Hindawi Publishing Corporation e Scientific World Journal Volume 2013, Article ID 678356, 7 pages http://dx.doi.org/10.1155/2013/678356 Research Article An Inhibitive Enzyme Assay to Detect Mercury and Zinc Using Protease from Coriandrum sativum Gunasekaran Baskaran, 1 Noor Azlina Masdor, 2 Mohd Arif Syed, 1 and Mohd Yunus Shukor 1 1 Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia 2 Biotechnology Research Centre, MARDI, P.O. Box 12301, 50774 Kuala Lumpur, Malaysia Correspondence should be addressed to Mohd Yunus Shukor; [email protected] Received 18 July 2013; Accepted 29 August 2013 Academic Editors: M. Y. Arica and B. C. Suedel Copyright © 2013 Gunasekaran Baskaran et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Heavy metals pollution has become a great threat to the world. Since instrumental methods are expensive and need skilled technician, a simple and fast method is needed to determine the presence of heavy metals in the environment. In this study, an inhibitive enzyme assay for heavy metals has been developed using crude proteases from Coriandrum sativum. In this assay, casein was used as a substrate and Coomassie dye was used to denote the completion of casein hydrolysis. In the absence of inhibitors, casein was hydrolysed and the solution became brown, while in the presence of metal ions such as Hg 2+ and Zn 2+ , the hydrolysis of casein was inhibited and the solution remained blue. Both Hg 2+ and Zn 2+ exhibited one-phase binding curve with IC 50 values of 3.217 mg/L and 0.727 mg/L, respectively. e limits of detection (LOD) and limits of quantitation (LOQ) for Hg were 0.241 and 0.802 mg/L, respectively, while the LOD and LOQ for Zn were 0.228 and 0.761 mg/L, respectively. e enzyme exhibited broad pH ranges for activity. e crude proteases extracted from Coriandrum sativum showed good potential for the development of a rapid, sensitive, and economic inhibitive assay for the biomonitoring of Hg 2+ and Zn 2+ in the aquatic environments. 1. Introduction Human activity in the last few decades has led to global contamination by organic and inorganic compounds [1, 2]. e presence of the pollutants generated from industrial and agriculture activities in the waterways has been identified to produce potential harmful effect on the aquatic living organisms and the food webs [3–6]. Nowadays, heavy metal contamination is considered to be among the most serious environmental problems. Heavy metals are any inorganic metallic compounds that can exert their toxicity via binding to the thiol group and disulfide bond that contribute to the stability of the enzyme [7]. e metals have high affinity to the disulfide bridge between two cysteine residues in any protein compound. Heavy metals are very dangerous to living organ- isms especially for humans since they can cause DNA damage and exert carcinogenic effects. In Malaysia, Juru Industrial Estate is renowned for releasing elevated concentrations of heavy metals into agricultural and aquaculture areas [8]. In addition, Alina et al. [9] reported that fishes in the coastal areas of Malaysia were contaminated by heavy metals. Hence, it is essential to monitor heavy metals in Juru area. e use of classical methods such as atomic absorption spectroscopy is expensive, requires highly-trained operators, complicated sample pretreatment, and needs a long measuring period [8]. erefore, simple and fast techniques are really needed for the detection of heavy metals in the environment. Inhibitive enzyme assays have been long developed to detect toxicants like heavy metals because of their rapid and economic approach. Recent works on the development of inhibitive enzyme assays involved the use of proteases such as papain, bromelain, and trypsin to detect heavy metals [8, 10, 11]. In general, bioassay is nonspecific towards a particular heavy metal, but it can be used as an early monitoring system [12, 13]. In this study, a novel source of protease from a
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Hindawi Publishing CorporationThe Scientific World JournalVolume 2013 Article ID 678356 7 pageshttpdxdoiorg1011552013678356
Research ArticleAn Inhibitive Enzyme Assay to Detect Mercury and Zinc UsingProtease from Coriandrum sativum
Gunasekaran Baskaran1 Noor Azlina Masdor2 Mohd Arif Syed1 and Mohd Yunus Shukor1
1 Department of Biochemistry Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia43400 Serdang Selangor Malaysia
2 Biotechnology Research Centre MARDI PO Box 12301 50774 Kuala Lumpur Malaysia
Correspondence should be addressed to Mohd Yunus Shukor mohdyunusupmedumy
Received 18 July 2013 Accepted 29 August 2013
Academic Editors M Y Arica and B C Suedel
Copyright copy 2013 Gunasekaran Baskaran et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
Heavy metals pollution has become a great threat to the world Since instrumental methods are expensive and need skilledtechnician a simple and fast method is needed to determine the presence of heavy metals in the environment In this study aninhibitive enzyme assay for heavy metals has been developed using crude proteases from Coriandrum sativum In this assay caseinwas used as a substrate and Coomassie dye was used to denote the completion of casein hydrolysis In the absence of inhibitorscasein was hydrolysed and the solution became brown while in the presence of metal ions such as Hg2+ and Zn2+ the hydrolysisof casein was inhibited and the solution remained blue Both Hg2+ and Zn2+ exhibited one-phase binding curve with IC
50values
of 3217mgL and 0727mgL respectively The limits of detection (LOD) and limits of quantitation (LOQ) for Hg were 0241 and0802mgL respectively while the LOD and LOQ for Zn were 0228 and 0761mgL respectively The enzyme exhibited broad pHranges for activity The crude proteases extracted from Coriandrum sativum showed good potential for the development of a rapidsensitive and economic inhibitive assay for the biomonitoring of Hg2+ and Zn2+ in the aquatic environments
1 Introduction
