Analytical Biochemistry 414 (2011) 226–231

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Analytical Biochemistry

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An enzyme immobilized microassay in capillary electrophoresisfor characterization and inhibition studies of alkaline phosphatases

Jamshed Iqbal ⇑Department of Pharmaceutical Sciences, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan

a r t i c l e i n f o a b s t r a c t

Article history:Received 25 January 2011Received in revised form 17 March 2011Accepted 18 March 2011Available online 23 March 2011

Keywords:Alkaline phosphataseCapillary electrophoresisDynamic surface coatingImmobilized enzyme reactorMicroreactor

0003-2697/$ - see front matter � 2011 Elsevier Inc. Adoi:10.1016/j.ab.2011.03.021

⇑ Fax: +92 992 383441.E-mail address: drjamshed@ciit.net.pk

1 Abbreviations used: ALP, alkaline phosphatase; TNAphosphatase; 4-NPP, 4-nitrophenylphosphate; 4-NP, 4alkaline phosphatase; CIAP, calf intestinal alkalineelectrophoresis; UV, ultraviolet; EMMA, electrophoretiAttoPhos, [2,20-bibenzothiazol]-6-hydroxy-benzathiazomotic flow; PB, polybrene; EDTA, ethlylenediaminetetramethyl)aminomethane; 4-APP, 4-aminophenylphosphLOQ, limit of quantification; PO4

�3, inorganic phospha

A simple and fast dynamically coated capillary electrophoretic method was developed for the charac-terization and inhibition studies of alkaline phosphatases (EC 3.1.3.1). An inside capillary enzymaticreaction was performed, and hydrolysis of the substrate 4-nitrophenylphosphate to 4-nitrophenolwas measured. Fused-silica capillary surface was dynamically modified with polycationic polybrenecoating. By reversal of the electroosmotic flow (EOF), analysis time was reduced up to 3 min as theanionic analytes were migrated in the same direction as the EOF. Furthermore, the sensitivity ofthe method was increased using electroinjection through high-field amplified injection. The baselineseparation of 4-nitrophenylphosphate and 4-nitrophenol was achieved by employing 50 mM sodiumphosphate as the running buffer (pH 8.5), 0.0025% polybrene, and a constant voltage of �15 kV,and the products were detected at 322 nm. Under the optimized conditions, a good separation withhigh efficiency was achieved. The new method was applied to study enzyme kinetics and inhibitorscreening. Km and Ki values obtained with the new CE method were compared well with the standardspectrophotometric method. Dynamic coating of fused-silica capillary gave fast and reproducible sep-aration of substrate and product. The method can be easily optimized for inhibition studies of otherisozymes.

� 2011 Elsevier Inc. All rights reserved.

Alkaline phosphatase (ALP,1 EC 3.1.3.1) is an isozyme family in calcification such as idiopathic infantile arterial calcification and

mammals involved in catalyzing the hydrolysis of phosphomono-esters, releasing phosphate and alcohol [1]. This isozyme familyis composed of two groups: tissue-specific ALPs (placental, intesti-nal, and germ cell) [2] and tissue-nonspecific alkaline phosphatase(TNAP) [2,3]. Intestinal ALP is a tissue-restricted isozyme of theALP superfamily [4]. It has a role in fat absorption in the gastro-intestinal tract and as a gut mucosal defense factor where itsexpression coincides with the stage of colonization by commensalbacterial flora [2]. TNAP is essential for proper skeletal mineraliza-tion. Its role is to hydrolyze inorganic pyrophosphate, a potent cal-cification inhibitor. The inhibitors of TNAP could be implementedin a drug therapy for osteoarthritis [5]. New and novel potentialinhibitors of TNAP are also required to probe the causativemechanisms, or treat the pathology, of diseases caused by medial

ll rights reserved.

P, tissue-nonspecific alkaline-nitrophenol; PLAP, placental

phosphatase; CE, capillarycally mediated microanalysis;le phosphate; EOF, electroos-acetic acid; Tris, tris(hydroxy-ate; LOD, limit of detection;te.

end-stage renal diseases [6]. Easy and fast methods are requiredfor the screening of compound libraries of potential new inhibitorsof ALPs.

