ARBOLEDA, J. W.

GROSSI DE SÁ, M. F.

JIMÉNEZ, A. V.

English

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www.elsevier.com/locate/yabioANALYTICALBIOCHEMISTRYAnalytical Biochemistry 366 (2007) 113–115Notes & TipsDetection of a-amylase inhibitors by a zymography method,performed in isoelectric focusing electrophoretic PhastGelsArnubio Valencia-Jiménez a,b,c,*, Jorge W. Arboleda V a,b, Maria Fátima Grossi-de-Sá aa Embrapa-Cenargen, PqEB, W5 Norte Final, 70770-900, Brası́lia, DF, Brazilb Universidade de Brası́lia, Asa Norte, 70910-900, Brası́lia, DF, Brazilc Universidad de Caldas, Manizales, Calle 65 26-10, ColombiaReceived 22 March 2007Available online 7 April 2007Different electrophoretic methods for the analysis ofa-amylase inhibitor isoforms have been tested and someof these methods include immunostaining after electropho-resis requiring specific antibodies which, in some cases, givefalse signals due to cross reactivity with other closelyrelated proteins. However, zymography techniques havebeen shown to be effective tools for visualizing, isolating,and characterizing natural enzyme inhibitors [1–3]. Manyof these zymography methods are based on the separationof proteinaceous inhibitors on an SDS–polyacrylamide gelcontaining the protein substrate copolymerized into the gel.Usually, the amylase inhibitors are tested on unique-per-cent native-polyacrylamide gels copolymerized with starch.After electrophoresis, the gel is washed and incubated withthe target amylase solution to allow the substrate to bedigested by the target amylase. The undigested amylasesubstrate remains where the inhibitor molecules are locatedand can be stained by using a KI/I2 solution, which pro-duces dark blue bands on a light background [4,5].A similar zymography method was useful in searchingfor identification of mutants in pigeonpea (Cajanus cajanL. Millisp) containing diverse isoforms of a-amylase inhib-itors; however the method was not useful to separate sim-ilar molecular-weight inhibitors with small differences intheir isoelectric points [5]. We developed a simple reversezymography method, performed in IEF1 gels which wereincubated in a soluble starch solution and then stained witha KI/I2 solution, allowing a better resolution and separa-tion of amylase inhibitors than the traditional native orSDS-based zymograms. By using this methodology, a widerange of quantities of plant amylase inhibitors could be0003-2697/$ - see front matter  2007 Elsevier Inc. All rights reserved.doi:10.1016/j.ab.2007.04.005* Corresponding author. Fax: +57 68 781500/781554.E-mail address: avalbioquimica@yahoo.com (A. Valencia-Jiménez).1 Abbreviation used: IEF, isoelectric focusing.selectively detected, allowing detection of new inhibitorswith small differences in their isoelectric point.Finely ground bean seeds of Phaseolus vulgaris L andP. coccineus were 3· defatted by shaking them with acetone(1:1 w/v) for 10 min and then decanting. The defatted flour(10 g) was mixed with 50 ml of 0.1 M NaCl and continu-ously stirred for 180 min at 4 C. The soluble proteins inthe slurry were obtained by centrifugation at 20,000g for60 min and the supernatant was dialyzed extensivelyagainst water to allow protein precipitation. After centrifu-gation (20,000g for 20 min), the supernatant was freeze-dried. The resulting powder was used as initial source ofinhibitor for zymography. Zymography technique wasdeveloped by using a PhastSystem instrument (GE Health-care Life Sciences) operating with PhastGel Media IEF3–9, which allows separation at a pH range from 3 to 9.Mixtures of pI markers between pH 3.5 and 9.3 were used.Electrophoresis was performed on a thermostatic platemaintaining the temperature at 15 C. Samples were runas described by the manufacturer (Separation TechniqueFile No. 100). In the IEF inhibitor zymogram, after elec-trophoresis the gel was incubated in 1.0% soluble starchin 10 mM Na-citrate, pH 5.0, containing 20 mM NaCland 20 mM CaCl2 for 1 h at 4 C. The gel was rinsedquickly with the same buffer and incubated at 30 C for20 min with human salivary amylase dissolved with theactivity buffer. After this, the gel was rinsed with waterand then the starch–gel was stained with an iodine/iodide(0.5% I2 and 5% KI) solution for 10 min. This treatmentproduced dark blue bands on a light background. To detectprotein bands, a part of the gel was stained in a Coomassiebrilliant blue solution prepared by adding one tablet ofPhastGel Blue R (GE Healthcare) to 400 ml of bleachingsolution (30% methanol and 10% acetic acid) for 30 minand then washing with the bleaching solution until the114 Notes & Tips / Anal. Biochem. 