Human activity in the last few decades has led to globalcontamination by organic and inorganic compounds [1 2]The presence of the pollutants generated from industrial andagriculture activities in the waterways has been identifiedto produce potential harmful effect on the aquatic livingorganisms and the food webs [3ndash6] Nowadays heavy metalcontamination is considered to be among the most seriousenvironmental problems Heavy metals are any inorganicmetallic compounds that can exert their toxicity via bindingto the thiol group and disulfide bond that contribute to thestability of the enzyme [7]Themetals have high affinity to thedisulfide bridge between two cysteine residues in any proteincompound Heavymetals are very dangerous to living organ-isms especially for humans since they can causeDNAdamageand exert carcinogenic effects In Malaysia Juru IndustrialEstate is renowned for releasing elevated concentrations of
heavy metals into agricultural and aquaculture areas [8] Inaddition Alina et al [9] reported that fishes in the coastalareas ofMalaysia were contaminated by heavymetals Henceit is essential to monitor heavy metals in Juru area The useof classical methods such as atomic absorption spectroscopyis expensive requires highly-trained operators complicatedsample pretreatment and needs a long measuring period [8]Therefore simple and fast techniques are really needed for thedetection of heavy metals in the environment
Inhibitive enzyme assays have been long developed todetect toxicants like heavy metals because of their rapid andeconomic approach Recent works on the development ofinhibitive enzyme assays involved the use of proteases suchas papain bromelain and trypsin to detect heavy metals [810 11] In general bioassay is nonspecific towards a particularheavy metal but it can be used as an early monitoring system[12 13] In this study a novel source of protease from a
2 The Scientific World Journal
Table 1 Enzyme activity of different plant crude extracts
Samples Percentage of activity ()Negative control 0 plusmn 05
Averrhoa carambola (Star fruit) 10 plusmn 10
Ipomoea batatas (Sweet potato) 12 plusmn 05
Solanum melongena (Brinjal) 15 plusmn 15
Mangifera similis (Mango) 14 plusmn 05
Cucumis sativus (Cucumber) 49 plusmn 10
Coriandrum sativum (Coriander) 95 plusmn 10
Citrus aurantiifolia (Lime) 21 plusmn 05
Murraya koenigii (Curry leaf) 32 plusmn 10
Positive control 100 plusmn 0
All data are expressed as mean plusmn SEM
local plant for detection of mercury (Hg) and zinc (Zn) wasinvestigated
2 Materials and Methods
21 Preparation of Buffer Solutions Thebufferswere preparedaccording to the methods of Dawson et al [14] Adjustmentto the pH of buffer was made using NaOH (5N) and HCl(5N)
22 Bradford Dye Binding Assay Bradford reagent was pre-pared by mixing Coomassie Brilliant Blue G-250 (100mgSigma St Louis USA) with 95 ethanol (50mL) and 85phosphoric acid (100mL) [15] The solution was made up to1 L and stirred overnight The solution was filtered throughfilter paper (Whatman Filter Paper No 1 GE HealthcarePittsburgh PA USA) and stored in dark bottles [16 17]
23 Preparation of Casein Solution Casein was preparedaccording to the method of Shukor et al [11] Casein (2 gSigma St Louis USA) was dissolved in deionised water(100mL) and the pH was adjusted to 80 The casein stocksolution was then incubated overnight at 60∘C under a mildstirring condition The solution was filtered through severallayers of cheesecloth [18] and the filtrate was centrifuged at10000timesg for 10min The protein concentration of casein inthe supernatant wasmeasured by Bradford dye-binding assayusing BSA (Sigma St Louis USA) as a standard
24 Preparation of Heavy Metals Solutions Silver arseniccadmium cobalt chromium copper mercury nickel leadand zinc stock solutions were purchased fromMerck Darm-stadt Germany The working solutions (10mgL 5mgL25mgL 10mgL and 05mgL) were prepared using deion-ized water and stored in acid-washed polypropylene contain-ers
25 Extraction of Plant Protease Extraction of plant pro-teases was carried out according to the modified method ofJiang et al [19] Chopped plant tissues were immersed in
sodium phosphate buffer (50mM pH 7) for 2 days in thechiller Homogenization buffer was added (1 ratio of plant 3ratios of chilled buffer) and the mixture was blended for 20 sat high speed followed by a 10min of cooling periodThe cycleof blending and coolingwas repeated until plant sampleswerehomogenized The products were sieved and centrifuged at10000timesg for 15min at 4∘C The pellet and supernatant werefurther assayed to determine proteases activity
26 Optimization of Enzyme Assay The optimization ofenzyme concentrations substrate concentrations pH tem-perature and incubation time was employed to obtain anoptimum absorbance for the detection of heavy metals Theabsorbance range between 03 and 09 is the ideal differencefrom the blank to ensure visible color changes [8] This isimportant for qualitative detection of heavy metals since itis not possible to bring spectrophotometer to the field
27 Protease Inhibition Studies Protease inhibitive assayfor heavy metals was performed as described by Shukoret al [8] Protective reagents like EDTA and DTT wereremoved to enhance the sensitivity towards heavy metals[7] Protease (100 120583L) was added to sodium carbonate buffer(20120583L 50mM pH 90) in a microcentrifuge tube followedby the addition of heavy metals (20 120583L) For the controldeionized water (20 120583L) was used instead of heavy metalsCasein (60 120583L) was added to the mixture after 20min ofincubation at room temperature An aliquot (20120583L) waswithdrawn and mixed with Bradford dye-binding reagent(200120583L) in amicrotiter plate wellThemixture was incubatedfor 5min and the absorbancewas designated as time zeroTheremaining solution was incubated at 35∘C for 20min Afterthe incubation period another aliquot (20 120583L) was collectedand treated in the same manner as the aliquot at time zeroThe absorbance at 595 nm was measured using a microtiterplate reader (Stat Fax 3200 Microplate Reader AwarenessTechnology Inc USA) The values for the IC
50(inhibitory
concentration 50) were calculated using the nonlinearregression analysis for one-phase binding model using theGraphPad PRISM 5 Software Means and standard deviationwere determined based on three independent experimentalreplicates
28 Collecting of Environmental Samples (Field Trials) Sam-ples were collected from aquatic environments from severalindustrial outlets that release heavy metals products such aspristine areas and galvanized metals factories In this study2 states in Malaysia were targeted for the sampling worksPrai and Bukit Tengah Industrial Areas (Penang) and EndauRompin National Park (Johor) Water samples were takenapproximately 20ndash30 cm from the surface of the water Thecollected samples were placed in the acid-washed HDPEbottles Several drops of concentrated nitric acid (HNO