The common methods used for high-throughput screening ofALP inhibitors involve potentiometric [7], colorimetric [8], fluori-metric [9], or luminescent [3,6,10] assays. However, the colorimet-ric method with 4-nitrophenylphosphate (4-NPP) as the substrateis the most widely used method to follow ALP assay. The enzymeconverts 4-NPP to 4-nitrophenol (4-NP), which is highly coloredwith an absorption maximum at 405 nm [11]. Recently, a lumines-cent assay using CDP-Star (2-chloro-5-(4-methoxyspiro{1,2-dioxe-tane-3,20(50-chloro)-tricyclo-[ 3.3.1.13.7 ]-decan}-4-yl)-1-phenylphosphate disodium salt) substrate was developed for the screen-ing of inhibitors of placental alkaline phosphatase (PLAP) and calfintestinal alkaline phosphatase (CIAP) [3,12]. However, these as-says have serious drawbacks, including the fact that suitable color-imetric or fluorimetric substrate is required to generate a signal;such substrates are also quite expensive. These methods are alsolimited because of interferences that can arise from compounds(e.g., inhibitors) that either absorb or fluoresce at wavelengths sim-ilar to the reagent or quench fluorescence. Therefore, spectroscopicassays are not suitable for the screening of complex mixtures.

Capillary electrophoresis (CE) has been used extensively forthe separation of charged and uncharged compounds as an

Microassay in CE for alkaline phosphatases / J. Iqbal / Anal. Biochem. 414 (2011) 226–231 227

alternative to routine spectroscopic methods [13–19]. CE is a fast,economical, and efficient separation technique that requiresconsiderably small volumes of samples [14]. The separations areperformed in an uncoated fused-silica column, which is cheapand easy to prepare. During recent years, CE has been used mostfrequently for monitoring enzymatic reactions [13–15,20–23].Enzyme assays based on CE are rapid and automatic, and theyrequire only small volumes of biochemical reagents, which isespecially useful when enzyme and substrates are very expensive.Hence, CE-based drug screening methods are playing a key role inidentification of drug leads. Recently, we have developed severalenzyme assays for characterization and inhibition studies [13–18]. In CE assays, various types of detectors have been used tomeasure the enzymatic reaction products, including ultraviolet(UV) [24], fluorimetric [25,26], and electrochemical [24,27]. Dur-ing recent years, CE-based on- and off-column ALP assays havebeen described by several groups [25,28–31]. An on-column,CE-based electrophoretically mediated microanalysis (EMMA) ofALP was employed using both electrochemical and spectrophoto-metric detection [24]. The CE-based EMMA method with electro-chemical detection system had a limitation in the use ofhigh-ionic-strength buffers and MgCl2. This method has also beencriticized for excessive analysis time because the product (4-NPP)of the enzyme reaction had a long migration time. Another CEmethod [27] was developed for the determination of ALP isoen-zymes in individual fibroblast cells of mouse bone marrow bycombining CE with an on-capillary enzyme-catalyzed reactionand electrochemical detection. The assay had higher sensitivitywith a purified product, but any contamination by other electro-active species present in buffer would result in unacceptablenoise levels. Different classes of ALP inhibitors have been investi-gated by CE with laser-induced fluorescence (CE–LIF) using on-column EMMA enzymatic reaction mode. A significant limitationto the method was that it required a fluorogenic substrate, Atto-Phos ([2,20-bibenzothiazol]-6-hydroxy-benzathiazole phosphate),for the measurement of enzymatic reaction [25,31]. Most of theCE-based ALP assays have been faulted for poor peak shapesand longer analysis times. The longer migration times of 4-NPand 4-NPP in uncoated fused-silica capillaries [32] are due tothe migration of these anionic analytes in the direction oppositeto the electroosmotic flow (EOF, counterelectroosmotic) and soare detected rather late [33]. To decrease the analysis time dras-tically and reverse the direction of the EOF, polycationic bufferadditives are used. A reversal of the EOF is achieved just bydynamically coating the negatively charged inner surface of afused-silica capillary with a layer of polycations [15,16]. On-column enzyme immobilized screening assays offer severalintrinsic advantages over the off-line screening assays, includingthe following: (i) the cost of the enzyme is reduced because itcan be reused several times; (ii) automation of the assay is easilyachieved; (iii) stability of the enzyme can be improved; and (iv)enzymatic reaction and separation steps can be performed withthe same capillary. Very recently, several enzyme immobilizedmicroreactors have been developed [34–36] to obtain advantagesof this nascent technology.

