Luminol–hydrogen peroxide chemiluminescence produced by sweet potato peroxidase

92 ORIGINAL RESEARCH I. S. Alpeeva and I. Y. Sakharov

Copyright © 2006 John Wiley & Sons, Ltd. Luminescence 2007; 22: 92–96DOI: 10.1002/bio

Luminescence 2007; 22: 92–96Published online 6 November 2006 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/bio.931 ORIGINAL RESEARCH

Luminol–hydrogen peroxide chemiluminescenceproduced by sweet potato peroxidase

Inna S. Alpeeva1 and Ivan Yu. Sakharov1,2*1Chemical Enzymology Department, Faculty of Chemistry, The M.V. Lomonosov Moscow State University, Moscow, 119992, Russia2Division of Chemistry, The G.V. Plekhanov Russian Economic Academy, Stremyanny per. 28, 113054, Moscow, Russia

Received 20 December 2005; revised 24 April 2006; accepted 2 May 2006

ABSTRACT: Anionic sweet potato peroxidase (SPP; Ipomoea batatas) was shown to efficiently catalyse luminol oxidation byhydrogen peroxide, forming a long-term chemiluminescence (CL) signal. Like other anionic plant peroxidases, SPP is able to cata-lyse this enzymatic reaction efficiently in the absence of any enhancer. Maximum intensity produced in SPP-catalysed oxidation ofluminol was detected at pH 7.8–7.9 to be lower than that characteristic of other peroxidases (8.4–8.6). Varying the concentrationsof luminol, hydrogen peroxide and Tris buffer in the reaction medium, we determined favourable conditions for SPP catalysis(100 mmol/L Tris–HCl buffer, pH 7.8, containing 5 mmol/L hydrogen peroxide and 8 mmol/L luminol). The SPP detection limitin luminol oxidation was 1.0 × 10−14 mol/L. High sensitivity in combination with the long-term CL signal and high stability isindicative of good promise for the application of SPP in CL enzyme immunoassay. Copyright © 2006 John Wiley & Sons, Ltd.

KEYWORDS: peroxidase; sweet potato; chemiluminescence; luminol; hydrogen peroxide

Copyright © 2006 John Wiley & Sons, Ltd.

(SbP) are highly stable (7–10). Furthermore, like ARP,these peroxidases are catalytically highly active towardsluminol, producing high and long-term luminescencesignals (11, 12). For SbP and PTP, there is no need touse enhancers to increase the efficiency of luminoloxidation. The above data suggest that the anionic plantperoxidases are promising enzymes for immunoassayswith CL detection. In this paper we describe an applica-tion of a novel anionic plant peroxidase isolated fromsweet potato (SPP) in the catalysis of CL oxidation ofluminol by hydrogen peroxide. A comparative analysisdemonstrated that among all the peroxidases studied,SPP showed the highest sensitivity in the enzymaticoxidation of luminol.

MATERIALS AND METHODS

Sweet potato peroxidase (RZ 3.4) with a specificactivity of 4900 U/mg was purified to homogeneity asdescribed previously (13). Horseradish peroxidase (spe-cific activity 1100 U/mg, RZ 3.0) was purchased fromBiozyme (UK) and used without further purification.Luminol–HCl, p-iodophenol and Tris were from Sigma(USA), H2O2 (30%) from Merck (Germany). The con-centration of peroxidases was measured by monitoringA403 using ε403 = 102 000 mol/L/cm.

Catalytic luminol oxidation was assayed as follows:25 µL 2.5–500 mmol/L hydrogen peroxide and 25 µL5.0–400 mmol/L luminol (both reagents were prelimi-narily dissolved in Tris buffer at relevant pH andconcentration) were mixed with 200 µL 10–100 mmol/L

INTRODUCTION

Peroxidase (EC 1.1.11.7) is one of the most widespreadplant enzymes. Commercially available horseradishperoxidase isozyme c (HRP-C) is currently broadlyapplied in enzyme-linked immunosorbent assay(ELISA) applied in clinical, food and environmentalmeasurements. HRP-C is used as an enzyme label forimmunochemical reagents. Multiple detection methodsfor enzyme activity of peroxidase-labelled immunore-agents were applied, including colorimetry, fluorimetryand chemiluminescence (CL) (1). CL detection is mark-edly more sensitive than other methods. Luminol isthe peroxidase substrate commonly used in CL assays,but its HRP-C-catalysed oxidation appeared to havelow effectiveness (2). The application of enhancerssomewhat increased the reaction efficiency in CLimmunoassays (3, 4).