3)
were added to extract heavy metals that bound to othercompounds in the samplesThe sampleswere filtered by using045 120583m filter membrane and finally assayed in inhibitiveprotease assay and analyzed by ICP-OES
The Scientific World Journal 3
Table 2 Summary of optimization results for Coriandrum sativum proteases in comparison to other proteases
Optimization parameter Optimum conditions for proteasesCoriandrum sativum Papaina Bromelainb Trypsinc
Enzyme concentration (mgmL)Substrate concentration (mgmL)Temperature (∘C)Incubation time (min)pH
04504253520
8ndash95
010130305ndash7
0102540305-6
010140305ndash7
aShukor et al [8]bShukor et al [10]cShukor et al [11]
Table 3 IC50 values of Coriandrum sativum proteases for Hg and Zn in comparison to other protease assays
IC50 (mgL)Heavymetals
Coriandrum sativumproteases Papaina Bromelainb Trypsinc Immobilized
Ureased 15min Microtoxd 48-hour Daphniamagnad
Hg 3217 024ndash062 013ndash016 1576ndash1704 033 0029ndash005 0005ndash021Zn 0727 211 mdash 48ndash67 146 027ndash29 054ndash51aShukor et al [8]bShukor et al [10]cShukor et al [11]dJung et al [13]
Table 4 LOD and LOQ values for the proteases from Coriandrumsativum
Metals Regression model 1198772 LOD (mgL) LOQ (mgL)
Hg One-phase binding 0966 0241 0802Zn One-phase binding 0984 0228 0650
3 Results and Discussion
31 Screening towards Plant Protease Identification of theplant source that gives the highest enzyme activity is verycrucial in this study since bioassay is only practical forenzyme that shows high enzymatic activity The significantcolor changes of the Bradford reagent are only observable ifthe protease has high activity Table 1 shows the percentageactivity between plant sources Commercialized enzymepapain (Sigma EC 34222 lot no 32K2619 St Louis USA)was used as a positive control and enzyme was replaced withdeionized water for the negative control
Among all plant samples Coriandrum sativum gave thehighest enzyme activity Although high enzyme activity doesnot correlate with the sensitivity of the enzyme towardsheavy metals this plant source was used since the activityis sufficiently high for inhibitive assay The inhibitive assay isonly valid if the difference in absorbance ismore than 02 aftera maximum incubation for 1 h [8] Coriandrum sativum hassenescence-related serinyl protease which is involved in thephysiological role and metabolic activities of the plant [19]
32 Optimized Conditions for Coriandrum sativum Pro-tease Activity The optimum concentration of Coriandrumsativum proteases was at 45120583gmLThe optimum concentra-tion was higher compared to previous optimization studies
on other proteases such as papain bromelain and trypsin asshown in Table 2The optimized concentration would ensurethat degradation was complete before any autodigestion ofproteases takes place Substrate casein gave optimum activityat 425120583gmL Higher concentration of the substrate is notdesired since it would take a longer time for the casein tobe digested The optimized temperature was at 35∘C lowercompared to the proteases such as bromelain and trypsin [1011] Thus it does not require extra heating process since it isalmost similar to the environmental temperature inMalaysiaThe highest enzyme activity was noted at incubation periodof 20min and at the pH range between 8 and 95 in sodiumcarbonate buffer The broad range of pH is favored forassaying environmental samples since it can bear deviationin samplesrsquo pH [11]
33 Heavy Metals Studies The Coriandrum sativum proteaseactivity was inhibited by two heavymetals mercury and zincat 1mgL as shown in Figure 1The other heavymetals did notshow significant inhibition towards the enzyme activity thusthe solution remained brown in color This is because Brad-ford reagent is unable to stain digested casein which is lessthan 2 kDa [10] Mercury and zinc were observed to exhibitdark blue color after the completion of the assay indicatingthat both heavymetals inhibit the activity of the proteaseThemechanism of metal inhibition on enzyme activity especiallyby mercury is through binding of the sulfhydryl groupsin cysteine proteases and through destruction of tertiarystructure in which the disulfide bridges were destroyed andthe casein became unfit to the active site of the enzyme [8]Therefore Bradford reagent stains the undigested casein andthe solution changed to dark blue in color The Coriandrumsativum proteases probably contain a mixture of serine [19]
4 The Scientific World Journal
Table 5 Protease inhibitive assay for samples from Prai Industrial Area
Sample GPS location Percentage activity () Concentration of heavy metals in samples (mgL) by ICP-OESZn Hg
1 N05∘20871015840 E100∘246921015840 9359 plusmn 01 004 plusmn 005 nd2 N05∘20871015840 E100∘246921015840 134 plusmn 03 12530 plusmn 033 nd3 N05∘208621015840 E100∘2467410158401015840 9854 plusmn 02 021 plusmn 012 nd4 N05∘208361015840 E100∘251771015840 2254 plusmn 05 763 plusmn 005 minus 9 nd5 N05∘202241015840 E100∘263021015840 9880 plusmn 07 006 plusmn 003 nd6 N05∘219831015840 E100∘240231015840 3880 plusmn 09 089 plusmn 001 nd7 N05∘219671015840 E100∘240441015840 9398 plusmn 01 014 plusmn 002 nd8 N05∘20871015840 E100∘246921015840 946 plusmn 01 1454 plusmn 013 nd9 N05∘20871015840 E100∘246921015840 2521 plusmn 08 397 plusmn 004 nd10 N05∘208621015840 E100∘246741015840 3366 plusmn 02 350 plusmn 002 nd11 N05∘196991015840 E100∘261291015840 1921 plusmn 03 874 plusmn 009 nd12 N05∘196991015840 E100∘261291015840 682 plusmn 03 1200 plusmn 002 152 plusmn 006
13 N05∘196991015840 E100∘261291015840 877 plusmn 08 1580 plusmn 013 nd14 N05∘202631015840 E100∘257741015840 9994 plusmn 09 002 plusmn 001 nd15 N05∘202631015840 E100∘257741015840 9939 plusmn 04 003 plusmn 003 nd16 N05∘201351015840 E100∘269251015840 339 plusmn 03 3865 plusmn 018 374 plusmn 009
17 N05∘211531015840 E100∘260731015840 9481 plusmn 05 002 plusmn 002 nd18 N05∘211531015840 E100∘260731015840 9742 plusmn 07 009 plusmn 001 nd19 N05∘211531015840 E100∘260731015840 9811 plusmn 01 002 plusmn 001 nd20 N05∘200911015840 E100∘252691015840 9746 plusmn 02 006 plusmn 002 nd21 N05∘211321015840 E100∘250811015840 9924 plusmn 02 071 plusmn 001 nd22 N05∘204721015840 E100∘268911015840 1605 plusmn 03 1084 plusmn 006 nd23 N05∘203871015840 E100∘244291015840 495 plusmn 04 005 plusmn 001 nd24 N05∘202631015840 E100∘257741015840 209 plusmn 07 1269 plusmn 003 414 plusmn 015