The aim of this investigation was to develop immobilized en-zyme microreactors on fused-silica capillary column for the char-acterization and inhibition studies of ALPs, that is, CIAP andTNAP. For improved separation performance, fused-silica capillarysurface was dynamically coated with polybrene (PB). Samples wereintroduced into capillary by electroinjection, which resulted in 10fold higher sensitivity of the CE–UV-based assay as compared withnormal pressure injection. The assay is simple, fast, and easy forautomation; furthermore, it should be easy to adapt the assay foruse with other isozymes employing negatively charged substratesand products.

Materials and methods

Materials

Magnesium chloride hexahydrate, ethlylenediaminetetraaceticacid (EDTA) disodium salt, tris(hydroxymethyl)aminomethane(Tris), sodium hydroxide, hydrochloric acid, zinc chloride, 1,5-di-methyl-1,5-diazaundecamethylene hexadimethrine bromide(polybrene, PB), glycerol, and dipotassium hydrogen phosphatewere purchased from Sigma (Taufkirchen, Germany). ALP (EC3.1.3.1) from calf intestine, TNAP from porcine kidney, levamisole,disodium 4-NPP, 4-NP, zinc chloride, sodium arsenate, and theoph-ylline were obtained from Sigma–Aldrich (Steinheim, Germany).

CE apparatus

All experiments were carried out on a P/ACE MDQ capillaryelectrophoresis system (Beckman Instruments, Fullerton, CA,USA) equipped with a UV detection system. On-line detectionwas achieved with a diode array detector. Instrument control anddata collection were performed by the P/ACE MDQ software 32KARAT obtained from Beckman Coulter (Fullerton, CA, USA). eCAPuncoated fused-silica capillaries (Beckman Coulter) of 75 lm i.d.,375 lm o.d., and 30 cm total length (20 cm from the injection sideto the detector) were used for separation. The capillary wasthermostated by liquid cooling at 37 �C, and the temperature ofthe sample storing unit was also adjusted to 37 �C. The separationwas performed using an applied constant voltage of �15 kV and adata acquisition rate of 8 Hz. Analytes were detected using directUV absorbance at 322 nm. The separation buffer was 50 mM boratebuffer (pH 9.5) containing 0.0025% (w/v) PB. Sample injectionswere made at the cathodic side of the capillary. Between the runs,capillary was rinsed for 2 min with separation buffer, which wasessential to obtain reproducible results.

Immobilization of ALPs onto capillary column

A new capillary column was flushed with 0.1 M NaOH solutionfor 15 min and then with deionized water for 5 min. Then the cap-illary column was purged with 0.25% (w/v) PB solution for 5 min,which covalently anchors to the silanol groups of the capillary wall.A 0.25% PB solution produced a positively charged coating coveringthe capillary wall completely. Subsequently, a plug of CIAP was in-jected into capillary by applying pressure at 10 psi for 5 s. Beforeenzyme assays, un-immobilized enzyme was removed from thecapillary by rinsing it with running buffer for 5 min. For immobili-zation of TNAP, capillary column was flushed with 0.1 M NaOHsolution for 30 min and then with deionized water for 15 min toremove CIAP from the capillary column. The above coating prepa-ration protocol was repeated for development of TNAP coatedcapillary.

Preparation of standard solutions for method validation

4-NPP and 4-NP were dissolved in deionized water to obtain10 mM stock solutions. These were further diluted to obtain 1-mM solutions in enzyme assay buffer (50 mM Tris–HCl [pH 9.5],5 mM MgCl2, and 0.1 mM ZnCl2). The 1 mM solutions were furtherdiluted in the reaction buffer as required for the standard calibra-tion curves and the enzyme inhibition assays.

