modified ferrous oxidation-xylenol orange assay for lipoxygenase activity in rice grains

Analytical MethodsA modified ferrous oxidation-xylenol orange assay for lipoxygenase activity in ricegrainsTarinee Timabud a ,Jirawat Sanitchon b ,Paweena Pongdontri a ,⇑a Department of Biochemistry,Faculty of Science,Khon Kaen University,Khon Kaen 40002,ThailandbDepartment of Plant Science and Agricultural Resources,Faculty of Agriculture,Khon Kaen University,Khon Kaen 40002,Thailanda r t i c l e i n f o Article history:Received 26September 2012Received in revised form 23April 2013Accepted 11May 2013Available online 18May 2013Keywords:Ferrous oxidation-xylenol orange (FOX)assayLipoxygenaseHydroperoxy fatty acid product Rice graina b s t r a c tFerrous oxidation-xylenol orange assay reagent was reformulated by using spectral analysis of ferric–xylenol orange complex to detect low concentrations of lipoxygenase rice grain products.Reducing the levels of ferrous sulphate and xylenol orange in the FOX reagent enabled the detection of low concentra-tions of hydroperoxy fatty acid derived from lipoxygenase activity in the range of 0.1–1.5l M.Protein,substrate and time courses of the modified FOX assay were studied to determine lipoxygenase activity in rice grain.The assay was also applicable as a high throughput technique for comparisons of lipoxyge-nase activity from various rice varieties.This has important implications for rapid screening for low-lipoxygenase containing rice cultivars in rice breeding program and grain quality during storage.Ó2013Elsevier Ltd.All rights reserved.1.IntroductionLipoxygenases (E.C.1.13.11.12),or LOX,are a group of iron con-taining dioxygenases that catalyse the hydroperoxidation of poly-unsaturated fatty acids that contain a penta-1,4-cis -diene system.The formation of a conjugated 1,3diene product from a 1,4diene substrate is the basis of most assays for lipoxygenase activity (Tay-lor &Morris,1983).LOX activity can be determined by direct detection of the LOX product,hydroperoxide fatty acid,using an in-creased absorption at 234nm or indirectly by a decrease in oxygen consumption as measured by an oxygen electrode (Axelrod,Chesebrough,&Laakso,1981).Various colorimetric methods have been developed for LOX activity measurement.These methods are based on oxidation reac-tion of a dye by hydroperoxide fatty acids to generate a coloured product,such as the iodometric assay (Hicks &Gebecki,1979),the ferrous oxidation-xylenol orange (FOX)assay (Waslidge &Hayes,1995)and the oxidative coupling of MBTH with DMAB (An-thon &Barrett,2001).Oxidisation of methylene blue or beta-caro-tene by the LOX products has also been established for visual inspection by a rapid assay of lipoxygenase (Romero &Barrett,1997).Some of these methods have been applied in determining LOX activity from various animal and plant samples,as well as li-pid hydroperoxides present in a number of food samples,with detection limit of at least 11l M for the iodometric assay and for the oxidative coupling of MBTH with DMAB (Delong et al.,2002)and 5l M for the FOX assay (Bou,Codony,Tres,Decker,&Guardi-ola,2008).Rice LOXs have been proposed to be involved in deterioration of rice grain during storage.By catalysing hydroperoxidation reaction of linoleic acid,a major fatty acid in the grain (Zhou,Blanchard,Helliwell,&Robards,2003),the LOX products undergo additional enzymatic reactions to form aldehydes,ketones and alcohols,which results in undesirable flavours in the grains.The volatile compounds not only reduce the quality of the rice,but they also at-tract insects that damage the grains and decrease seed germination (Zhang et al.,2007).In rice embryos,there are three isozymes found in most rice varieties;i.e.,LOX-1,LOX-2and LOX-3(Ida,Masaki,&Morita,1983),which have optimal pH’s at 9.0,6.0and 6.6,respectively (Zhang et al.,2007).These LOXs are suggested to play different roles during grain storage and seed development (Ida et al.,1983;Ohta,Ida,Mikami,&Morita,1986;Ren et al.,2008)by producing different products that finally yield different compounds (Zhang et al.,2007).Among the rice LOX isozymes,LOX-3is the most active during grain storage (Ida et al.,1983)and plays an important role in producing specific odours and col-ours (Suzuki,Yasi,Matsukura,&Terao,1996).Zhang et al.