All data are expressed as mean plusmn SEMnd not detected
Table 6 Protease inhibitive assay for samples from Bukit Tengah Industrial Area
Sample GPS location Percentage activity () Concentration of heavy metals in samples (mgL) by ICP-OESZn Hg
1 N05∘204471015840 E100∘264031015840 639 plusmn 04 1865 plusmn 003 nd2 N05∘206651015840 E100∘263641015840 9684 plusmn 07 nd nd3 N05∘206011015840 E100∘264271015840 387 plusmn 05 1342 plusmn 001 nd4 N05∘206401015840 E100∘264701015840 3247 plusmn 09 nd 236 plusmn 001
5 N05∘189471015840 E100∘263481015840 4870 plusmn 10 nd 157 plusmn 002
All data are expressed as mean plusmn SEMnd not detected
and cysteine proteases Serine proteases are known to beinhibited by zinc [11] while cysteine proteases are stronglyinhibited by mercury [8] Therefore the inhibitive enzymeassay using Coriandrum sativum is a potential candidate forbiomonitoring of Hg and Zn in aquatic environments
34 Comparison of theHalfMaximal Inhibitory Concentration(IC50) Values of Proteases In terms of sensitivity the IC
50
values are often used as a comparison among established
assays [4]The comparison of IC50values of proteases studied
so far with other types of assays is summarized in Table 3 ForHg Coriandrum sativum proteases showed better sensitivitycompared to trypsin with the IC
50value of 3217mgL
(Figure 2) Meanwhile the IC50
value for Zn is 0727mgL(Figure 3) which is more sensitive than other assays likepapain bromelain trypsin and immobilized urease andwithin the range of Microtox andDaphnia magna assaysThelower IC
50value denotes the higher effectiveness of protease
in inhibiting heavy metalsThe advantages of inhibitive assay
The Scientific World Journal 5
Heavy metals
0
10
20
30
40
50
60
70
80
90
100
Enzy
me a
ctiv
ity (
)
Ag As Cd Co Cu Hg Ni Pb Zn ControlCr3+
Figure 1 The effect of heavy metals at 1mgL on Coriandrum sativum proteases activity All data are expressed as mean plusmn SEM
Table 7 Protease inhibitive assay for samples from Endau Rompin
Samples GPS location Percentage activity () Concentration of heavy metals in samples (mgL) by ICP-OESZn Hg
1 N02∘306741015840 E103∘213871015840 9749 plusmn 03 nd nd2 N02∘308021015840 E103∘210861015840 9768 plusmn 02 nd nd3 N02∘307831015840 E103∘211401015840 9593 plusmn 05 nd nd4 N02∘307841015840 E 103∘21021015840 9953 plusmn 07 nd ndAll data are expressed as mean plusmn SEMnd not detected
0
20
40
60
80
100
0 2 4 6 8 10 12 14 16
Enzy
me a
ctiv
ity (
)
Concentration of Hg (mgL)
Figure 2 in which the disulfide bridges were destroyed and thecasein became unfit to the active site of the enzymeInhibitionof proteolytic activity of Coriandrum sativum protease by Hgusing Coomassie brilliant blue assay Data is generated using thenonlinear regression analysis for one-phase bindingmodel using theGraphPad PRISM 5 Software All data are expressed asmeanplusmn SEM
in comparison with Microtox are that it is inexpensive andsuitable for the detection of heavy metals in field trials sinceit is based on colorimetric changes which do not requirephotometer Meanwhile Daphnia magna assays have broadrange of IC
50value and need a longer period of incubation
48 h compared to inhibitive assaywhich only requires 20min
0
20
40
60
80
100
0 05 1 15 2 25 3 35 4
Enzy
me a
ctiv
ity (
)
Concentration of Zn (mgL)
Figure 3 Inhibition of proteolytic activity of Coriandrum sativumprotease by Zn using Coomassie brilliant blue assay Data isgenerated using the nonlinear regression analysis for one-phasebinding model using the GraphPad PRISM 5 Software All data areexpressed as mean plusmn SEM
35 Limits of Detection (LOD) and Limit of Quantitation(LOQ) for Hg and Zn The LOD and LOQ values for Hgand Zn detected by coriander proteases are shown in Table 4LOD is the lowest concentration of heavy metal that caninhibit the enzyme activity which is at least three times thestandard deviation of the blank at 119910-intercept [20] The LODvalues of Hg and Zn are 0241 and 0228mgL respectively
6 The Scientific World Journal
Meanwhile LOQ indicates the minimal concentration ofheavymetal that can produce observable color changes to theBradford assay LOQ is ten times the value of the standarddeviation of themean blank value [20]The LOQvalues ofHgand Zn are 0802 and 0761mgL respectivelyThe LOQ valuefor Hg was lower and about the same for Zn compared tothe trypsin assay 135 and 061mgL respectively [11] Thusproteases from this plant have a promising potential to beused as an inhibitive assay for Hg and Zn
36 Field Trials Samples were collected from Prai andBukit Tengah Industrial Areas and Endau Rompin NationalPark The samples were analyzed using the inhibitive assayand the results were validated with ICP-OES Twelve outof 24 samples from Prai Industrial Area gave more than50 inhibition to the protease activity (Table 5) ICP-OESresults showed that the positive samples contain extremelyhigh concentration of Hg and Zn with some exceeding themaximum permissible limit (MPL) for Hg and Zn 0001and 5mgL respectively as outlined by the Department ofEnvironment (DOE) [21] LOQ value for Hg 0802mgL ismuch higher than the MPL for Hg in aquatic environmentas outlined by the DOE of Malaysia 0001mgL Howeverthis assay can be used in monitoring Hg levels in industrialsites since these areasmostly contain very high concentrationof Hg Previous studies carried out in the same area alsoreported that the area is highly polluted with heavy metals[8 10] indicating a continuous trend
Four out of 5 samples from Bukit Tengah Industrial Area(Table 6) gave positive inhibitory result on enzyme activitydue to the presence of Hg and Zn as determined by ICP-OESSince galvanizedmetal factories can be found in this area it issuspected that these types of industries are responsible for theelevatedHg andZn levelThus a fast and inexpensivemethodfor monitoring these heavy metals is urgently needed
Endau Rompin is the one of the largest national park inMalaysia with pristine water and a diverse mixture of floraand fauna Four distinct samples were taken from this areaand the results showed that therewas no significant inhibitoryeffect on enzyme activityThe results obtained from ICP-OESalso proved the absence of heavy metals (Table 7) indicatinga good correlation between inhibitive assay and instrumentalmethod
4 Conclusion
