In-gel detection of urease activity by nitroprusside–thiol reaction

UREASE ACTIVITY STAINING WITH NITROPRUSSIDE-THIOL 99

Copyright © 2007 John Wiley & Sons, Ltd. Phytochem. Anal. 19: 99–103 (2008)DOI: 10.1002.pca

Phytochemical AnalysisPhytochem. Anal. 19: 99–103 (2008)Published online 24 July 2007 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/pca.1012

In-gel Detection of Urease Activity by Nitroprusside–ThiolReaction

VINEET SHARMA,1,2 RAJESH CHAUDHARY,2 J. M. KHURANA1 and K. MURALIDHAR2*1 Department of Chemistry, University of Delhi, Delhi-7, India2 Department of Zoology, University of Delhi, Delhi-7, India

Received 20 May 2006; Revised 17 May 2007; Accepted 17 May 2007

Abstract: A new staining method for urease activity in non-denaturing polyacrylamide gels is described. The increase in local pH ofthe gel, resulting from ureolytic activity of urease, causes a purple red coloured band after incubation of the polyacrylamide gelwith urea. Staining of urease activity using this method is very specific for catalytically active urease even in crude preparations.Detection of urease activity by this method is rapid, simple and economical. The described method is also more sensitive thanexisting methods of urease staining. A minimum of 0.25 mU of urease activity can be detected after 5 min of incubation with thesubstrate. The method has been used to demonstrate the presence of different charge isoforms of urease in a member of the plantfamily Cucurbitaceae. Copyright © 2007 John Wiley & Sons, Ltd.

Key words: In-gel staining; urease activity; pH increase; Cucurbitaceae.

* Correspondence to: K. Muralidhar, Department of Zoology, University ofDelhi, Delhi-7, India.E-mail: [email protected]/grant sponsor: University Grants Commission, New Delhi.Contract/grant sponsor: Council of Scientific and Industrial Research,New Delhi.

INTRODUCTION

Urease (urea amidohydrolase; E.C.3.5.1.5.) is a nickel-dependent metallo-enzyme, ubiquitously present inbacteria, selected fungi, algae, certain invertebrates andalmost the entire plant kingdom (Fahmey et al., 1994;Mobley et al., 1995; Das et al., 2002; Krajewska et al.,2003). The presence of urease activity in soils iscontinuously exploited in the widespread agriculturalpractice of urea based fertilizer application to enhancecrop yields (May and Douglas, 1976; Qin and Cabral,2002). Urease of many pathogenic organisms includingHelicobacter pylori has been implicated in the hostpathology and this has stimulated much industrial andmedical research (Mobley et al., 1995; Moncrief et al.,1995; Qin and Cabral, 2002). Urease is also of wideecological interest, since urea has been recognised as apollutant (Martino et al., 2003). As a further extension,immobilised urease has application in urea removaldevices and biosensors (Qin and Cabral, 2002).

Urease catalyses the hydrolysis of urea at an acceler-ated rate to yield ammonia and carbamate. Carbamateis unstable and spontaneously decomposes to yielda second molecule of ammonia and carbonic acid(Mobley et al., 1995; Moncrief et al., 1995).

The released products, carbonic acid and ammoniaare in equilibrium with their ions resulting in a netincrease in pH of the reaction (Mobley et al., 1995;Krajewska et al., 2003).

H2CO3 H+ + HCO3

−

2NH3 + 2H2O 2NH4+ + 2OH−

This increase in pH has been previously used to as-say urease and served as a method to localise ureaseactivity in non-denaturing gels. Other methods involveeither precipitation with lead (Shaik et al., 1980), silverstaining (De Martin Llano et al., 1989) or modifiedFishbein’s reagent (Witte and Escobar, 2001) to localiseurease in the native polyacrylamide gels.