(2007)and Tang,Zhang,Liu,Yu,and Yuejin (2009)have shown that vola-tiles emitted from LOX-3containing rice varieties attract stored rice insects more than LOX-3-free varieties.These results support the hypothesis that the absence of LOX-3in rice grains might re-duce oxidative deterioration during storage.Therefore,develop-ment of a screening method to compare LOX activity in rice grains from different varieties would help rice breeders identify low-LOX varieties.0308-8146/$-see front matter Ó2013Elsevier Ltd.All rights reserved./10.1016/j.foodchem.2013.05.037Corresponding author.Tel./fax:+6643342911.E-mail address:paweena@kku.ac.th (P.Pongdontri).To select a colorimetric method for determining LOX activity from a number of rice grains,the ferrous oxidation-xylenol orange method,or FOX assay,has been useful as there are fewer steps re-quired and it is suitable for different plant samples(Pinto,Tejeda, Duque,&Macias,2007).In principle,the LOX products,dissolved in methanol,oxidise Fe2+to Fe3+in the acidic solution of the FOX re-agent.The resulting Fe3+subsequently forms a complex with xyle-nol orange,Fe3+–XO,which absorb strongly at540–600nm(Bou et al.,2008).Here,we report a modified FOX assay to determine LOX activity in rice grains.Slight modification of the FOX reagent,as described by Pinto et al.(2007),was made to successfully detect low concen-tration of LOX product in rice in the range of0.3–1l M generated at the optimal pH of rice LOX-3(pH6.6).The modified FOX assay was able to detect the LOX product in1-ml and0.25-ml reactions and allowed for conversion into a high throughput assay for LOX activ-ity in rice grains.2.Materials and methods2.1.ChemicalsLinoleic acid was obtained from Fluka.Perchloric acid,were supplied from Ajax Finechem(UNIVAR,Australia).Bis–Tris(Sigma, USA)was a gift from Dr.Atit Silsirivanit,Faculty of Medicine,Khon Kaen University.The3,30-bis[N,N-di(carboxyethyl)aminomethyl]-o-cresolsulfonephthalein tetrasodium salt(xylenol orange)was purchased from Sigma(St.Louis,MO,USA).Ferric sulphate was ob-tained from BDH Chem(BDH,England).Ferrous sulphate was ob-tained from Carlo Erba(CARLO ERBA,Spain).All other chemicals were purchased from the supplier and were of at least analytical grade.2.2.Rice materialsAll paddy rice grains(Khao Dok Mali105,Khao Rai Keknoi Phi-sanulok,Khao Khun Wang Chiangmai,Khao Rai Banhad Khon Kaen, Khao Sanpahsak6Mae Tang Chiangmai and Khao Mae Wang Chiangmai)were cultivated from June to October2009,harvested in November2009and stored at12–15°C before analysis in November2010–July2011.2.3.Extraction of crude LOX from rice grainSixty rice grains(approximately1.2–1.3g)were de-husked,and the crude LOX was extracted by grinding with a mortar and pestle on ice with4ml of extraction buffer(100mM Bis–Tris,pH6.6,con-taining0.1%Tween20and1mM EDTA).The extracts were trans-ferred into1.5-ml tubes and centrifuged at12,000rpm(Labquip 1000series,England)for25min.The crude LOX extracts were col-lected and protein content was determined using the Bradford method with BSA as a standard(Bradford,1976),whilst LOX activ-ity was determined as described below.2.4.Preparation of NH4-linoleateThe linoleic acid substrate(10mM)was prepared in the form of NH4-linoleate(Pongdontri&Hills,2001)to obtain a soluble form of linoleic acid for the LOX assay.Linoleic acid(0.0286g)was com-bined with23.5l l of concentrated NH4OH and76.5l l of distilled water and incubated at60°C for30min until the solution was clear;if the solution did not become clear,additional NH4OH was gradually added.To remove excess ammonia,nitrogen gas was blown over reaction mixture.Tween20(200l l)was added,and the solution was mixed and the total volume was adjusted to 10ml with100mM Bis–Tris,pH6.6.2.5.Spectral study of the Fe3+–XO complexTo study appropriate concentrations of the Fe3+and XO for determining rice LOX product,mixtures of Fe3+and xylenol orange at different concentrations were prepared in1-ml reaction volume composed of110mM perchloric acid in methanol/water(9:1)and 