Talanta 53 (2000) 6974

The use of simulation in the EPR spin probe technique fordetection of irradiated seeds

M. Maral Sunnetcioglu a,*, Dilek Dadayl b

a Department of Physics Engineering, Faculty of Engineering, Hacettepe Uni6ersity, 06532 Beytepe, Ankara, Turkeyb Department of Physics, Faculty of Art and Science, Zonguldak Karaelmas Uni6ersity, 67100 Zonguldak, Turkey

Received 28 September 1999; received in revised form 8 February 2000; accepted 14 February 2000

Abstract

An electron paramagnetic resonance (EPR) spin probe study of irradiated wheat seeds was performed dependingon irradiation dose. The structural changes in the membrane integrity were followed using aqueous solutions of4-hydroxy-TEMPO (TANOL) spin probe and a line broadening material. In the studies dry seed embryos were keptin these solutions for 150 min. The spectra were recorded at various times of air drying process. The simulation ofthese spectra indicated a decrease in the water content of the embryos depending on the increasing irradiation dose.This indicates the increase in the permeability of the membranes as a result of the radiation damage. From the decaycurves it is possible to determine about irradiation dose, however, this approach is not very successful at closeirradiation doses. 2000 Elsevier Science B.V. All rights reserved.

Keywords: Electron paramagnetic resonance; Nitroxide spin probes; Irradiated wheat; Simulation of electron paramagneticresonance spectra

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1. Introduction

Irradiation of food for preservation is a com-mon technique accepted by many countries. Thiscan be utilized for various purposes such as killingbacteria, viruses, insects or to delay the ripeningof some fruits. The permitted limit of irradiationdose in foods was stated as 10 kGy [1]. Therefore,it is necessary to develop convenient methodscapable to distinguish between irradiated foodand unirradiated one and also to establish the

amount of irradiation dose [2]. Frequently usedtechniques are thermoluminescence and electronparamagnetic resonance (EPR). After the firstapplications in 1970s [35], EPR is now a leadingtechnique in the search of irradiated foods [610].This technique is based on the presence of asubstantial amount of free radicals in the inter-ested food. The radicals produced, as a result ofg-irradiation, usually have a limited lifetime andin storage the numbers of radicals decay to theirbackground values. The lifetime of free radicals inseeds is approximately 30 days [1114]. EPR spinprobe technique is an important tool to investi-gate samples, which have insufficient amount of

* Corresponding author. Tel.: 90-312-2977224; fax: 90-312-2352550:2354341.

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M.M. Sunnetcioglu, D. Dadayl : Talanta 53 (2000) 697470

free radicals for direct EPR applications [15,16].Paramagnetic probes are introduced into the sam-ple and they transfer valuable information aboutthe dynamic and structural changes in their envi-ronments without perturbing this environment.This technique has a wide application in biologi-cal systems such as model membranes, blood cells,proteins [1721]. Recently, spin probe techniquewas applied to the investigation of plant seeds[22]. After this first application the technique wasutilized to test the viability of seeds [2326]. Itwas first applied to the investigation of irradiatedseeds in 1997 [27] and further studies followed[28,29].

The aim of the current study is to develop amore practical way for detection of irradiatedseeds using spin probe technique together withsimulation methods and to test the ability of thespin probe method for long storage times.

2. Experimental

Irradiated and control Durum wheat samples(c.v. Kunduru 1149), harvested in 1994, were usedin the studies. Prior studies, germination testswere carried out and 98% germination was ob-tained. Irradiation was performed at 1.0, 2.5, 5.0,10.0, 20.0 kGy doses using g-irradiation fromcalibrated 60Co source at Saraykoy Nuclear Re-search Institute, Ankara. The absorbed dose waschecked by Fricke dosimetry. For spin probestudies, after irradiation at May 1996, sampleswere stored in a steel cabinet at room temperaturefor approximately 2 years. Experiments were per-formed between September 1998 and July 1999.Dry embryos of wheat seeds were used in thestudies. Sample preparation was performed asstated in a previous study [25]. Aqueous solutions(10 mM) of 4-hydroxy-TEMPO (TANOL) wereprepared including the line broadening materialpotassium ferricyanide (K3Fe(CN)6) at 250 mMconcentration. The presence of potassium ferri-cyanide provides the disappearance of the signalfrom extracellular regions as a result of linebroad-ening. For damaged cells following the leakage itcauses an increase in the linewidth of the signalfrom intracellular regions. In our previous studies,

the irradiation dose dependence was followed viathe rehydration curves [27,29]. In this study, twodifferent methods were tried to shorten the spec-trometer times and, instead of recording a numberof spectra, for each sample only one spectrum wasrecorded.