Enzymatic activity assay on microplate

The standard spectrophotometric enzyme assay was performedas described previously [37,38] with minor modifications. In brief,

228 Microassay in CE for alkaline phosphatases / J. Iqbal / Anal. Biochem. 414 (2011) 226–231

activity of the ALP was determined using the enzymatic conversionof 4-NPP and measuring the absorbance of the yellow product (4-NP) at 405 nm with a 96-well microplate reader (BioTek ELx800,BioTek Instruments, Winooski, VT, USA). In a final volume of100 ll containing 50 mM Tris–HCl, 5 mM MgCl2, and 0.1 mM ZnCl2

(pH 9.5), 5 ll (0.25 U/ml) of appropriately diluted bovine intestinalALP dissolved in buffer (10 mM Tris–HCl, 5 mM ZnCl2, 0.2 mMMgCl2, and 50% glycerol, pH 7.0), and 10 ll of different concentra-tions of the inhibitor. The reaction was initiated by the addition of10 ll of substrate (10 mM 4-NPP). Incubation was performed for20 min at 37 �C, and the reaction was stopped by the addition of0.5 ml of 0.2 M NaOH. The enzymatic activity was calibrated with4-NP standards. Each analysis was repeated three times (tripli-cates) in three separate experiments.

Enzyme activity and inhibition assays of immobilized CIAP

The activity of CIAP immobilized microreactor was assayed bycarrying out enzyme activity assay and inhibitor screening. Forthe determination of Michaelis–Menten constants (Km) and maxi-mal velocity (Vmax), different concentrations of 4-NPP (10, 50,100, 150, 200, 250, 500, and 1000 lM) in reaction buffer (50 mMTris–HCl [pH 9.5], 5 mM MgCl2, and 0.1 mM ZnCl2) were injectedelectrokinetically at a voltage of �6 kV for 10 s into an ALP mic-roreactor. A separation voltage of �15 kV was applied for 1 minto start mixing of substrate with enzyme. Then the high voltagewas turned off, the substrate was incubated for 5 min, and�15 kV was applied again to separate the product (4-NP) fromthe unreacted substrate (4-NPP). The peak area of 4-NP was usedfor the measurement of enzyme activity and calculation of Km

and Ki values. For the inhibitor screening, various concentrationsof inhibitors in a fixed substrate concentration were injected intothe capillary; however, incubation and separation conditions re-mained the same. Under the applied conditions, less than 5% ofsubstrate was converted by the immobilized enzyme. The inhibi-tion percentage was calculated according to the reduction of thepeak area of the 4-NP. The Cheng–Prusoff equation [39] was usedto calculate the Ki values from the IC50 values determined by thenonlinear curve fitting program PRISM 4.0 (GraphPad Software,San Diego, CA, USA).

Enzyme inhibition assays of the immobilized TNAP

Enzyme inhibition activity of TNAP was studied using differentconcentrations of levamisole (inhibitor of TNAP) in a fixed sub-strate concentration. All conditions for injection and CE separationsfor the TNAP inhibition assays were similar to those for CIAP.

Table 1Limits of detection, limits of quantification, migration times, and linearity for 4-NPPand 4-NP determination.

Compound 4-NPP 4-NP

Limit of detection ± SD (lM) 1.21 ± 0.20 2.13 ± 0.10Limit of quantification ± SD (lM) 0.46 ± 0.11 0.75 ± 0.12Linearity of calibration curve (R2) 0.999 0.999Mean value of migration time ± SD

(min) (n = 12)1.28 ± 0.008 2.80 ± 0.02

% RSD of migration time (min) 0.62 0.71Regression equation y = 935.5x + 0.83,

Sy,x = 927y = 654.2x + 0.97,Sy,x = 643

Note. SD, standard deviation; RSD, relative standard deviation.