50mM Bis–Tris buffer pH6.6and a series of ferric sulphate con-centration of0.03125–16mM or xylenol orange0–150l M.The absorption spectra of the mixtures were measured in the range of340–700nm by UV-1800spectrophotometer(Shimadzu,Japan).2.6.Standard curve of hydroperoxide derivativeStandard linoleate hydroperoxide was prepared as previously described(Pinto et al.,2007)with slight modifications.Specifically, 2l g protein extract of crude soybean lipoxygenase was added to 1-ml of10mM NH4-linoleate in100mM borate buffer(pH9.0) to generate the hydroperoxide product,which was observed by monitoring the increase in the absorbance at234nm.After the absorbance reached a constant level,9ml of methanol was added to result in a10-fold dilution.The concentration of the hydroper-oxide was calculated using e=25mMÀ1cmÀ1.The standard curve of hydroperoxide was prepared either by detection at A234(Axelrod et al.,1981)or modified FOX.Serial con-centrations of linoleate hydroperoxide(0–1.5l M)were prepared in1ml of100mM Bis–Tris buffer pH6.6for A234measurement or in0.5ml of the buffer before adding0.5-ml2Âmodified FOX re-agent containing0.25mM ferrous sulphate,37.5mM xylenol or-ange,220mM perchloric acid in methanol(9:1).A550values were determined using UV–vis spectrophotometer(Jasco,England). Similar serial concentrations of the standard hydroperoxide were also prepared in0.125-ml100mM Bis–Tris buffer pH6.6in96-well microtiterplate and reacted with0.125-ml2Âmodified FOX reagent before measuring A550using a Benchmark plate reader (Biorad,USA).The absorbance values were plotted against the hydroperoxide concentration to give standard curves.2.7.Protein,substrate and time courses for modified FOX assay for LOX activity in rice grainThe ammonium linoleate substrate,in the range of0–5mM in either0.5-ml or0.125-ml volumes,was reacted with different amounts of crude LOX extract(0–5l g)in25l l of the extraction buffer.The reactions were performed for2min before being stopped by the addition of the2Âmodified FOX reagent.Blanks were also performed in parallel but in a slightly different sequence. The FOX reagent was added to the ammonium linoleate substrate prior to the addition of the crude enzyme to ensure complete Fe3+–XO complex in the reaction background occurred.The hydroperox-ide-oxidised Fe3+–XO complex from each reaction was detected by measuring the A550value,and a blank was subtracted to result in D A550.To determine the LOX reaction time course,the reaction mix-tures contained5mM ammonium linoleate and3.5l g of crude LOX in either0.5-ml or0.125-ml volumes.The reactions were stopped at1min intervals over the course of10min by the modi-fied FOX reagent and left for10min before determining D A550.To ensure Fe3+–XO complex was completely formed,the com-plex was monitored by A550measurement every minute after 2min rice LOX reactions of500l l or125l l volumes were stopped by the modified FOX reagent.Blanks were performed in parallel as described earlier,and the D A550was calculated for each time point.2406T.Timabud et al./Food Chemistry141(2013)2405–24112.8.LOX activity in the rice grainBy using A234,a25l l aliquot containing2l g of each crude LOX extract was added into1-ml1mM NH4-linoleate in100mM Bis–Tris Buffer pH6.6to start the reaction.The concentration of the hydroperoxy fatty acid product was monitored at234nm using a UV–vis spectrophotometer(Agilent8453,Germany).The LOX activity was expressed as the rate of the reaction(l M of hydroper-oxy linoleate/min/l g protein).For the modified FOX assay,a LOX reaction mixture of0.5or 0.125ml composed of NH4-linoleate at concentrations of1mM in100mM Bis–Tris buffer,pH6.6.Crude LOX extract from each rice variety(2l g)was added to start the LOX reaction.After 2min,the reactions were stopped by adding an equal volume of the2Âmodified FOX reagent and left for further10min to allow complete Fe3+–XO complex formation.Blanks were performed as described previously.D A550was calculated and converted to hydroperoxide fatty acid concentration by using standard curves. The LOX activity was also expressed as the rate of the reaction (l M of hydroperoxy linoleate/min/l g protein).2.9.Statistical