In the first method (method 1) certain amountof prepared solution (:0.5 cm high in the capil-lary) was taken into a 1 mm i.d. capillary andsingle dry embryos were kept in the solution for150 min. At the end of the waiting time, thesolution in the capillary was absorbed by a paperand the spectra of the samples recordedimmediately.

In the second method (method II) five dryembryos were soaked for 150 min in the solutionin a glass tube. After this time embryos weretaken onto glass plates using a paintbrush and leftto air-dry for 15 min. Then the spectra wererecorded.

All of the experiments were repeated at leastthree times. The spectra were recorded at roomtemperature by the use of a Varian E-9 and aBruker EMX X-band spectrometers using the fol-lowing spectral conditions; modulation amplitude,0.05 mT; microwave power, 2 mW.

3. Results and discussion

The recorded spectra for control and irradiatedsamples using both method I and method II indi-cate a change in the spectrum shape depending onirradiation dose. This change is especially effectiveat m1 1 high field line, which splits into lipidand aqueous parts [24,25]. The reason of theserelative changes in the spectra is due to changes inthe membrane permeability as a result of irradia-tion. In healthy cells the plasma membrane isimpermeable to potassium ferricyanide. Since thesignal from the solution is completely removed,recorded spectra consist of only lipid and polarsignals from intracellular regions, however, sincesome of the cells are damaged within embryo,polar signal consists of two parts. Therefore, theexperimental spectra can be simulated using thesum of the signals from three main regions.

M.M. Sunnetcioglu, D. Dadayl : Talanta 53 (2000) 6974 71

1. TANOL:water in healthy cells (waterpercentage).

2. TANOL:water line broadening material indamaged cells.

3. Lipid parts both in healthy and damaged cells(lipid percentage).

3.1. Simulation of spectra

In the calculation of the spectra, each line wasrepresented as a LorentzianGaussian sum. Inobtaining the first derivative line, the Gaussianpercentages represent the doubly integrated inten-sities [30]. Simulation of the spectra was com-pleted in three steps. First, the beginning values ofthe parameters were determined from a six-parameter fit of 10 mM TANOL:water and 10mM TANOL:soyabean oil samples. The fittedparameters were derivative line widths, g values,nitrogen hyperfine coupling constant (A) and thepercentage of the Gaussian line. In the secondstep, the signals from embryo were fitted by keep-ing g, A values and Gaussian percentages forwater and lipid parts constant. A 12-parameter fitwas performed for nine line widths, B0 main fieldvalue and for percentages of TANOL in aqueousand lipid environments. The remaining part repre-sents TANOL in aqueous regions including the

line broadening material. These calculations weredone to fine tune the line widths of TANOL inthe intracellular aqueous and lipid regions. Onlycontrol and 20 kGy samples were used since theyrepresent high and low water limits, respectively.After obtaining satisfactory results from theabove calculations, in the last step, all spectrawere simulated by keeping the line widths ofTANOL signals from undamaged intracellularaqueous parts and from lipid parts constant. Thewater, lipid percentages and the line widths ofTANOL signal in the aqueous regions includingthe line broadening material were left as variables.Calculations were performed using a FORTRANprogramme including Simplex method as a sub-routine [31].

The simulation results for method I andmethod II are given in Figs. 1 and 2, respectively.The average standard deviation in the simulationof the spectra was smean0.03. The line widths ofthe prepared solution including the line broaden-ing agent were found for 1, 0, 1, lines as1.52, 1.53, 1.5790.03 mT, respectively. As thissolution run into the intracellular regions, theseline widths decrease. The line widths from theregions including the line broadening materialdistributed about a mean value for each nitrogenhyperfine line. There is a method dependent dif-

Fig. 1. The spectra recorded with the samples prepared according to method I. Solid line, experimental; dotted line, calculated.