Results and discussion

Dynamic coating of capillary column for efficient separation

During recent years, several enzyme assays have been devel-oped on CE [16–18] because it is a fast, economical, and efficientseparation technique that requires considerably small volumes ofsamples. ALP studies have been carried out on CE using on- andoff-column enzyme reactions [24,25,31]. The most commonly usedsubstrates for ALP are 4-NPP and 4-aminophenylphosphate (4-APP). However, in uncoated fused-silica capillary, longer migrationtimes and broad peak shapes with peak tailing were observed forthe substrates and products of ALP. These compounds are highlybasic and strongly interact with the silanols on the capillary,resulting in tailing peaks. The use of a dynamic surface coatinghas been shown to improve the routine performance of CE in en-zyme analysis [16]. In the current work, we employed EOF modifier

PB to the buffer system. PB, which is a cationic surfactant, adsorbson the capillary wall surface by dynamic electrostatic interactionsbetween the positively charged tertiary ammonium ion and thenegatively charged Si–O� group [40,41]. The positively chargedcapillary wall produced a reversed EOF (from cathode to anode)that migrated to the same direction of the negatively chargedproducts of ALP enzymes. Therefore, the electrophoretic mobilitiesof the negatively charged 4-NPP and 4-NP were accelerated and,hence, the analysis times were shortened drastically (co-EOF elec-trophoresis). A short analysis time of less than 3 min was obtainedwith this method. This dynamic coating was not stable because itwas easily eluted at alkaline conditions of enzyme assay. Therefore,PB was added to the buffer electrolyte to provide a constant supplyof EOF modifier throughout the assay.

Quantitative determination of 4-NPP and 4-NP

The CIAP and TNAP activity and inhibition were determined bymeasuring the peak area of 4-NP. Method validation of CE mea-surements of 4-NPP and 4-NP was performed by electrokineticallyinjecting the different concentrations of 4-NPP and 4-NP in reac-tion buffer. Quantitative parameters of the method validation areprovided in Table 1. A strictly linear correlation between 4-NPPand 4-NP concentrations was found; a correlation coefficient (R2)of 0.999 for 4-NPP and 4-NP (n = 3) was calculated for a concentra-tion range from 0.05 to 50 lM. The limits of detection (LODs) weredetermined to be 1.21 and 2.13 lM for 4-NPP and 4-NP, respec-tively. The limits of quantification (LOQs) were found to be 0.46and 0.75 lM for 4-NPP and 4-NP, respectively. Standard deviationsof the method validation were quite low (Table 1).

Immobilization of enzymes on capillary column

A three-step process involves initially flushing the capillarywith 1 N NaOH solution to fully ionize the surface silanols and gen-erate a negatively charged surface. A polycationic PB solution inhigh concentrations (0.25% [w/v], prepared by dissolving 25 mgof PB in 10 ml of water) was then purged for 2 min through theuncoated fused-silica capillary. The multiple charged PB coats theentire capillary wall, making it strongly positively charged (seeFig. 1). ALP (0.2 U/ml) in enzyme dilution buffer (10 mM Tris–HCl, 5 mM ZnCl2, 0.2 mM MgCl2, and 50% glycerol [50 ml of glyc-erol in 50 ml of Tris–HCl buffer], pH 7.0) was then injected intothe capillary column at 10 psi for 5 s to coat the 5 cm part of thecapillary. Finally, the capillary was flushed with a running buffersolution that contained 0.002% PB (prepared by diluting 0.8 ml of0.25% PB in a 10 ml flask) at pH 9.5. The use of the fused-silica cap-illary as the support for enzyme immobilization offers benefitssuch as a large surface area/volume ratio within the capillary,which allows adequate interactions of the flowing substrate withthe immobilized enzyme and also offers a low-pressure drop

Fig.1. Schematic representation of dynamic coating process and on-capillary CIAPimmobilized enzymatic reaction. The capillary column was purged with PB for2 min, and then CIAP in enzyme assay buffer (10 mM Tris–HCl, 5 mM ZnCl2, 0.2 mMMgCl2, and 50% glycerol, pH 7.0) was injected into the capillary column at 10 psi for5 s to coat the 5 cm part of the capillary. For enzyme assays, 1 mM 4-NPP solutionwas injected into the enzyme microreactor for 5 s. After incubation for 5 min,reverse polarity was applied to separate the CIAP reaction product (4-NP). ALP, calfintestinal alkaline phosphatase; EOF, electroosmotic flow; PB, polybrene; S,substrate; P, product.