analysesThe data were expressed as the means±the standard deviation (SD).A statistical analysis was performed using a one-way analysis of variance(ANOVA).Comparisons of the means among the six rice varieties were performed using Duncan’s test,and a p value of less than0.05was considered significant.3.Results and discussion3.1.Spectral study of Fe3+–XO complexPreliminary analysis of crude LOX activity from grains of KDML105rice variety by monitoring hydroperoxy fatty acid prod-uct using UV absorption(e234=25mMÀ1cmÀ1)suggested that the product concentration was in the range of0.3–1l M within20min of observation in the presence of5mM substrate and1–5l g pro-tein in1-ml reaction.However,determination of the LOX activity by using FOX assay as described by Pinto et al.(2007)was not suc-cessful.The range of the rice LOX concentration was lower than the detection limit of FOX assay,5l M,which may be due to the for-mulation of the FOX reagent that contains110mM perchloric acid, 150l M xylenol orange,and2mM ferrous sulphate in a methanol/ water(9:1)mixture.It is likely that the concentration of the Fe3+–XO complex was too low in the presence of2mM ferrous sulphate and150l M xylenol orange.Therefore,spectral studies of the Fe3+–XO complex were determined using varied concentration of fer-rous sulphate and xylenol orange to check which concentrations of both compounds might be appropriate to determine the LOX activity.Initially we have varied concentrations of Fe3+(0–16mM)in 500l l of220mM perchloric acid and150l M xylenol orange in methanol/water(9:1)for10min,followed by adding500l l of 100mM Bis–Tris buffer pH 6.6.The absorption spectra of the Fe3+–XO complexes in the solutions were analysed within the UV–visible range of340–700nm.Fig.1a shows that the maximum absorption(k max)of the complex gradually shifted within a526–556nm range as the concentration of Fe3+increased.However, these complexes resulted in different k max values relative to the samples with the Fe3+-omitted FOX reagent,which exhibited peaks at480nm(Fig.1a).This change in k max indicated that Fe3+was bound to XO.The absorbance at550nm increased,at the rise of Fe3+concentration,in a hyperbolic fashion(Fig.1b).This result suggested that Fe3+at0.125mM was excessive for binding to XO and was used to formulate the modified FOX reagent.After the Fe3+concentration wasfixed at0.125mM,a suitable XO concentration was then investigated.A series of XO concentra-tions,in the range of2.34–150l M,was used to form complexes with Fe3+in the FOX reagent,which contained0.125mM Fe3+ and110mM perchloric acid in methanol/water(9:1)in1-ml reaction.Fig.2shows the spectra of the complex within theT.Timabud et al./Food Chemistry141(2013)2405–24112407340–700nm range with peaks at550nm(Fig.2a).These data indi-cated that the k max of the Fe3+–XO complex was550nm,which is an important characteristic of the complex.The absorbance in-creased in a XO-dependent manner(Fig.2b).The linearity indi-cated that the given concentration of0.125mM Fe3+was sufficient for the formation of the Fe3+–XO complex in the presence of 2.34–150l M XO.To select which concentration of XO was appropriate for routine analysis in the laboratory,we have chosen 18.75l M of XO concentration to limit the absorption value not to exceed most of the upper photometric range and a relative%error in concentration of laboratory instruments.We,therefore,have reformulated the FOX reagent from that de-scribed by Pinto et al.(2007)by lowering the concentration of fer-rous sulphate to0.125mM(from2mM)and xylenol orange to18.75l M(from150l M).Other components were unchanged;i.e.,110mM perchloric acid and methanol.The modified FOX re-agent was then prepared as2Âconcentration which was com-posed of0.25mM ferrous sulphate,37.5l M xylenol orange and 220mM perchloric acid in methanol(9:1).The modified2ÂFOXreagent was used to stop the LOX reaction and reacted with the LOX products.This reformulated FOX reagent was able to detect LOX activity from rice grains as shown in later experiments.Nota-bly thatfinal concentration of perchloric acid must be at110mM after stopping the LOX reaction,otherwise,the Fe3+–XO complex