An assay for detectingHg and Zn using proteases from a localplant has been successfully developed The optimizationsof enzyme and substrate concentration pH temperatureand incubation time were performed using the Bradford-protease-casein system In field trials samples obtained frompolluted and nonpolluted sites showed promising resultsThefindings showed that Prai and Juru Industrial Areas are highlypolluted with Hg and Zn The protease has a broad pH rangeand high sensitivity towardsHg andZn suggesting robustnessand suitability for biomonitoring field works For futurestudies it is recommended that the proteases be purifiedto increase the sensitivity towards the heavy metals This
study provides fundamental information for the developmentof rapid sensitive and economic inhibitive assay for thebiomonitoring of heavy metals in the environment
Acknowledgments
The authors would like to thank Shamala Salvamani andZakiuddin Sahlani for their technical support This researchwas funded by The Special Cradle Research DevelopmentFund (CRDF) from the Malaysian Technology DevelopmentCorporation
References
[1] S K Chaerun N P D Pangesti K Toyota andW BWhitmanldquoChanges inmicrobial functional diversity and activity in paddysoils irrigated with industrial wastewaters in Bandung WestJava Province IndonesiardquoWater Air and Soil Pollution vol 217no 1ndash4 pp 491ndash502 2011
[2] R K Sahu S Katiyar J Tiwari and G C Kisku ldquoAssessment ofdrain water receiving effluent from tanneries and its impact onsoil and plants with particular emphasis on bioaccumulation ofheavy metalsrdquo Journal of Environmental Biology vol 28 no 3pp 685ndash690 2007
[3] J K Katnoria S Arora R Bhardwaj andANagpal ldquoEvaluationof genotoxic potential of industrial waste contaminated soilextracts of Amritsar Indiardquo Journal of Environmental Biologyvol 32 no 3 pp 363ndash367 2011
[4] S Knasmuller E GottmannH Steinkellner et al ldquoDetection ofgenotoxic effects of heavy metal contaminated soils with plantbioassaysrdquoMutation Research vol 420 no 1ndash3 pp 37ndash48 1998
[5] V A Kulkarni V S Naidu and T G Jagtap ldquoMarine ecologicalhabitat a case study on projected thermal power plant aroundDharamtar creek Indiardquo Journal of Environmental Biology vol32 no 2 pp 213ndash219 2011
[6] M Oliveira M A Santos and M Pacheco ldquoGlutathioneprotects heavy metal-induced inhibition of hepatic microsomalethoxyresorufin O-deethylase activity in Dicentrarchus labraxLrdquo Ecotoxicology and Environmental Safety vol 58 no 3 pp379ndash385 2004
[7] M F Frasco D Fournier F Carvalho and L GuilherminoldquoImplementation of assay conditions for the use ofAchE activityas a biomarker of metal toxicityrdquo Biomarkers vol 10 no 5 pp360ndash375 2005
[8] Y Shukor N A Baharom F A Rahman M P Abdullah NA Shamaan and M A Syed ldquoDevelopment of a heavy metalsenzymatic-based assay using papainrdquo Analytica Chimica Actavol 566 no 2 pp 283ndash289 2006
[9] M Alina A Azrina A S Mohd Yunus S Mohd Zakiuddin HMohd Izuan Effendi and R Muhammad Rizal ldquoHeavy metals(mercury arsenic cadmium plumbum) in selected marine fishand shellfish along the straits of malaccardquo International FoodResearch Journal vol 19 no 1 pp 135ndash140 2012
[10] M Y Shukor N Masdor N A Baharom et al ldquoAn inhibitivedetermination method for heavy metals using bromelain acysteine proteaserdquo Applied Biochemistry and Biotechnology vol144 no 3 pp 283ndash291 2008
[11] M Y Shukor N A Baharom N A Masdor et al ldquoThedevelopment of an inhibitive determination method for zincusing a serine proteaserdquo Journal of Environmental Biology vol30 no 1 pp 17ndash22 2009
The Scientific World Journal 7
[12] S HanM Zhu Z Yuan and X Li ldquoAmethylene blue-mediatedenzyme electrode for the determination of trace mercury(II)mercury(I) methylmercury and mercury-glutathione com-plexrdquo Biosensors and Bioelectronics vol 16 no 1-2 pp 9ndash162001
[13] K Jung G Bitton and B Koopman ldquoAssessment of urease inhi-bition assays for measuring toxicity of environmental samplesrdquoWater Research vol 29 no 8 pp 1929ndash1933 1995
[14] R M C Dawson D C Elliott W H Elliott and K M JonesData for Biochemical Research Clarendon Press Oxford UK1969
[15] A Bensadoun and D Weinstein ldquoAssay of proteins in thepresence of interfering materialsrdquo Analytical Biochemistry vol70 no 1 pp 241ndash250 1976
[16] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976
[17] G L Ellman K D Courtney V Andres Jr and R MFeatherstone ldquoA new and rapid colorimetric determination ofacetylcholinesterase activityrdquo Biochemical Pharmacology vol 7no 2 pp 88ndash95 1961
[18] H M Farrell Jr E D Wickham and M L Groves ldquoEnvi-ronmental influences on purified casein disulfide interactionsrdquoJournal of Dairy Science vol 81 no 11 pp 2974ndash2984 1998
[19] W Jiang X Zhou Y Zhao and P Liu ldquoIdentification ofa senescence-related protease in coriander leavesrdquo ChineseScience Bulletin vol 47 no 13 pp 1096ndash1099 2002
[20] D A Armbruster M D Tillman and L M Hubbs ldquoLimit ofdetection (LOD)limit of quantitation (LOQ) comparison ofthe empirical and the statistical methods exemplified with GC-MS assays of abused drugsrdquo Clinical Chemistry vol 40 no 7pp 1233ndash1238 1994
[21] DOE Environmental Quality Report Department of Environ-ment Ministry of Science Technology and the EnvironmentMalaysia 2001
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International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
2 The Scientific World Journal
Table 1 Enzyme activity of different plant crude extracts
Samples Percentage of activity ()Negative control 0 plusmn 05
Averrhoa carambola (Star fruit) 10 plusmn 10
Ipomoea batatas (Sweet potato) 12 plusmn 05
Solanum melongena (Brinjal) 15 plusmn 15
Mangifera similis (Mango) 14 plusmn 05
Cucumis sativus (Cucumber) 49 plusmn 10
Coriandrum sativum (Coriander) 95 plusmn 10
Citrus aurantiifolia (Lime) 21 plusmn 05
Murraya koenigii (Curry leaf) 32 plusmn 10
Positive control 100 plusmn 0
All data are expressed as mean plusmn SEM
local plant for detection of mercury (Hg) and zinc (Zn) wasinvestigated
2 Materials and Methods
21 Preparation of Buffer Solutions Thebufferswere preparedaccording to the methods of Dawson et al [14] Adjustmentto the pH of buffer was made using NaOH (5N) and HCl(5N)
22 Bradford Dye Binding Assay Bradford reagent was pre-pared by mixing Coomassie Brilliant Blue G-250 (100mgSigma St Louis USA) with 95 ethanol (50mL) and 85phosphoric acid (100mL) [15] The solution was made up to1 L and stirred overnight The solution was filtered throughfilter paper (Whatman Filter Paper No 1 GE HealthcarePittsburgh PA USA) and stored in dark bottles [16 17]