We report here a specific method to stain for ureaseactivity using the nitroprusside–thiol reaction in non-denaturing polyacrylamide gels. Sodium nitroprussidereacts with thiol compounds at alkaline pH and pro-duces a purple red complex (Szacilowski et al., 2002).In the described method, sodium nitroprusside–thiolcomplex is used to stain urease, which produces apurple-red coloured band in native polyacrylamide gelelectrophoresis. The alkalinity required for the colourdevelopment derives from the action of urease on urea.

EXPERIMENTAL

Chemicals and standards. Canavalia ensiformis urease(Type III), used as a standard preparation, was purchasedfrom Sigma (St. Louis, MO, USA). Sodium nitroprussidewas obtained from Qualigens Fine Chemicals (Mumbai,India). β-Mercaptoethanol and dithiothreitol (DTT) were

PhytochemicalAnalysis

100 V. SHARMA ET AL.


obtained from Ferak (Berlin, Germany) and Sigma,respectively. Urea was obtained from E. Merck (Mumbai,India).

Urease purification and sample preparation. Ureasefrom dehusked Cajanus cajan (L.) Millsp. was partiallypurified according to the procedure of Das et al. (2002).Urease from other sources was extracted into 50 mM

phosphate buffer, pH 7.0, containing 50 mM sodiumchloride and 1 mM EDTA. At the time of homogenisation10 mM DTT and 0.1 mM phenylmethylsulphonyl fluoride(PMSF) were also added and the homogenate cen-trifuged twice at 15,000 g for 30 min at 4°C. Ureaseactivity was determined by the standard indophenolmethod. (Weatherburn, 1967; Witte and Escobar, 2001)One unit is defined as the amount of enzyme requiredto produce 1 μmol of ammonium ion/min at 37°C, pH 7.0.The protein concentration was determined by Lowry’smethod using BSA as a standard (Lowry et al., 1951).

Polyacrylamide gel electrophoresis. Electrophoresis wascarried out at room temperature under non-denaturingconditions using an Atto (Tokyo, Japan) electrophoresisunit. The stacking and separating gels contained 5% and8% acrylamide solution, respectively. The buffer systemof Laemmli (1970) was used. Sodium dodecyl sulphate(SDS) was replaced in each electrophoretic solution bydouble-distilled water and the urease samples were notheated. Following centrifugation, plant extracts weremixed with sample buffer and electrophoresis wasperformed for 4 h at constant voltage.

Zymogram analyses. At the end of electrophoresis, thegel was washed with double–distilled water for 10 minand then three times with 0.01 and 0.005 M acetatebuffer (pH 6.0) for 15 min each with gentle shaking.The washed gel was then immersed in 5 M urea solutionand incubated at 37°C for 5 min without shaking. Thegel was then transferred to a 1.5% sodium nitroprus-side solution and kept for 5 min. A volume (200 μL) offreshly prepared 1 M β-mercaptoethanol was added

immediately and mixed to obtain a final concentrationof 10 mM β-mercaptoethanol for the staining solution.The gel was gently shaken and a dark purple red colourdeveloped, which became intense within 5 min. Photo-graphs were taken on a BioRad (CA, USA) densitometerusing a green filter and were processed with QuantityOne software (BioRad, USA).

Staining of urease with lead acetate and modifiedFishbein’s method was performed as previously de-scribed (Shaik et al., 1980; Witte and Escobar, 2001).

RESULTS AND DISCUSSION

Sodium nitroprusside reacts with thiols and forms apurple colour complex in alkaline medium (Szacilowskiet al., 2002). Transformation of the yellow colour ofnitroprusside–thiol solution essentially requires alka-line pH to develop into the purple colour complex.The local alkalinity produced by ureolytic activity cantheoretically be used to develop purple colour due tonitroprusside–thiol reaction. In order to optimise thestandard conditions for zymogram development, a con-stant β-mercaptoethanol concentration was used (10 mM

final concentration), and the concentration of nitro-prusside solution was varied. The gel was allowed todevelop its final colour and densitometric analysis wasperformed. It was found that 1.5% final concentrationof nitroprusside was optimum for colour developmentat 10 mM concentration of β-mercaptoethanol inthe staining solution [Fig. 1(a)]. The effect of β-mercaptoethanol was also investigated by using thenitroprusside concentration at 1.5%. From Fig. 1(b), itis clear that the presence of 10 mM β-mercaptoethanoland 1.5% nitroprusside concentration in the stainingsolution was ideal and appeared to be optimum interms of sensitivity, high signal/background ratio andstability of the colour complex.