Fig.2. Michaelis–Menten plot for the CIAP enzymatic reaction of initial 4-NPPconcentrations with respect to the reaction velocity for the determination of Km andVmax values by using calf ALP immobilized in-capillary reaction. For enzyme activityassay, see Materials and methods. For CE conditions, see Fig. 3. Data pointsrepresent means ± standard deviations from three separate experiments, each runin duplicate. Using CIAP immobilized enzyme activity assay, the Km was 82 lm andthe Vmax was 0.96 nmol/min.

Fig.3. Typical electropherograms of CIAP online enzymatic reaction by enzyme immconcentration of 4-NPP was 1 mM, and 0.25 U/ml ALP was immobilized onto the capillai.d. � 30 cm (20 cm from outlet to detection window); separation voltage, 15 kV; detect(w/v) PB. In the inhibition assay, the concentration of inhibitor (PO4

�3) was 100 lM. Th

Microassay in CE for alkaline phosphatases / J. Iqbal / Anal. Biochem. 414 (2011) 226–231 229

across the capillary [42]. A power supply of �15 kV with switchedpolarity was employed, which causes anionic compounds to mi-grate toward the detector (anodic) end of the capillary. The separa-tion using the dynamically coated capillary showed Gaussiannontailing peaks with short migration times for substrate andproduct of enzyme.

Michaelis–Menten constant and maximal velocity determination usingCIAP immobilized enzyme reactor

Using the optimized conditions, Km and Vmax values for CIAPwere determined. Km values were obtained by using different con-centrations of the substrate 4-NPP. The Michaelis–Menten plot ofCIAP is depicted in Fig. 2. However, the enzyme velocity was deter-mined by measuring the peak area of the product (4-NP) of theenzymatic reaction. The Km value obtained with the CIAP immobi-lized enzyme reactor was 82 lM, whereas the Vmax value was0.96 nmol/min/mg enzyme. The Km value obtained with the CIAPimmobilized microreactor is in excellent agreement with a valueof calf ALP enzyme using a 96-well microplate reader. However,the Vmax value obtained with our newly established method is halfof the Vmax value (2 nmol/min/mg) using the spectrophotometricmethod. This could be due to longer incubation times in spectro-photometric assays, which was not possible in enzyme immobi-lized microreactor.

Inhibition of CIAP

For inhibition studies of immobilized CIAP, three standardinhibitors—inorganic phosphate (PO4

�3, a competitive inhibitor),theophylline (a reversible noncompetitive inhibitor), and arsenate(an irreversible inhibitor of CIAP) were analyzed to obtain theirdose–response curves. Fig. 3 shows typical electropherograms forALP control and inhibitor screening assays using CIAP enzymeimmobilized microreactor. Concentration–inhibition curves of allthree calf ALP inhibitors are depicted in Fig. 4. In Fig. 3, curve a rep-resents a control assay in which only substrate was present, andcurve b shows an ALP inhibition assay in which PO4

�3 (10 lM)was added. In that electropherogram, the peak area for product(4-NP) was significantly smaller compared with the control assay.Substrate 4-NPP has two negative charges and migrated first,whereas 4-NP has a single negative charge and migrated slightlylater at 2.80 min. The peaks for substrate and products appear in

obilization control assay (without inhibitor) (a) and inhibition assay (b). Thery column. The separation conditions were as follows: fused-silica capillary, 75 lmion at 322 nm; separation buffer, 50 mM borate buffer (pH 9.5) containing 0.0025%e same separation conditions were applied as in the control assay.