366 (2007) 113–115background of the gel was completely decolored. Theisoelectric points were calculated using standard proteinswith known isoelectric points and with software (ImageMaster VDS) provided by Pharmacia Biotech.To determine whether this zymography technique couldbe used as a powerful tool to find new a-amylase inhibitorsfrom plants, different accessions of P. vulgaris seeds werescreened for the presence of these kinds of enzymatic inhib-itors. In this experiment, two adjacent applications of pro-tein samples from bean seeds containing the inhibitor wereseparated by using IEF-PhastGel focused in pH 3.0 to 9.0ampholine gradients. One part of the PhastGel was visual-ized for protein and pI markers and the other part was usedto locate the a-AI activity bands. Fig. 1 shows that this opti-mal zymography technique gives clear patterns containing asharp blue a-AI band on the starch–gel background, becausethe amylase digests the starch everywhere in the gel, exceptwhere the inhibitor is located (Fig. 1, lanes 2a and 2b). It isvery important to point out that, after longer incubationtimes the a-AI band could gradually disappear. We foundtwo protein bands with isoelectric points (pI) of 4.7 and 5.0(Fig. 1, lanes 1b and 1d). Similar results were previously re-ported by Valencia Jimenez et al. [6] and Veronique et al.[7]. In those experiments, the isoelectric point for a-AI iso-form 1 was found to be 4.7 and 4.6, respectively. In addition,positions of the protein and inhibitory activity bands on theIEF PhastGel were identical, giving direct evidence that thea-amylase inhibitor was proteinaceous. a-AI-1, which inhib-its mammalian and some insect a-amylases, is frequentlyfound in many cultivated common bean varieties [8].P. coccineus has been reported to contain a-amylaseinhibitors [9–11], and we used our zymography methodol-ogy to detect the presence of this kind of a-amylase inhib-itor in two different accessions of scarlet runner bean. Byusing this new technique, it was possible to find not onlyFig. 1. Isoelectric focusing of a-amylase inhibitor from two accessions ofP. vulgaris. Lanes 1a and 1c, mixture of pI standard proteins, humancarbonic anhydrase B (pI 6.55), bovine carbonic anhydrase B (pI 5.85),ß-lactoglobulin A (pI 5.20), soybean trypsin inhibitor (pI 4.55),amyloglucosidase (pI 3.5). Lanes 1b and 1d, proteins from P. vulgarisCoomassie blue stained, showing the a-AI protein band. Lanes 2a and 2b,inhibitor zymogram. Isoelectric focusing was performed on PhastGel IEF3-9. The zymogram gel was specifically stained with iodine solution todetect a-amylase inhibitor activity bands.one but different a-AI-1 isoforms in some of the Phaseolusvarieties that were tested. The results (Fig. 2) show that ourmethodology produces a very clear zymogram image (darkblue band of a-amylase inhibitor bands on a white back-ground) and probes the high capacity of this technique toseparate and to elucidate, with a very good resolution,the different a-AI-1 isoforms that could be present in theplant sample. The size and intensity of the dark blue banddepend upon the protein concentration and activity ofa-amylase inhibitor in the starch–PhastGel. Of particularinterest is the different patterns found in the two seedP. coccineus accessions. The P. coccineus accession thatwas run in lane 1 shows a a-AI isoform (a-AI-1) with asuperior level of expression compared to those of theothers. In our opinion, any effort to purify a-amylaseinhibitors from this scarlet runner bean accession must beaddressed to this particular activity band