While developing the zymogram, β-mercaptoethanolwas used as a thiol compound for colour development.Formation of this zymogram with other commercially

Figure 1 Optimisation of reagents concentration for the zymogram development. Effects of different concentration of (a) sodiumnitroprusside in staining reagent containing 10 mM β-mercaptoethanol and (b) β-mercaptoethanol in staining reagent containing1.5% sodium nitroprusside.



method seems to be more sensitive to detect the cata-lytically active molecular aggregates in comparisonwith other sensitive zymograms of urease following thesame duration of activity staining [Fig. 3(b)]. In othermethods of urease staining, minimum detection limitsare reported to be 15 mU for silver and 600 mU for thelead acetate methods (Shaik et al., 1980; De Martinet al., 1989). A minimum of 25 μU of urease can bedetected by the modified Fishbein’s reagent after150 min of incubation of the gel with substrate, while2.5 mU can be detected within 5 min (Witte andEscobar, 2001). However, prolonged staining of ureaseactivity by modified Fishbein’s reagent may cause heavybackground staining depending on the proteins present(Witte and Escobar, 2001). The in-gel detection methodpresented here can detect a minimum of 0.25 mU after5 min of incubation of gel with urea, which provides aconsiderably shorter time and higher sensitivity withan added advantage of a clear background. No colourchange was observed in the rest of the gel, which leadsto the clear visualisation of urease activity bands. Thecolour formed after urease action was generally foundto be stable for a minimum of 60 min, but can be per-manently fixed by subsequent lead acetate treatment(Shaik et al., 1980).

A sensitivity profile of urease activity staining usingthe nitroprusside–thiol reaction is shown in Fig. 4,which clearly demonstrates that the described methodcan detect urease activity of 0.25 mU after incubationof the gel for 5 min with the substrate at 37°C.

This zymogram is very specific for catalytically activeurease only and no interference was observed from otherproteins. However, other deaminases may contribute tocolour development if their substrates are present inthe staining reagent. When equal amounts of protein ofheat-denatured urease, a native urease preparation fromCanavalia ensiformis of Sigma chemical company, ourown purified Cajanus cajan urease and other proteinswere electrophoresed and stained, the purple coloured

Figure 2 Effect of different thiol compounds in the develop-ment of zymogram. Equal units of enzyme were loaded andthe zymogram was developed by the use of different thiol com-pounds at same concentration.

available thiol compounds such as dithiothreitol andL-cysteine instead of β-mercaptoethanol could also beperformed under similar conditions. However a higherintensity of the purple coloured complex was obtainedwith the use of β-mercaptoethanol in comparison todithiothreitol or L-cysteine (Fig. 2).

In the present method, the polyacrylamide gel wasincubated with a solution of urea, and this was fol-lowed by incubation with β-mercaptoethanol–nitroprussidesolution. After an incubation of 5 min purple colouredbands developed corresponding to the site of ureaseactivity. The bands that developed due to urease activ-ity were found to be at the same site [Fig. 3(a)] as foundin the case of other published methods of urease activ-ity staining (Shaik et al., 1980; Witte and Escobar,2001). Densitometric analysis clearly revealed that inthe present method, the colour yield was higher thanin other methods and hence making this method com-paratively more sensitive in the detection of the polymericnature of urease [Fig. 3(b)]. Two bands of catalyticallyactive urease aggregate were clearly visible after 5 minof incubation with substrate at 37°C. The present

Figure 3 (a) Native PAGE of Canavalia ensiformis urease developed with different in-gel detection methods for same time length.Equal units of urease activity were loaded and stained with modified Fishbein’s reagent (Witte and Escobar, 2001), lead acetatemethod (Shaik et al., 1980) and thiol nitroprusside method. (b) Densitometric analysis of urease activity stained with modifiedFishbein’s reagent (L1), lead acetate (L2) and nitroprusside–thiol method (L3).