Fig.4. Concentration-dependent inhibition of CIAP, theophylline (.), arsenate (N),and PO4

�3 (�) determined by CIAP immobilized on-capillary enzyme reaction usinga substrate concentration of 1 mM 4-NPP, a reaction buffer consisting of 5 mMZnCl2, 0.2 mM MgCl2, 50% glycerol, and 10 mM Tris–HCl (pH 9.8), and variousconcentrations of inhibitor. For separation conditions, see Fig. 3. Data pointsrepresent means ± standard deviations from three separate experiments, each runin duplicate.

Table 2Determination of Ki values for standard inhibitors of CIAP with enzyme immobilizedCE method.

Inhibitor Ki ± SEM (lM)

ALP immobilized CE 96-well plate reader Literaturevalue

PO4�3 2.41 ± 0.02 3.11 ± 0.03 1.55 [43]a

Theophylline 50.2 ± 3.6 96.1 ± 0.05 90.0 [31]b

Arsenate 8.91 ± 1.7 0.261 ± 0.02 1.67 [43]a

Note. Values represent means ± standard errors (SEM) of three separate experi-ments. For CE conditions, see Fig. 3.

a Human intestinal enzyme and 4-NPP substrate were used, and the reaction wasmonitored using a spectrophotometer.

b Calf intestinal enzyme and AttoPhos fluorogenic substrate were used, and assaywas performed on CE.

230 Microassay in CE for alkaline phosphatases / J. Iqbal / Anal. Biochem. 414 (2011) 226–231

the electropherogram in less than 3 min, clearly proving that thenew enzyme assay method is simple and very fast. The mechanismof inhibition of phosphate was determined at CIAP immobilizedmicroreactor. Enzyme kinetics was investigated using differentconcentrations of substrate at two fixed concentrations of inhibi-tor: 0.1 and 1.0 lM. It was found that the Vmax value did not varysignificantly in the presence of the inhibitor. However, the Km va-lue increased with increasing phosphate concentrations, indicatingthat phosphate showed a competitive mechanism of inhibition.The Lineweaver–Burk plots for phosphate are shown in Fig. 5.The Ki values of the standard CIAP inhibitors tested on enzymeimmobilized microreactor were close to the Ki values obtained bythe standard spectrophotometric assays performed in the currentinvestigation as well as to the literature values (Table 2).

Inhibition of TNAP

An enzyme inhibition assay of immobilized TNAP was carriedout using levamisole as an uncompetitive standard inhibitor. The

Fig.5. Lineweaver–Burk plot of inhibition of CIAP by phosphate determined by CIAPimmobilized on-capillary enzyme reaction. Concentrations of PO4

�3: d, 0 lM; N,1.0 lM; j, 0.1 lM. For separation conditions, see Fig. 3. Data points representmeans ± standard deviations from three separate experiments, each run induplicate.

Ki value was calculated by injecting seven concentrations of theinhibitor, and each analysis was repeated three times (triplicates)in two separate experiments. The calculated Ki value of TNAPwas 23 lM, which was very close to the literature value [37] (datanot shown).

Conclusions

An in-line enzyme bioreactor was developed inside the capillarycolumn for characterization of CIAP and inhibition studies of CIAPand TNAP. To improve the separation performance of enzyme bio-reactor, the capillary surface was dynamically coated using PB. Incoated capillaries, enzyme assay times were drastically reducedby shortening the migration times of substrate and product. PBcoating produced higher repeatability for migration times (relativestandard deviation values < 1%) in comparison with the uncoatedcapillary. The quantitative analysis of the enzymatic reactionscan be carried out in less than 3 min. Therefore, it will allow thescreening of compound libraries to identify potent and selectiveinhibitors of ALP isozymes. In addition, lower detection limits ofreactants were obtained by applying electroinjection at high field.The values of the Michaelis–Menten constant (Km) and inhibitionconstant (Ki) were close to those obtained in the current studyusing a 96-well microplate reader and literature values. ALP immo-bilized bioreactor allows reuse of the enzyme for multiple assays,which saves on reagent costs by reducing the consumption of re-agents. Furthermore, it is fast, easy to prepare, and regeneratesthe enzyme bioreactor.

Acknowledgment

This work was financially supported by the Higher EducationCommission (HEC) of Pakistan under the National Research Sup-port Program for Universities.

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