M.M. Sunnetcioglu, D. Dadayl : Talanta 53 (2000) 697472

Fig. 2. The spectra recorded with the samples prepared according to method II. Solid line, experimental; dotted line, calculated.

ference in the mean values. For 1, 0, 1 linesthe line widths were obtained as 0.75, 0.66, 0.8790.06 mT for method I and 0.52, 0.45, 0.6490.06mT for method II, respectively. The dose andmethod dependent changes in the water percent-age can be followed from the spectra. Since inmethod I the spectra were recorded immediatelyafter the soaking in Fig. 1, the water signal issuperior in the spectra of 05 kGy irradiationdose range and even at 10 and 20 kGy. In Fig. 2,however, the water percentages are lower. Theresults of simulations justify these observations.The spectra of the samples in method II wererecorded after 1 (Fig. 3) and 2.5 h. It was stillpossible to follow the dose dependence but aconsideration of the spectra indicated the presenceof an additional signal of the probe from slowmotional region. To clarify the additional signalthe difference spectrum of 24 h air-dried sampleand lipid signal was drawn (Fig. 4). The linewidth of the lipid signal was calculated from step2 (Table 1).

The decay of water percentage and lipid per-centage with increasing irradiation dose wasshown in Fig. 5A and B for method I and methodII, respectively. In both methods there is littlechange in the lipid percentage with increasingirradiation dose, however, there is a percentagedifference for lipid parts in these two methods.The source of this might be migration of some

Fig. 3. The recorded spectra of the samples prepared as inmethod II but air dried for 1 h.

M.M. Sunnetcioglu, D. Dadayl : Talanta 53 (2000) 6974 73

Fig. 4. The sample prepared as in method II, air dried for 24h. (a) The recorded spectrum of the 5 kGy sample prepared asin method II but air dried for 24 h (solid line), the lipid signalcalculated from step 2 (dotted line). (b) The difference spec-trum. (c) Smoothed difference spectrum.

Fig. 5. The change in the water (squares) and lipid (triangles)percentages depending on irradiation dose. (A) Method I, (B)method II.

spin probes in aqueous environments towardslipid parts as a result of dehydration.

The decay of water percentage is exponential inboth methods and from the exponential fit thefollowing results were obtained.

method I y5.537.1 expx

5.2

method II y3.437.2 expx

4.2

Here, y is the water percentage (%) and x is theirradiation dose (kGy). The decay in method II israpid relative to method I. From these curves itmight be possible to obtain the irradiation dose ofan unknown sample, however, as it can be seen

Table 1The fitted values of experimental parameters from step 1 and step 2

Sample g (Step 1)Line widths (mT) (step 2) A (mT) (step 1)

1 1m1 0

1.689TANOL:water (10 mM) 0.160 0.140 0.175 2.00551.5432.00600.1400.115TANOL:lipid (10 mM) 0.110

M.M. Sunnetcioglu, D. Dadayl : Talanta 53 (2000) 697474

from Figs. 13 air drying time is highly critical.Any time until 1 h might be used but in relativestudies the waiting times of the samples must bethe same.

In conclusion, the above results indicated theapplicability of our spin probe technique to thecereals even after long storage times past afterirradiation. The suggested new methods arepromising in shortening the spectrometer time,however, at close irradiation doses these methodsare not satisfactory. Currently studies on the seedshave been carried out to widen the applicability ofthe spin probe technique to various seeds.

Acknowledgements

We thank Hacettepe University for the financialsupport of the project 97 01 602 005. We alsowish to thank Professor Bekir Aktas from GebzeYuksek Teknoloji Enstitusu for the use of theirlaboratories for some control measurements.

References

[1] Anonymous, 1981. Wholesomeness of Irradiated Food,Report of Joint FAO:IAEA:WHO Expert Committee,World Health Organization, Geneva. Technical ReportSeries 659.

[2] K.W. Bogl, Appl. Radiat. Isot. 40 (1989) 1203.[3] J.A.P. Boshard, D.E. Holmes, L.H. Piette, Int. J. Appl.