23 Preparation of Casein Solution Casein was preparedaccording to the method of Shukor et al [11] Casein (2 gSigma St Louis USA) was dissolved in deionised water(100mL) and the pH was adjusted to 80 The casein stocksolution was then incubated overnight at 60∘C under a mildstirring condition The solution was filtered through severallayers of cheesecloth [18] and the filtrate was centrifuged at10000timesg for 10min The protein concentration of casein inthe supernatant wasmeasured by Bradford dye-binding assayusing BSA (Sigma St Louis USA) as a standard
24 Preparation of Heavy Metals Solutions Silver arseniccadmium cobalt chromium copper mercury nickel leadand zinc stock solutions were purchased fromMerck Darm-stadt Germany The working solutions (10mgL 5mgL25mgL 10mgL and 05mgL) were prepared using deion-ized water and stored in acid-washed polypropylene contain-ers
25 Extraction of Plant Protease Extraction of plant pro-teases was carried out according to the modified method ofJiang et al [19] Chopped plant tissues were immersed in
sodium phosphate buffer (50mM pH 7) for 2 days in thechiller Homogenization buffer was added (1 ratio of plant 3ratios of chilled buffer) and the mixture was blended for 20 sat high speed followed by a 10min of cooling periodThe cycleof blending and coolingwas repeated until plant sampleswerehomogenized The products were sieved and centrifuged at10000timesg for 15min at 4∘C The pellet and supernatant werefurther assayed to determine proteases activity
26 Optimization of Enzyme Assay The optimization ofenzyme concentrations substrate concentrations pH tem-perature and incubation time was employed to obtain anoptimum absorbance for the detection of heavy metals Theabsorbance range between 03 and 09 is the ideal differencefrom the blank to ensure visible color changes [8] This isimportant for qualitative detection of heavy metals since itis not possible to bring spectrophotometer to the field
27 Protease Inhibition Studies Protease inhibitive assayfor heavy metals was performed as described by Shukoret al [8] Protective reagents like EDTA and DTT wereremoved to enhance the sensitivity towards heavy metals[7] Protease (100 120583L) was added to sodium carbonate buffer(20120583L 50mM pH 90) in a microcentrifuge tube followedby the addition of heavy metals (20 120583L) For the controldeionized water (20 120583L) was used instead of heavy metalsCasein (60 120583L) was added to the mixture after 20min ofincubation at room temperature An aliquot (20120583L) waswithdrawn and mixed with Bradford dye-binding reagent(200120583L) in amicrotiter plate wellThemixture was incubatedfor 5min and the absorbancewas designated as time zeroTheremaining solution was incubated at 35∘C for 20min Afterthe incubation period another aliquot (20 120583L) was collectedand treated in the same manner as the aliquot at time zeroThe absorbance at 595 nm was measured using a microtiterplate reader (Stat Fax 3200 Microplate Reader AwarenessTechnology Inc USA) The values for the IC
50(inhibitory
concentration 50) were calculated using the nonlinearregression analysis for one-phase binding model using theGraphPad PRISM 5 Software Means and standard deviationwere determined based on three independent experimentalreplicates
28 Collecting of Environmental Samples (Field Trials) Sam-ples were collected from aquatic environments from severalindustrial outlets that release heavy metals products such aspristine areas and galvanized metals factories In this study2 states in Malaysia were targeted for the sampling worksPrai and Bukit Tengah Industrial Areas (Penang) and EndauRompin National Park (Johor) Water samples were takenapproximately 20ndash30 cm from the surface of the water Thecollected samples were placed in the acid-washed HDPEbottles Several drops of concentrated nitric acid (HNO
3)
were added to extract heavy metals that bound to othercompounds in the samplesThe sampleswere filtered by using045 120583m filter membrane and finally assayed in inhibitiveprotease assay and analyzed by ICP-OES
The Scientific World Journal 3
Table 2 Summary of optimization results for Coriandrum sativum proteases in comparison to other proteases
Optimization parameter Optimum conditions for proteasesCoriandrum sativum Papaina Bromelainb Trypsinc
Enzyme concentration (mgmL)Substrate concentration (mgmL)Temperature (∘C)Incubation time (min)pH
04504253520
8ndash95
010130305ndash7
0102540305-6
010140305ndash7
aShukor et al [8]bShukor et al [10]cShukor et al [11]
Table 3 IC50 values of Coriandrum sativum proteases for Hg and Zn in comparison to other protease assays
IC50 (mgL)Heavymetals
Coriandrum sativumproteases Papaina Bromelainb Trypsinc Immobilized
Ureased 15min Microtoxd 48-hour Daphniamagnad
Hg 3217 024ndash062 013ndash016 1576ndash1704 033 0029ndash005 0005ndash021Zn 0727 211 mdash 48ndash67 146 027ndash29 054ndash51aShukor et al [8]bShukor et al [10]cShukor et al [11]dJung et al [13]
Table 4 LOD and LOQ values for the proteases from Coriandrumsativum
Metals Regression model 1198772 LOD (mgL) LOQ (mgL)
Hg One-phase binding 0966 0241 0802Zn One-phase binding 0984 0228 0650
3 Results and Discussion
31 Screening towards Plant Protease Identification of theplant source that gives the highest enzyme activity is verycrucial in this study since bioassay is only practical forenzyme that shows high enzymatic activity The significantcolor changes of the Bradford reagent are only observable ifthe protease has high activity Table 1 shows the percentageactivity between plant sources Commercialized enzymepapain (Sigma EC 34222 lot no 32K2619 St Louis USA)was used as a positive control and enzyme was replaced withdeionized water for the negative control
Among all plant samples Coriandrum sativum gave thehighest enzyme activity Although high enzyme activity doesnot correlate with the sensitivity of the enzyme towardsheavy metals this plant source was used since the activityis sufficiently high for inhibitive assay The inhibitive assay isonly valid if the difference in absorbance ismore than 02 aftera maximum incubation for 1 h [8] Coriandrum sativum hassenescence-related serinyl protease which is involved in thephysiological role and metabolic activities of the plant [19]
32 Optimized Conditions for Coriandrum sativum Pro-tease Activity The optimum concentration of Coriandrumsativum proteases was at 45120583gmLThe optimum concentra-tion was higher compared to previous optimization studies