Later, it was found that anionic peroxidase fromArthromyces ramonus (ARP) in the absence ofenhancers showed a 500-fold increase in luminol oxida-tion compared to HRP-C (5). Although this enzymeis highly active towards luminol, its low stability underthe analysis conditions did not allow the use of ARPin immunoassay (6). In contrast, the anionic plantperoxidases from palm tree leaves (PTP) and soya beans

*Correspondence to: I. Y. Sakharov, Chemical Enzymology Depart-ment, Faculty of Chemistry, The M.V. Lomonosov Moscow StateUniversity, Moscow, 119992 Russia.E-mail: [email protected]

Contract/grant sponsor: INTAS, Russia; Contract/grant number:03-55-2428.


Sweet potato peroxidase-catalysed CL ORIGINAL RESEARCH 93 ORIGINAL RESEARCH 93

Tris–HCl, pH 7.6–9.0, in wells of transparent polystyrenestrips (MaxiSorp plates, NUNC, Denmark) for enzymeimmunoassay. Then the enzymatic reaction was initiatedby adding 10 µL peroxidase solution soluble in Tris–HClat the relevant pH and concentration. The CL kineticswas measured for 40 min at room temperature on aLumiScan luminometer (Immunotech, Russia). The CLsignal formed in the absence of enzyme was used as acontrol.

RESULTS AND DISCUSSION

Recently the anionic sweet potato peroxidase waspurified and characterized by Castillo Leon et al. (13).The enzyme is a glycoprotein with a molecular mass of37 000 Da and pI 3.5. It belongs to a family of secretoryplant peroxidases. Like other peroxidases, it catalysesthe oxidation of luminol by the ‘ping-pong’ mechanism:

E + H2O2 → EI + H2O (1)

EI + A → EII + A. (2)

EII + A + H+ → E + A. + H2O (3)

where E is the ferric enzyme (resting state), EI andEII are compounds I and II, the oxidized intermediatesof peroxidase which are by two and one oxidationequivalents above the resting state, respectively. Theradical product of luminol oxidation is then convertedinto 3-aminophthalate, and this process results in lightemission (14).

Determination of favourable conditions for SPP-catalysed oxidation of luminol by hydrogen peroxiderevealed the maximal CL at pH 7.8–7.9 (Fig. 1). Thisvalue is lower than those for the same reaction catalysed

by PTP and SbP and for the CL-enhanced reactioncatalysed by HRP-C (pH 8.4–8.6) (6, 11, 12).

Varying the luminol concentration, we found thatthe CL signal increased with luminol concentration,reaching saturation at 5 mmol/L luminol (Fig. 2). Similardependence was observed in PTP catalysis (11).

Earlier in the study of luminol oxidation by PTP andSbP, we reported that the concentration of Tris bufferaffected the CL intensity. Furthermore, a character ofthese dependencies differed for these enzymes. So, whilein the case of PTP the intensity increased with decreas-ing the buffer concentration and reached its maximumin 10–20 mmol/L Tris solutions (11), we observed areverse dependence for SbP, where the light intensityincreased with the buffer concentration reaching thesaturation in 60 mmol/L Tris buffer (12). Our study ofthe influence of buffer concentration on the emissionintensity caused by luminol oxidation in the presence ofSPP showed the observed effect to be similar to that forSbP (Fig. 3).

Hydrogen peroxide is an oxidative substrate forperoxidases and produces compound I from a restingform of peroxidase (Eq.1). However, at high H2O2 con-centrations, inactivation of plant and fungal peroxidasesoccurs, giving less active compound III and the inactivecompound P-670 (15, 16). Therefore, this substrate isknown as a suicidal one. The second substrate, in ourcase luminol, may protect the peroxidase active site fromthe inactivating action of H2O2. Since the optimal con-centration of H2O2 strongly depends on the chemicalnature of substrate and its concentration, we varied thehydrogen peroxide concentration in feed. The maximumCL signal was shown to be at around 5 mmol/L H2O2

(Fig. 4). Further increase in the concentration of thissubstrate attenuated the light intensity.

Previously it was reported that in the absence ofenhancers, HRP-C catalysed the luminol oxidation with

Figure 1. Effect of pH in reaction medium on CL intensityproduced through SPP-catalysed oxidation of luminol byhydrogen peroxide. Conditions: [SPP] = 1.2 × 10−12 mol/L;[H2O2] = 8 mmol/L; [luminol] = 10 mmol/L; 100 mmol/L Trisbuffer; CL intensity was recorded 15 min after the start ofthe reaction.

Figure 2. Effect of luminol concentration in reaction mediumon CL intensity produced through SPP-catalysed oxidationof luminol by hydrogen peroxide. Conditions: [SPP] = 0.7 ×10−12 mol/L; [H2O2] = 5 mmol/L; 100 mmol/L Tris buffer,pH 7.8; CL intensity was recorded 15 min after the start ofthe reaction.



low efficiency (2). The addition of enhancers notably in-creased the light intensity compared with unenhancedreactions (4, 17, 18). Contrary to HRP-C, SPP doesnot alter the activity in the presence of p-iodophenol(a frequently used enhancer) at concentrations of0.5–2.0 mmol/L. The ability to efficiently catalyse theluminol oxidation in the absence of enhancers appearsto be a feature of all anionic peroxidases (6, 11, 12, 19).Thus, from the obtained results, in further study we used100 mmol/L Tris buffer, pH 7.8, containing 5 mmol/Lhydrogen peroxide and 8 mmol/L luminol as favourableconditions for the SPP-catalysed oxidation of luminol.

