See discussions, stats, and author profiles for this publication at:
/publication/226379285
A simple colorimetric method for determination of hydrogen peroxide in plant tissues
ARTICLE in PLANT GROWTH REGULATION · NOVEMBER 2006
Impact Factor: 1.67 · DOI: 10.1007/s10725-006-9000-2
CITATIONS 47READS 822
5 AUTHORS, INCLUDING:
Zhenfei Guo
South China Agricultural University
42 PUBLICATIONS 1,035 CITATIONS
SEE PROFILE
All in-text references underlined in blue are linked to publications on ResearchGate, letting you access and read them immediately.Available from: Zhenfei Guo Retrieved on: 14 March 2016
自行车棚制作Abstract A simple colorimetric method for determination of hydrogen peroxide in plant materials is described.The method is based on hydrogen peroxide producing a stable red product in reaction with 4-aminoantipyrine and phenol in the presence of peroxidase.Plant tissues was ground with trichloroacetic acid (5%w/v)and extracts were adjusted to pH 8.4with ammonia solution.Activated charcoal was added to the homogenate to remove pigments,antioxidants and other interfering substances.The colorimetric reagent (pH 5.6)consisted of 4-aminoantipyrine,phenol,and peroxidase.With this method,we have determined the hydrogen peroxide concen-tration in leaves of eight species which ranged from 0.2to 0.8l mol g –1FW.Changes in hydro-gen peroxide concentration of Stylosanthes gui-anensis in response to heat stress are also analyzed using this method. Keywords 4-aminoantipyrine ÆHydrogen peroxide ÆPeroxidase ÆPlant tissue
Introduction
Hydrogen peroxide (H 2O 2)is one of the major reactive oxygen species (ROS)in plant tissues.It is produced in chloroplasts and mitochondria via electron transport,where oxygen is reduced to superoxide which is further dismuted into H 2O 2spontaneously or catalyzed by superoxide dismutase (Asada 1999;Moller 2001).H 2O 2pro-duction in plant cells is also catalyzed by glycol-late oxidase in peroxisomes (Noctor et al.2002),membrane bound NADPH oxidase (Jiang and Zhang 2003)and oxalate oxidase (Hu et al.2003).When plants are subject to environment stress,it accumulates and leads to oxidative damage (Asada 1999).Accumulating evidence suggests that H 2O 2is a key signaling molecule involved in plant response to both biotic and abiotic stresses,such as pathogen attacks,extreme temperatures,drought,excessive radiation,ozone and wounding (Neill et al.2002;Foyer et al.1997;Prasad et al.
1994;Orozco-Ca
´rdenas et al.2001;Wohlgemuth et al.2002).Therefore it is frequently important to determine hydrogen peroxide concentration in plant tissues.
There are several analytical methods for determination of H 2O 2,including spectrophoto-metric (Patt采果器
erson et al.1984),chemiluminescent (Pe
´rez and Rubio 2006;Warm and Laties 1982)and fluorometric (Genfa and Dasgupta 1992).Patterson’s spectrophotometric method has been
B.Zhou ÆJ.Wang ÆZ.Guo (&)ÆH.Tan
College of Life Science,South China Agricultural University,Guangzhou 510642,China e-mail:zhfguo@
B.Zhou ÆX.Zhu
Physiological Laboratory for South China Fruits,College of Horticulture,South China Agricultural University,Guangzhou 510642,China
Plant Growth Regul (2006)49:113–118DOI 10.1007/s10725-006-9000-2
123
ORIGINAL PAPER
导电
银胶A simple colorimetric method for determination of hydrogen peroxide in plant tissues
Biyan Zhou ÆJihua Wang ÆZhenfei Guo ÆHuoquan Tan ÆXiaochuan Zhu
Received:19January 2006/Accepted:10July 2006/Published online:29September 2006ÓSpringer Science+Business Media B.V.2006
used for estimation of H2O2in plant extracts (Aroca et al.2003;Vaidyanathan et al.2003). However,we failed to detect any H2O2in Stylo-santhes guianensis,an important pasture legume, by using Patterson’s spectrophotometric method, suggesting that some substances in plant tissues might interfere with the assay.This has led us to find a novel method suitable for this pasture legume as well as other plant materials.
Glucose oxidase/peroxidase system is widely used for determination of glucose in blood (Trinder1969).In this reaction,glucose is con-verted to gluconic acid and H2O2,catalyzed by glucose oxidase.H2O2is subsequently reacted with4-aminoantipyrine and phenol which pro-duces a stable red product in the presence of per-oxidase.The objective of this study is to establish an easy and rapid protocol for determination of H2O2in plant tissues using this colorimetric approach.