derived from the LOX product cannot properly form corresponded to previous report(Hicks&Gebecki,1979).To successfully deter-mine rice LOX activity by the FOX assay,a suitable buffer system for performing LOX reactions is necessary.Highly polar buffer sys-tems should not be used,for instance,phosphate,Hepes,Mes or Mops buffers.Precipitates were formed after adding2ÂFOX re-agent(data not shown).This can be explained by methanol,con-taining in the modified FOX reagent,which decreased polarity in the FOX assay.It can be avoided by using less polar buffer system such as Tris–HCl buffer in pH range of7.5–9.0or Bis–Tris buffer in pH range of5.8–7.3.In addition,the LOX reaction performed at pH more than8also interrupted the FOX assay(data not shown).High alkali pH of the LOX reaction could change the oxidation state of the Fe2+in the FOX reagent to Fe3+forming Fe3+–XO in high pH conditions,but not by the LOX product.3.2.Evaluation of the modified FOX reagent for low concentration of hydroperoxy fatty acid measurementTo validate the modified FOX reagent to determine LOX activity from rice grains,which generate hydroperoxide product at low concentration in the range of0.3–1l M,wefirstly used standard hydroperoxy fatty acid in the range of0–1.5l M in1-ml reaction volume.Linearity of the absorbance at550nm in the range of0–1.5l M showed that the modified FOX reagent was able to detect hydroperoxy fatty acid at low concentration(Fig.3).The hydroper-oxide standard curve by the modified FOX assay agreed well with that of A234.To ensure that Fe3+–XO complex derived from low2408T.Timabud et al./Food Chemistry141(2013)2405–2411concentration of the hydroperoxide was formed,analysis of their spectra in the range of 340–700nm was made.They appeared their k max of the Fe 3+–XO complex at 550nm,which were different from that of the blanks (data not shown).In order to prepare a high throughput assay for LOX activity from rice grains by the modified FOX method,we also performed the reaction in 0.25ml by mixing 0.125-ml standard hydroperox-ide with equal volume of 2Âmodified FOX reagent in 96-well microplate,left for 10min and measured the A 550by a microplate reader (Benchmark,Biorad,USA).A linear standard curve was also obtained,although higher absorbance values than that of 1-ml reaction volume were observed (Fig.3).3.3.Activity assay optimisation of LOX in rice grains using the modified FOX reagentOptimal concentrations of protein,substrate and reaction peri-od to determine LOX activity were analysed by protein,substrateand time courses.Initially,crude LOX from KDML105rice grains (0–5l g)and the linoleate substrate (0–5mM)were reacted in either 0.5-ml or 0.125-ml reactions,and left for 2min,before stop-ping the LOX activity by adding 2Âmodified FOX reagent contain-ing 0.25mM ferrous sulphate,37.5l M xylenol orange and 220mM perchloric acid in methanol (9:1).The reactions were fur-ther incubated for 10min to allow formation of Fe 3+–XO complex before measuring A 550values.Fig.4a and b show that saturated substrate concentrations for LOX activity determination were be-gun at 4mM and 4.5mM for 1-ml and 0.25-ml assay reactions,respectively.Protein concentrations of both reaction volumes were optimised at 3.5l g.To check if a 2min period was appropriate for the LOX activity assay by the modified FOX method,the LOX reaction time course was determined in the presence of 3.5l g crude LOX from KDML105rice grain and 5mM substrate.The blank measurements were subtracted and as shown in Fig.5a the rate of the reactions in 1-ml and 0.25-ml volumes were similar (0.045and 0.0490.00.05.10.15.20.25.3001234512345P ro t ei nc on ce nt r at i on N H4-l i n o l ea t e c on c e n tr a ti o n (μM )0.00.02.04.06.08.10.121234512345P ro te i nc on ce nt r at i on H 4-l i n o l ea t e c on c e n tr at i o n (μΜ)a b and substrate courses of LOX activity in KDML105modified FOX assay.