Radiat. Isot. 22 (1971) 316.[4] N.J.F. Dodd, A.J. Swallow, F.J., Ley. Radiat. Phys.

Chem. 26 (1985) 51.[5] M.F. Desrodiers, Appl. Radiat. Isot. 47 (1996) 1621.[6] C.R. Hunter, D.R. Hunter, G.J. Troup, Search 19 (1988)

198.[7] A.R. Forrester, D.B. McKay, Pure Appl. Chem. 65 (2)

(1990) 307.[8] B.J. Tabner, V.A. Tabner, Radiat. Phys. Chem. 41 (3)

(1993) 545.

[9] J. Raffi, C. Hasbany, G. Lesgards, D. Ochin, Appl.Radiat. Isot. 47 (11:12) (1996) 1633.

[10] E.M. Steward, R. Gray, Appl. Radiat. Isot. 47 (11:12)(1996) 1629.

[11] G.J. Troup, J.R. Pilbrow, D.R. Hutton, C.R. Hunter,G.L. Wilson, Appl. Radiat. Isot. 40 (1012) (1989) 1223.

[12] J. Raffi, J.-P. Agnel, S.R. Kassis, Sci. Aliments 7 (4)(1987) 657.

[13] E.P. Munoz, E. Adem, G. Burillo, R.V. Gleason, H.S.Murrieta, Radiat. Phys. Chem. 43 (4) (1994) 311.

[14] H.S. Murrieta, E.P. Munoz, E. Adem, G. Burillo, M.Vazquez, E.B. Cabrera, Appl. Radiat. Isot. 47 (11:12)(1996) 1657.

[15] L.J. Berliner, Spin Labeling; Theory and Applications,Academic Press, New York, 1976, pp. 14.

[16] N. Kocherginsky, H.M. Swartz, Nitroxide Spin Labels,Reactions in Biology and Chemistry, CRC Press, BocaRaton, FL, 1995, pp. 125.

[17] M.P. Gierula, W.K. Subczynski, A. Kusumi, Biochemie73 (1991) 1311.

[18] K. Strazalka, W.I. Gruszecki, Biochim. Biophys. Acta1194 (1994) 138.

[19] S.R. Wassall, L. Wang, R.C.Y. McCabe, W.D. Ehringer,W. Stillwell, Chem. Phys. Lipids 60 (1991) 29.

[20] P. Muller, A. Zachowski, Y. Beuzard, P.F. Devaux,Biochim. Biophys. Acta 1151 (1993) 7.

[21] P. Cooper, J. Kudynska, H.A. Buckmaster, R. Kudynski,Biochim. Biophys. Acta 1139 (1992) 70.

[22] J. Kutcher, T. Hanke, B. Ebert, J. Hellebrant, Stud.Biophys. 111 (23) (1986) 105.

[23] A.I. Smirnov, H.A. Golovina, O.E. Yakimchenko, S.I.Aksyonov, Y.S. Lebedev, J. Plant Physiol. 140 (1992) 447.

[24] E.A. Golovina, A.N. Tikhonov, Biochim. Biophys. Acta1190 (1994) 385.

[25] M.M. Sunnetcioglu, D. Dadayl, R. Sungur, G. Bingol, J.Plant. Physiol. 151 (1997) 196.

[26] E.A. Golovina, A.N. Tikhonov, F.A. Hoekstra, PlantPhysiol. 114 (1997) 383.

[27] D. Dadayl, M.M. Sunnetcioglu, H. Koksel, S. Celik,Cereal Chem. 74 (4) (1997) 375.

[28] M.M. Sunnetcioglu, D. Dadayl, S. Celik, H. Koksel,Cereal Chem. 75 (6) (1998) 875.

[29] M.M. Sunnetcioglu, D. Dadayl, S. Celik, H. Koksel,Appl. Radiat. Isot. 50 (1999) 557.

[30] L.J. Berliner, J. Reuben, Biological Magnetic Resonance,vol. 8, Plenum Press, New York, 1989, pp. 77102.

[31] J.A. Nelder, R. Mead, Comput. J. 7 (1965) 308.

.