Gilman / Analytical Biochemistry 307 (2002) 226–234 231
fractional activity ¼ ðI1 I0Þ=ðI2 I0Þ; ð4Þwhere I1 (Fig. 7) is the activity of alkaline phosphataseafter interacting with EDTA, I2 is the activity of alkalinephosphatase before interacting with EDTA, and I0 is thebaseline fluorescence. Fig. 8 presents the fractional ac-tivity versus EDTA concentration from 10lM to4.0mM. The dotted line is the fractional activity meas-ured for control assays (Fig. 1B). Fractional activityvalues above this line indicate activation, and values be-low this line indicate inhibition. Concentrations of EDTAat 1.0mM and higher irreversibly inhibit alkaline phos-phatase. Concentrations from 20 to 400lM EDTA acti-vate alkaline phosphatase. The maximum activation isobserved at 40lM EDTA. EDTA is thought to irrevers-ibly inhibit alkaline phosphatase by binding to the zincbound to the enzyme and then removing the zinc, leavingthe enzyme inactivated [39,41]. The mechanism by whichEDTA activates alkaline phosphatase is unknown.
Activation of alkaline phosphatase by EDTAhas beenreported for human placental alkaline phosphatase at
concentrations of 10–100mM EDTA [42], for Gastro-thylax crumenifer and Cotylophoron orientale alkalinephosphatases at 1.0mM EDTA [43], and for Ascarissuum alkaline phosphatase at 10mM EDTA [44]. How-ever, the alkaline phosphatases used in all three of thesestudies were in crude preparations, and it is not clearwhat other enzymes were present in these preparations.Only one of the alkaline phosphatase preparations wasfrom a mammalian source (human placental), and thisshowed inhibition at low EDTA concentrations (10lMto 1mM) and activation at high EDTA concentrations(10–100mM) [42]. This is the opposite of what is ob-served here for calf intestinal alkaline phosphatase whereactivation is observed at low EDTA concentrations (20–400lM), and inhibition is observed at high EDTA con-centrations (1.0mM or higher). It has also been reportedthat purified human placental alkaline phosphatase isonly inactivated by EDTA (5–40mM) and not activatedby EDTA at any concentration [45].
One possible explanation for these results, unrelatedto the ability of EDTA to chelate zinc, is ionic strengtheffects of the EDTA injection zone on alkaline phos-phatase [46,47]. To determine whether or not the ionicstrength of the EDTA zone is causing the observed in-hibition and activation of alkaline phosphatase, thesame experiments have been performed with NaCl re-placing EDTA. The ionic strength of the NaCl solutionsis the same as the EDTA solutions used in the experi-ments presented in Fig. 8. These experiments (data notshown) indicate that the change in ionic strength due toEDTA does not cause the observed inhibition of alka-line phosphatase. Injections of NaCl result in fractionalactivities above the control value at all concentrationstested. However, the activity increase is less than thatobserved for EDTA at all ionic strengths, and the NaCleffect increases with increasing ionic strength. For ex-ample, at an ionic strength of 0.66mM, the fractionalactivity of the enzyme increases by 4% for NaCl and32% for EDTA. At an ionic strength of 1.32mM, thefractional activity increases by 4% for NaCl and 18% forEDTA. At an ionic strength of 26.5mM, the activity ofthe enzyme increases by 17% for NaCl and decreases by83% for EDTA. It has been reported that the addition ofsome salts, including NaCl, increases the activity ofE. coli alkaline phosphatase [46]. However, activationof the magnitude observed at low EDTA concentrationshere cannot be attributed to only ionic strength.
Conclusions
Inhibition and activation of alkaline phosphatase havebeen studied by an on-column CE method. Using thisapproach, reversible inhibition, irreversible inhibition,and activation can be readily distinguished. Competitiveand noncompetitive reversible inhibitors can be analyzed
Fig. 8. A plot of fractional activity versus concentration. Fractional
activity values below the control value (0:98� 0:02) indicate irrevers-
ible inhibition of alkaline phosphatase by EDTA, and values above the
control value indicate alkaline phosphatase activation by EDTA. The
dashed line is a reference to the control value.
Fig. 7. The experimental conditions are the same as Fig. 6 except the
concentration of EDTA was 0.030mM. I0, I1, and I2 are defined in the
text.
232 A.R. Whisnant, S.D. Gilman / Analytical Biochemistry 307 (2002) 226–234
quantitatively using Michaelis–Menten treatment of theCE data. The potential of this CE approach for studyingenzyme activity and inhibition is demonstrated by theobservation of activation and irreversible inhibition ofalkaline phosphatase by EDTA. Both effects are appar-ent from only visual inspection of electropherogramsfrom CE enzyme inhibition assays. Future studies willinclude a more detailed study of the effect that EDTA hason alkaline phosphatase activity and continued devel-opment of mathematical descriptions of these assays toexpand the quantitative information that can be obtainedfrom electropherograms.
This CE method is applicable to any combination ofenzyme and inhibitor, provided they have different elec-trophoretic mobilities. On rare occasions where thesemobilities are equal, modest changes to the pH of theseparation buffer should result in a mobility difference. Asignificant limitation is that the sensitive experimentsshown here depend on the availability of a fluorogenicsubstrate for LIF detection. Although custom instru-ments have been used in this work, these experiments willalso work in commercial CE instruments with LIF de-tectors. In fact, autoinjectors and more sophisticatedthermostating systems should lead to better quantitativeresults and increased sample throughput.
Compared to enzyme assays performed in microplatewells, CE enzyme inhibition assays offer more informa-tion per experiment. Visual inspection of an electro-pherogram will indicate whether an enzyme has beeninhibited reversibly, irreversibly, or activated. Elec-trophoretic enzyme inhibition assays in microfabricateddevices can also distinguish between reversible and irre-versible inhibition based on the shape of electrophero-grams [23]. These assays also consume 104 less enzymeper experiment compared to the microplate assays usedhere. Fifty injections from a single 50lL sample used forCE will reduce the enzyme concentration by only 1%.Each microplate well used in this work contained 200lLof the same enzyme solution and could be used for onlyone experiment. Although each CE experiment providessubstantially more information than an experiment in asingle well of a microplate, the CE method is serial innature, and about 12 experiments can be performed eachhour under the current conditions.
Acknowledgment
The authors thank Jason Pittman for his help withinstrumentation and useful suggestions.
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