(a)in 1-ml assay volume,(b)in Time(min)2468A 550 (total volume 0.25 ml)A 550 (total volume 1 ml)0.00.05.10.15.20.25A 550 (total volume 0.25 ml)A 550 (total volume 1 ml)a bT.Timabud et al./Food Chemistry 141(2013)2405–24112409D A550/min).A slightly higher D A550value was observed in the1-ml than in the0.25-ml assay volumes.Therefore,2min period was appropriate to determine rate of reaction for the LOX activity assay.To assess if the Fe3+–XO complex may not be completely formed within10min,time course of the complex formation was also per-formed as described in materials and methods.D A550values ofwere calculated and showed that the complex was stably formed from5min(Fig.5b)and indicated that Fe3+–XO complex was fully detected in previous experiments.To summarise,in this study a modified FOX reagent was pre-pared to determine LOX activity in rice grains.Appropriate concen-trations of substrate and protein were determined as well as the LOX reaction period and duration of Fe3+–XO complex formation. However,the given concentrations of the substrate(5l M)usually gave high background colour,hindering accurate absorbance read-ings.We,therefore,used saturated substrate concentration derived from LOX activity determined by A234,1mM(data not shown),to perform LOX reactions for the modified FOX assay.Consequently, crude LOX protein had to be reduced to2l g protein,in order to give an appropriate rate of reaction which yielded correct amount of product for the modified FOX assay.parison of LOX activity from rice grain from six varietiesLOX activity of different rice grains was determined by using the A234values in comparison to the modified FOX assay of 0.25ml assay volume.Standard curves of hydroperoxy linoleate in the range of0–1.5l M(Fig.3)were used for determination of the LOX activity expressed as l M hydroperoxy linoleic acid/min/ l g protein.A statistical analysis showed that there were threegroups of rice grains according to the LOX activity determined from the A234values(Fig.6).This measurement was able to divide the rice grains into three groups according to the LOX activity as determined by both meth-ods.Nevertheless,the hydroperoxy linoleic acid product detected using the FOX assay was higher than that detected using the A234 values.It is possible that this difference is due to different reaction rates.The quantities of substrate and enzyme in the1-ml reaction were higher than those in the0.25-ml reactions of the modified FOX assay.Slower reaction rates are normally detected in the pres-ence of lower concentrations of the substrate and enzyme. Although the FOX assay has been reported to be a sensitive method to examine many biological peroxides,endogenous peroxide in the reaction was negligible in the blank containing the same amount of substrate and enzyme.In the blank solution,the substrate reacted with the FOX reagent prior to the addition of the crude enzyme solution ensuring that there was no LOX activity in the blank. Therefore,the D A550revealed actual hydroperoxy linoleate product of LOX in this study.4.ConclusionsThe FOX reagent based on Pinto et al.(2007)was reformulated by lowering concentrations of ferrous sulphate and xylenol orange in order to detect low concentrations of hydroperoxy fatty acid,a product of rice grain LOX reaction.Spectral studies of Fe3+–XO complex suggested that the2Âmodified FOX reagent composed of0.25mM ferrous sulphate,37.5l M xylenol orange and 220mM perchloric acid in methanol(9:1),would give effectivefi-nal concentration of each component for the detection of low con-centrations of hydroperoxy fatty acid present in the LOX reaction. Comparison of the modified FOX assay,in1-ml and0.25-ml assay volumes,with detection of hydroperoxide by A234was performed by using standard hydroperoxide and suggested that the modified FOX assay was a potent method for detection of0.1–1.5l M of the standard hydroperoxy linoleate.The modified FOX assay was also able to develop a high throughput technique for comparing LOX activity of rice grains from various cultivars.A similar trend to the A234values of the LOX activity from six varieties was observed, which divided LOX-containing rice grains into three groups.This is a promising technique to determine the total LOX activity in rice grains for the selection of low-LOX cultivars in breeding programs and rapid testing for grain quality during storage. 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