A comparison of kinetic curves of luminol oxidationby hydrogen peroxide catalysed by HRP-C and SPP ispresented in Fig. 5. For enhanced CL reaction (ECR) inthe presence of HRP-C, the intensity reached its maxi-mum for a short time and then quickly dropped (curve

Figure 3. Effect of Tris buffer concentration on CL intensityproduced through SPP-catalysed oxidation of luminol byhydrogen peroxide. Conditions: [SPP] = 0.7 × 10−12 mol/L;[luminol] = 10 mmol/L; [H2O2] = 5 mmol/L; Tris buffer, pH7.8; CL intensity was recorded 15 min after the start of thereaction.

Figure 4. Effect of H2O2 concentration in the reactionmedium on CL intensity produced through SPP-catalysedoxidation of luminol by hydrogen peroxide. Conditions:[SPP] = 0.7 × 10−12 mol/L; [luminol] = 10 mmol/L; 100 mmol/LTris buffer, pH 7.8; CL intensity was recorded 15 min after thestart of the reaction.

Figure 5. Kinetic curves of CL intensity through luminoloxidation by hydrogen peroxide in presence of HRP-C (a) andSPP (b). Conditions: a, [HRP-C] = 1.8 × 10−10 mol/L; [H2O2] =1.5 mmol/L; [luminol] = 2 mmol/L; 50 mmol/L Tris buffer, pH8.4; [p-iodophenol] = 1 mmol/L; b, [SPP] = 1.2 × 10−12 mol/L;[H2O2] = 8 mmol/L; [luminol] = 4 mmol/L; 100 mmol/L Trisbuffer, pH 7.8.

a). SPP shows the alternative character (curve b). It canbe seen that the SPP-induced CL signal increased at alower rate than in the case of HRP-C. However, afterreaching the maximum intensity, this value little changedwith time. Since the accuracy of analysis depends on theaccuracy of measurements, we believe that the use ofSPP in CL immunoassay is more preferable than HRP-C. Note that, like SPP, other anionic plant peroxidasesalso produce a long-term CL signal (11, 12).

In favourable conditions the detection limit was evalu-ated for SPP corresponding to the enzyme concentrationrequired for CL twice that of the CL of the same solu-tion but without the enzyme. The value obtained for thedetection limit for SPP (10 fmol/L) (Fig. 6, insert) is theleast among those reported previously for HRP-C(30 fmol/L for ECR) (18), PTP (2 pmol/L) (11), SbP(300 fmol/L) (12) and tobacco peroxidase (100 fmol/L)(19). For all peroxidases except HRP-C, the detectionlimit was measured in the absence of enhancers. Alsonote that SPP concentration vs. light intensity is linearin a broad interval of the enzyme concentration (0.2–2.0 pmol/L). The mean standard curve of SPP for CLdetection under favorable conditions is presented inFig. 6. The relative standard deviation (RSD) and therecovery values at the SPP concentrations 0.2, 1.0 and2 pmol/L were 6 and 100.5 ± 1.0%, 11 and 107.0 ± 6.5%,and 9.3 and 108.1 ± 7.8%, respectively. The best fittingof the experimental data was reached with the linearfunction y = 2.6x − 3943 (r2 = 0.996, n = 5).

Thus, the results obtained demonstrated that SPP isthe most sensitive peroxidase in luminol oxidation. Alsonote that, although HRP-C is one of the most stablebiocatalysts compared with other enzymes, the stabilityof SPP is higher than that of HRP (20). This featureof SPP is important for its practical application.Therefore, the combination of molecular and catalytic


Sweet potato peroxidase-catalysed CL ORIGINAL RESEARCH 95 ORIGINAL RESEARCH 95

characteristics obtained for SPP promises well for itsuse in CL enzyme immunoassay. The synthesis of activeimmunoconjugates labelled by SPP and their applicationin CL ELISA for determination of sulphanilamides willbe the subject of further work.

Acknowledgement

The authors thank INTAS (Grant No. 03-55-2428) forsupport of this work.

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Figure 6. Calibration curve for determination of SPP using the reaction of luminol oxidation. Conditions:[H2O2] = 8 mmol/L; [luminol] = 10 mmol/L; 100 mmol/L Tris buffer, pH 7.8; CL intensity was recorded 10 min after thestart of the reaction.



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Luminol–hydrogen peroxide chemiluminescence produced by sweet potato peroxidase