Materials and methods
Plant materials and chemicals
Rice(Oryza sativa L.),Stylosanthes guianensis, alfalfa(Medicaga sativa L.)and tobacco(Nicoti-ana tabacum L.)seedlings were grown in a greenhouse as described(Huang and Guo2005; Zhou et al.2005).Litchi(Litchi chinensis Sonn.), longan(Dimocarpus longana Lour.),banana (Musa acuminata Colla(AAA))and taro(Col-ocasia esculenta)were grown in thefield at the South China Agriculture University.Heat stress treatment was applied to Stylosanthes guianensis when moved into a growth chamber for a12h photoperiod(160l mol m–2s–1PPFD)at38°C. In each case fully expanded second leaves were selected for H2O2determination.
Catalase and4-aminoantipyrine were purchased from Sigma.Peroxidase was obtained from Bo Ao Biochemical Company(Shanghai,China).All other chemicals were of analytic grade obtained from Guangzhou Chemical Factory(China).
Standardization of hydrogen peroxide
Hydrogen peroxide was standardized as described by Aebi(1974)and Patterson et al.(1984).H2O2(30%,w/v)was diluted to approximately10mM in50mM phosphate buffer(pH7.0).The diluted H2O2solution was standardized by its absorbance at240nm,by measuring against blanks fro
m which H2O2had been removed by addition of excessive catalase.Absorbance due to the addi-tion of catalase was corrected with a blank of catalase alone in the buffer.The molar absorp-tivity of hydrogen peroxide at240nm and pH7.0 was40M–1cm–1.
Determination of H2O2
Hydrogen peroxide was extracted from plant tis-sues as described by Patterson et al.(1984).Fresh leaves(0.5g)were frozen in liquid nitrogen and ground to powder in a mortar with pestle,together with5ml of5%TCA and0.15g activated char-coal.The mixture was centrifuged at10,000g for 20min at4°C.The supernatant was adjusted to pH8.4with17M ammonia solution and thenfil-tered.Thefiltrate was divided into aliquots of1ml. To one of these,the blank was added8l g of cat-alase and then kept at room temperatures for 10min.To both aliquots with and without cata-lase,1ml of colorimetric reagent was added.The reaction solution was incubated for10min at30°C. Absorbance at505nm was determined spectro-photometrically(Model UV-2010,Hitachi, Japan).The colorimetric reagent contained10mg of4-aminoantipyrine,10mg of phenol,5mg of peroxidase(150U mg–1),dissolved in50ml of 100mM acetic acid buffer(pH5.6).
Results and discussion
An absorbance spectrum of the colorimetric products was measured from450nm to600nm and revealed a peak at505nm(Fig.1),suggest-ing the optimum colorimetric wavelength for the red reaction product was505nm.In order to establish the optimum amount of colorimetric reagent used in the assay medium,five concen-trations of reaction reagents were tested and the result are shown in Table1.The absorbance of colorimetric products increased with the increase of the concentration of colorimetric reagent. Treatment A and B had a similar absorbance.
123
Treatment C had 3.7%lower absorbance than that of Treatment B,but had 10.8%or 26.4%higher absorbance than Treatment D or Treat-ment E,respectively,although the concentration of colorimetric reagent increased significantly from Treatment E to Treatment C.Therefore,the concentration of colorimetric reagent in Treat-ment C should be sufficient to initiate the color-imetric reaction with H 2O 2.
For determination of the stability of reaction product,absorbance at 505nm of the product was recorded immediately after mixing 100l M of H 2O 2solution with the colorimetric reagent.The absorbance showed a slight decline at the first 5min and maintained stability at least to 20min (data not shown).Absorbance of the reaction
product could be consistently measured within 10to 20min after initiating the reaction.
Colorimetric reaction was affected by pH.Absorbance of the reaction product increased with the increase in the pH of the filtrate obtained from rice leaf extracts (Fig.2).TCA,the acidic extraction solution,inhibited the action of both catalase and peroxidases in plant tissues (Patter-son et al.1984),but the pH of the extract should be adjusted to above 8.0to give an adequate coloration.Although the reaction solution had the highest absorbance at pH 9.0,the blank which was added catalase had deep color at pH 9.0,but colorless at pH 8.4;pH 8.4was therefore adopted for future H 2O 2assay.