because of thedemonstrated abundance of this inhibitory activity band.Other zymography methods tested to detect a-amylaseinhibitors in plant seed sources, based on starch–gels, werealways run on homogeneous starch–gels needing, in somecases, additional experimental steps which require thatthe initial polyacrylamide gel must be sandwiched withother starch–gels [12], generating a probable diminutionof inhibitory activity. The results show that, under theconditions of our assay, the zymography technique usingIEF PhastGels is a promising tool to screen plant extractsfor inhibitory activity.In summary, we have developed a simple starch–Phast-Gel method very useful for detecting a-amylase inhibitorsfrom natural sources, differing not only in their molecularweights, but also in their isoelectric points. Our methodol-ogy shows that it is possible to separate these proteinaceousinhibitors while keeping their biological activity under verygood resolution conditions. By using this methodologyFig. 2. Isoelectric focusing of a-amylase inhibitors from two differentaccessions of P. coccineus seeds. Lanes 1 and 2, a-AI zymograms showingthe activity protein bands. Electrophoresis was performed on PhastGelIEF 3-9.Notes & Tips / Anal. Biochem. 366 (2007) 113–115 115some characteristics of a-AIs from common and scarletrunner bean seeds are revealed.References[1] Q.T. Lea, N. Katunuma, Detection of protease inhibitors by areverse zymography method, performed in a tris (hydroxymethyl)aminomethane–Tricine buffer system, Anal. Biochem. 324 (2004)237–240.[2] G.R. Bushell, P. Ghosh, J.S. Hanspal, Detection of proteaseinhibitors using substrate-containing sodium dodecyl sulfatepoly-acrylamide gel electrophoresis, Anal. Biochem. 132 (1983) 288–293.[3] D. Michcaud, D. Cantin, A. Raworth, Vrain, Assessing the stabilityof cystatin/cystein complex using denaturing gelatin-polyacrylamidegel electrophoresis, Electrophoresis 17 (1996) 74–79.[4] N.P. Chougule, A.P. Giri, V.K. Hivrale, P.J. Chhabda, M.S.Kachole, Identification of amylase inhibitor deficient mutants inpigeonpea ( Cajanus cajan (L.) Millisp.), Biochem. Genet. 42 (2004)165–180.[5] A.P. Giri, M.S. Kachole, Amylase inhibitors of pigeonpea (Cajanuscajan) seeds, Phytochemistry 47 (1998) 197–202.[6] A. Valencia-Jimenez, A.E. Bustillo, G.A. Ossa, M.J. Chrispeels,a-amylases of the coffee berry borer (Hypothenemus hampei) and theirinhibition by two plant amylase inhibitors, Insect Biochem. Mol. Biol.30 (2000) 207–213.[7] L.B-A. Véronique, B.G. Coralie, P. Françoise, R. Pierre, Character-ization and functional properties of the a-amylase inhibitor (a-AI)from kidney bean (Phaseolus vulgaris) seeds, Biochim. Biophys. Acta1343 (1997) 31–40.[8] M.F. Grossi-de-Sa, M.J. Chrispeels, Molecular cloning of bruchid(Zabrotes subfasciatus) alpha-amylase cDNA and interactions of theexpressed enzyme with bean amylase inhibitors, Insect Biochem. Mol.Biol. 27 (1997) 271–281.[9] J.J. Pueyo, A. Delgado-Salinas, Presence of alpha-amylase inhibitorin some members of the subtribe phaseolinae (phaseoleae: fabaceae),Am. J. Bot. 84 (1997) 1008–1012.[10] M. Ishimoto, T. Sato, M.J. Chrispeels, K. Kitamura, Bruchidresistance of transgenic azuki bean expressing seed a-amylaseinhibitor of common bean, Entomol. Exp. Appl. 79 (1996) 309–315.[11] A.P. Railene, A.N.B. João, M.deS. Maria Cristina, B.deO.N.Osmundo, Z.F. Edson Luiz, V.J. Arnubio, G de S. Maria Fátima,An a-amylase inhibitor gene from Phaseolus coccineus encodes aprotein with potential for control of coffee berry borer (Hypothenemushampei), Phytochemistry 67 (2006) 2009–2016.[12] R.J. Weselake, A.W. Macgregor, R.D. HILL, Purification andcharacteristics of an endogenous a-amylase inhibitor from barleykernels, Plant Physiol. 73 (1983) 1008–1012.

Detection of alfa-amylase inhibitors by a zymography method, performed in isoelectric focusing electrophoretic PhastGels.

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Detection of alfa-amylase inhibitors by a zymography method, performed in isoelectric focusing electrophoretic PhastGels.

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