on other proteases such as papain bromelain and trypsin asshown in Table 2The optimized concentration would ensurethat degradation was complete before any autodigestion ofproteases takes place Substrate casein gave optimum activityat 425120583gmL Higher concentration of the substrate is notdesired since it would take a longer time for the casein tobe digested The optimized temperature was at 35∘C lowercompared to the proteases such as bromelain and trypsin [1011] Thus it does not require extra heating process since it isalmost similar to the environmental temperature inMalaysiaThe highest enzyme activity was noted at incubation periodof 20min and at the pH range between 8 and 95 in sodiumcarbonate buffer The broad range of pH is favored forassaying environmental samples since it can bear deviationin samplesrsquo pH [11]
33 Heavy Metals Studies The Coriandrum sativum proteaseactivity was inhibited by two heavymetals mercury and zincat 1mgL as shown in Figure 1The other heavymetals did notshow significant inhibition towards the enzyme activity thusthe solution remained brown in color This is because Brad-ford reagent is unable to stain digested casein which is lessthan 2 kDa [10] Mercury and zinc were observed to exhibitdark blue color after the completion of the assay indicatingthat both heavymetals inhibit the activity of the proteaseThemechanism of metal inhibition on enzyme activity especiallyby mercury is through binding of the sulfhydryl groupsin cysteine proteases and through destruction of tertiarystructure in which the disulfide bridges were destroyed andthe casein became unfit to the active site of the enzyme [8]Therefore Bradford reagent stains the undigested casein andthe solution changed to dark blue in color The Coriandrumsativum proteases probably contain a mixture of serine [19]
4 The Scientific World Journal
Table 5 Protease inhibitive assay for samples from Prai Industrial Area
Sample GPS location Percentage activity () Concentration of heavy metals in samples (mgL) by ICP-OESZn Hg
1 N05∘20871015840 E100∘246921015840 9359 plusmn 01 004 plusmn 005 nd2 N05∘20871015840 E100∘246921015840 134 plusmn 03 12530 plusmn 033 nd3 N05∘208621015840 E100∘2467410158401015840 9854 plusmn 02 021 plusmn 012 nd4 N05∘208361015840 E100∘251771015840 2254 plusmn 05 763 plusmn 005 minus 9 nd5 N05∘202241015840 E100∘263021015840 9880 plusmn 07 006 plusmn 003 nd6 N05∘219831015840 E100∘240231015840 3880 plusmn 09 089 plusmn 001 nd7 N05∘219671015840 E100∘240441015840 9398 plusmn 01 014 plusmn 002 nd8 N05∘20871015840 E100∘246921015840 946 plusmn 01 1454 plusmn 013 nd9 N05∘20871015840 E100∘246921015840 2521 plusmn 08 397 plusmn 004 nd10 N05∘208621015840 E100∘246741015840 3366 plusmn 02 350 plusmn 002 nd11 N05∘196991015840 E100∘261291015840 1921 plusmn 03 874 plusmn 009 nd12 N05∘196991015840 E100∘261291015840 682 plusmn 03 1200 plusmn 002 152 plusmn 006
13 N05∘196991015840 E100∘261291015840 877 plusmn 08 1580 plusmn 013 nd14 N05∘202631015840 E100∘257741015840 9994 plusmn 09 002 plusmn 001 nd15 N05∘202631015840 E100∘257741015840 9939 plusmn 04 003 plusmn 003 nd16 N05∘201351015840 E100∘269251015840 339 plusmn 03 3865 plusmn 018 374 plusmn 009
17 N05∘211531015840 E100∘260731015840 9481 plusmn 05 002 plusmn 002 nd18 N05∘211531015840 E100∘260731015840 9742 plusmn 07 009 plusmn 001 nd19 N05∘211531015840 E100∘260731015840 9811 plusmn 01 002 plusmn 001 nd20 N05∘200911015840 E100∘252691015840 9746 plusmn 02 006 plusmn 002 nd21 N05∘211321015840 E100∘250811015840 9924 plusmn 02 071 plusmn 001 nd22 N05∘204721015840 E100∘268911015840 1605 plusmn 03 1084 plusmn 006 nd23 N05∘203871015840 E100∘244291015840 495 plusmn 04 005 plusmn 001 nd24 N05∘202631015840 E100∘257741015840 209 plusmn 07 1269 plusmn 003 414 plusmn 015
All data are expressed as mean plusmn SEMnd not detected
Table 6 Protease inhibitive assay for samples from Bukit Tengah Industrial Area
Sample GPS location Percentage activity () Concentration of heavy metals in samples (mgL) by ICP-OESZn Hg
1 N05∘204471015840 E100∘264031015840 639 plusmn 04 1865 plusmn 003 nd2 N05∘206651015840 E100∘263641015840 9684 plusmn 07 nd nd3 N05∘206011015840 E100∘264271015840 387 plusmn 05 1342 plusmn 001 nd4 N05∘206401015840 E100∘264701015840 3247 plusmn 09 nd 236 plusmn 001
5 N05∘189471015840 E100∘263481015840 4870 plusmn 10 nd 157 plusmn 002
All data are expressed as mean plusmn SEMnd not detected
and cysteine proteases Serine proteases are known to beinhibited by zinc [11] while cysteine proteases are stronglyinhibited by mercury [8] Therefore the inhibitive enzymeassay using Coriandrum sativum is a potential candidate forbiomonitoring of Hg and Zn in aquatic environments
34 Comparison of theHalfMaximal Inhibitory Concentration(IC50) Values of Proteases In terms of sensitivity the IC
50
values are often used as a comparison among established
assays [4]The comparison of IC50values of proteases studied
so far with other types of assays is summarized in Table 3 ForHg Coriandrum sativum proteases showed better sensitivitycompared to trypsin with the IC
50value of 3217mgL
(Figure 2) Meanwhile the IC50
value for Zn is 0727mgL(Figure 3) which is more sensitive than other assays likepapain bromelain trypsin and immobilized urease andwithin the range of Microtox andDaphnia magna assaysThelower IC
50value denotes the higher effectiveness of protease
in inhibiting heavy metalsThe advantages of inhibitive assay
The Scientific World Journal 5
Heavy metals
0
10
20
30
40
50
60
70
80
90
100
Enzy
me a
ctiv
ity (
)
Ag As Cd Co Cu Hg Ni Pb Zn ControlCr3+
Figure 1 The effect of heavy metals at 1mgL on Coriandrum sativum proteases activity All data are expressed as mean plusmn SEM
Table 7 Protease inhibitive assay for samples from Endau Rompin
Samples GPS location Percentage activity () Concentration of heavy metals in samples (mgL) by ICP-OESZn Hg
1 N02∘306741015840 E103∘213871015840 9749 plusmn 03 nd nd2 N02∘308021015840 E103∘210861015840 9768 plusmn 02 nd nd3 N02∘307831015840 E103∘211401015840 9593 plusmn 05 nd nd4 N02∘307841015840 E 103∘21021015840 9953 plusmn 07 nd ndAll data are expressed as mean plusmn SEMnd not detected
0
20
40
60
80
100
0 2 4 6 8 10 12 14 16
Enzy
me a
ctiv
ity (
)
Concentration of Hg (mgL)