102 V. SHARMA ET AL.


band developed only in the two lanes containing Can-avalia ensiformis and Cajanus cajan urease (Fig. 5).

Urease is present in almost all plants with relativelyhigher abundance in the members of the families Legu-minaceae and Cucurbitaceae (Fahmey et al., 1994; Daset al., 2002). Urease, because of its universal presencein the plant kingdom, has immediate taxonomic value.An initial purification scheme of this enzyme urgentlyrequires a rapid and economical screening system toconfirm the presence of urease in different plants andtheir parts. Screening of urease activity by means ofa zymogram can also be used to monitor the relativeexpression of this enzyme in different plant species.It has been demonstrated that the semi-quantitativeestimation of urease level in seeds of different membersof the Cucurbitaceae can be performed using the presentmethod [Fig. 6(a)]. Equal amounts of protein wereloaded in each case and the gel was incubated withurea for the same length of time. The colour developedwas analysed by densitometry, which revealed thatthe highest level of urease activity were observed inthe seeds of Trichosanthes dioica Roxb., followed byCucumis melo L., Cucumis sativus L., Luffa aegyptiacaMill. and Lagenaria siceraria (Molina) Standl. in de-creasing order [Fig. 6(b)].

Zymogram, or activity staining in non-denaturingpolyacrylamide gel, is an established method to dem-onstrate the presence of different catalytically activemolecular species of urease (De Martin et al., 1989).Urease activity staining in native PAGE can be used tounderstand the polymeric status of this enzyme underdifferent ionic strengths, pH and storage conditions.

Figure 6 Native-PAGE showing the relative abundance ofurease in seeds of different cucurbits. (a) Equal amount ofprotein was loaded in each case which included Luffa aegy-ptiaca (L1), Trichosanthes dioica (L2), Cucumis melo (L3),Cucumis sativus (L4) and Lagenaria siceraria (L5). (b) Densito-metric analysis of the gel, confirming the presence of size/charge isoforms of seed urease.

Presence of different forms of urease in different plantscan easily be demonstrated by the zymogram method.In Fig. 6(a), urease activity was observed at differentlocations in the gel; which could be attributed to thenaturally occurring isoforms of urease in different plantseeds belonging to the same family. A similar observa-tion was noticed earlier by Fahmey et al. (1994).

The present work reports a new, sensitive and in-expensive method to assay urease in polyacrylamidegels after separation on PAGE under native and non-denaturing conditions. This method is specific, timesaving and easy to perform and may be used to demon-strate charge/size isoforms of urease, which will helpin the better understanding of the distribution of ureasein the plant kingdom especially for taxonomic studies.

AcknowledgementsFinancial assistance of the University Grants Commis-sion, New Delhi (Junior Research Fellowship) to V.S.and Council of Scientific and Industrial Research, NewDelhi (Junior and Senior Research Fellowship) to R.C.are gratefully acknowledged.

Figure 4 Native PAGE of a dilution series of Canavalia ensiformisurease; 0.25–100 mU of the enzyme was electrophoresed andstained after 5 min of incubation with substrate at 37°C.

Figure 5 Native-PAGE showing selectivity of nitroprusside–thiol method of activity staining, for catalytically active urease.Equal protein amounts of bovine serum albumin (L1), heat-killed urease (L2), Cajanus cajan urease (L3), ova albumin(L4), Canavalia ensiformis urease (L5) and horseradish peroxi-dase (L6) were loaded and stained for urease activity.



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In-gel detection of urease activity by nitroprusside–thiol reaction

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