The effect of catalase quantity on the color of the blank was tested.The absorbance of the reac-tion product decreased with the increase in cata-lase quantity.7l g of catalase produced zero absorbance,suggesting this quantity of enzyme was sufficient to remove all H 2O 2(Table 2).Thus 8l g catalase was used as the standard blank assay.In order to determine if the above colorimetric reaction was effective for quantification of H 2O 2,known standard concentrations of H 2O 2were as-sayed.Absorbance of the colorimetric products showed a highly linear positive correlation to a wide range of H 2O 2concentrations (Fig.3).For
Table 1Effect of 5concentrations of colorimetric reagent on the absorbance of the reaction product at 505nm Treatment Absorbance at 505nm A 0.167±0.001B 0.161±0.001C 0.155±0.001D 0.140±0.002E
0.123
±0.00
H 2O 2solution (60l M)was mixed by equal volumes of colorimetric reagent.50ml of colorimetric reagents contained 40mg 4-aminoantipyrine,40mg phenol and 20mg peroxidase in Treatment A;20mg 4-aminoantipyri ne,20mg phenol and 10mg peroxidase in Treatment B;10mg 4-aminoantipyrine,10mg phenol and 5mg peroxi-dase in Treatment C;5mg 4-aminoantipyrine,5mg phenol and 2.5mg peroxidase in Treatment D,and 2.5mg 4-aminoantipyrine,2.5mg phenol and 1.25mg peroxidase in Treatment E.Values are means ±SE (n =
3)
0.00
0.050.100.150.200.25图像
拼接器
混凝土
隧道湿喷机
0.30Wavelength (nm)
A b s o r b a n c e
Fig.1Absorbance spectrum of colorimetric reaction product.5%(w/v)TCA containing 100l M H 2O 2was adjusted to pH 8.4with 17M ammonia solution and mixed by equal volume of colorimetric reagent.Absorbance of the reaction product at wavelengths from 450nm to 600nm was measured by spectrophotometer
123
quantification of H 2O 2in most of the plant mate-rial used here,we used a calibration range of 0–100l M of H 2O 2.When H 2O 2was added to the extracts of rice and litchi leaves after adjusted to pH 8.4prior to quantification of H 2O 2,the absor-bance of the colorimetric products was also highly li
nearly correlated to H 2O 2concentration (Fig.4).Ascorbic acid and reduced glutathione are important antioxidants and can scavenge H 2O 2via ascorbate-peroxidase or react directly with H 2O 2(Noctor and Foyer 1998).They can also be ex-tracted from plant tissues in 5%TCA (Law et al.1983;Zhou et al.2005).The effect of ascorbic acid (2.5l M)on the determination of H 2O 2was to suppress absorbance by 52%.10l M of ascorbic acid was sufficient to make the colorimetric prod-ucts undetectable (Table 3).This shows
that
00.20.40.60.81
1.20
50
100
150
200
250
300
350
400
Concentrations of hydrogen peroxide (µM)
A b s o r b a n c e a t 505 n m
Fig.3Standard curve of absorbance vs.hydrogen perox-ide concentration.Values represent the means of 3
replicates and bars indicate SE
Table 2Absorbance of colorimetric reaction product by the H 2O 2solution added different amount of catalase Amount of catalase (l g)Absorbance at 505nm 00.175±0.00810.079±0.00430.028±0.00150.011±0.001
708
5%TCA containing 60l M H 2O 2was adjusted to pH 8.4with 17M ammonia solution.The adjusted TCA solution without H 2O 2was served as a blank.The solution was divided into aliquots of 1ml.0,1,3,5,7and 8l g of catalase was added respectively to the aliquots and kept at room temperatures for 10min,after which 1ml of colorimetric reagent was added to the solution.Absorbance was determined at 505nm.Values are means ±SE (n =3)
Conc 00.050.10.150.20.250.30.350.40
20
40
60
80
100
120
Concentrations of hydrogen peroxide (µM)
A b s o r b a n c e a t 505 n m
A
B
0.050.10.150.20.250.30.350.4A b s o r b a n c e a t 505
Fig.4Absorbance of the colorimetric products vs.added hydrogen peroxide concentration in the extract of rice (A )and litchi leaves (B ).Fresh leaves (2g)were frozen in liquid nitrogen and ground in a mortar and pestle in 20ml of 5%TCA.Activated charcoal was added to the homogenate and mixed thoroughly.The mixture was centrifuged.The supernatant was adjusted to pH 8.4with 17M ammonia solution.Then the adjusted extract was filtered.Hydrogen peroxide was added to the filtrate.The filtrate with 0–100l M hydrogen peroxide was colored by colorimetric reagent.Values represent the means of 3replicates and bars indicate SE
Table 3Absorbance of colorimetric reaction product by the H 2O 2solution in different concentration of ascorbic acid
Ascorbic acid concentration (l M)Absorbance at 505nm 00.175±0.0022.50.085±0.00340.056±0.0021
0012.5
5%TCA containing 60l M H 2O 2was adjusted to pH 8.4with 17M ammonia solution.Then ascorbic acid was added to the solution to reach 2.5l M,5l M,10l M and 12.5l M,respectively and then mixed with colori-metric reagent.Absorbance of the colorimetric product was determined at 505nm.Values are means ±SE (n =3)
123