Figure 2 in which the disulfide bridges were destroyed and thecasein became unfit to the active site of the enzymeInhibitionof proteolytic activity of Coriandrum sativum protease by Hgusing Coomassie brilliant blue assay Data is generated using thenonlinear regression analysis for one-phase bindingmodel using theGraphPad PRISM 5 Software All data are expressed asmeanplusmn SEM
in comparison with Microtox are that it is inexpensive andsuitable for the detection of heavy metals in field trials sinceit is based on colorimetric changes which do not requirephotometer Meanwhile Daphnia magna assays have broadrange of IC
50value and need a longer period of incubation
48 h compared to inhibitive assaywhich only requires 20min
0
20
40
60
80
100
0 05 1 15 2 25 3 35 4
Enzy
me a
ctiv
ity (
)
Concentration of Zn (mgL)
Figure 3 Inhibition of proteolytic activity of Coriandrum sativumprotease by Zn using Coomassie brilliant blue assay Data isgenerated using the nonlinear regression analysis for one-phasebinding model using the GraphPad PRISM 5 Software All data areexpressed as mean plusmn SEM
35 Limits of Detection (LOD) and Limit of Quantitation(LOQ) for Hg and Zn The LOD and LOQ values for Hgand Zn detected by coriander proteases are shown in Table 4LOD is the lowest concentration of heavy metal that caninhibit the enzyme activity which is at least three times thestandard deviation of the blank at 119910-intercept [20] The LODvalues of Hg and Zn are 0241 and 0228mgL respectively
6 The Scientific World Journal
Meanwhile LOQ indicates the minimal concentration ofheavymetal that can produce observable color changes to theBradford assay LOQ is ten times the value of the standarddeviation of themean blank value [20]The LOQvalues ofHgand Zn are 0802 and 0761mgL respectivelyThe LOQ valuefor Hg was lower and about the same for Zn compared tothe trypsin assay 135 and 061mgL respectively [11] Thusproteases from this plant have a promising potential to beused as an inhibitive assay for Hg and Zn
36 Field Trials Samples were collected from Prai andBukit Tengah Industrial Areas and Endau Rompin NationalPark The samples were analyzed using the inhibitive assayand the results were validated with ICP-OES Twelve outof 24 samples from Prai Industrial Area gave more than50 inhibition to the protease activity (Table 5) ICP-OESresults showed that the positive samples contain extremelyhigh concentration of Hg and Zn with some exceeding themaximum permissible limit (MPL) for Hg and Zn 0001and 5mgL respectively as outlined by the Department ofEnvironment (DOE) [21] LOQ value for Hg 0802mgL ismuch higher than the MPL for Hg in aquatic environmentas outlined by the DOE of Malaysia 0001mgL Howeverthis assay can be used in monitoring Hg levels in industrialsites since these areasmostly contain very high concentrationof Hg Previous studies carried out in the same area alsoreported that the area is highly polluted with heavy metals[8 10] indicating a continuous trend
Four out of 5 samples from Bukit Tengah Industrial Area(Table 6) gave positive inhibitory result on enzyme activitydue to the presence of Hg and Zn as determined by ICP-OESSince galvanizedmetal factories can be found in this area it issuspected that these types of industries are responsible for theelevatedHg andZn levelThus a fast and inexpensivemethodfor monitoring these heavy metals is urgently needed
Endau Rompin is the one of the largest national park inMalaysia with pristine water and a diverse mixture of floraand fauna Four distinct samples were taken from this areaand the results showed that therewas no significant inhibitoryeffect on enzyme activityThe results obtained from ICP-OESalso proved the absence of heavy metals (Table 7) indicatinga good correlation between inhibitive assay and instrumentalmethod
4 Conclusion
An assay for detectingHg and Zn using proteases from a localplant has been successfully developed The optimizationsof enzyme and substrate concentration pH temperatureand incubation time were performed using the Bradford-protease-casein system In field trials samples obtained frompolluted and nonpolluted sites showed promising resultsThefindings showed that Prai and Juru Industrial Areas are highlypolluted with Hg and Zn The protease has a broad pH rangeand high sensitivity towardsHg andZn suggesting robustnessand suitability for biomonitoring field works For futurestudies it is recommended that the proteases be purifiedto increase the sensitivity towards the heavy metals This
study provides fundamental information for the developmentof rapid sensitive and economic inhibitive assay for thebiomonitoring of heavy metals in the environment
Acknowledgments
The authors would like to thank Shamala Salvamani andZakiuddin Sahlani for their technical support This researchwas funded by The Special Cradle Research DevelopmentFund (CRDF) from the Malaysian Technology DevelopmentCorporation
References
[1] S K Chaerun N P D Pangesti K Toyota andW BWhitmanldquoChanges inmicrobial functional diversity and activity in paddysoils irrigated with industrial wastewaters in Bandung WestJava Province IndonesiardquoWater Air and Soil Pollution vol 217no 1ndash4 pp 491ndash502 2011
[2] R K Sahu S Katiyar J Tiwari and G C Kisku ldquoAssessment ofdrain water receiving effluent from tanneries and its impact onsoil and plants with particular emphasis on bioaccumulation ofheavy metalsrdquo Journal of Environmental Biology vol 28 no 3pp 685ndash690 2007
[3] J K Katnoria S Arora R Bhardwaj andANagpal ldquoEvaluationof genotoxic potential of industrial waste contaminated soilextracts of Amritsar Indiardquo Journal of Environmental Biologyvol 32 no 3 pp 363ndash367 2011
[4] S Knasmuller E GottmannH Steinkellner et al ldquoDetection ofgenotoxic effects of heavy metal contaminated soils with plantbioassaysrdquoMutation Research vol 420 no 1ndash3 pp 37ndash48 1998
[5] V A Kulkarni V S Naidu and T G Jagtap ldquoMarine ecologicalhabitat a case study on projected thermal power plant aroundDharamtar creek Indiardquo Journal of Environmental Biology vol32 no 2 pp 213ndash219 2011
[6] M Oliveira M A Santos and M Pacheco ldquoGlutathioneprotects heavy metal-induced inhibition of hepatic microsomalethoxyresorufin O-deethylase activity in Dicentrarchus labraxLrdquo Ecotoxicology and Environmental Safety vol 58 no 3 pp379ndash385 2004
[7] M F Frasco D Fournier F Carvalho and L GuilherminoldquoImplementation of assay conditions for the use ofAchE activityas a biomarker of metal toxicityrdquo Biomarkers vol 10 no 5 pp360ndash375 2005
[8] Y Shukor N A Baharom F A Rahman M P Abdullah NA Shamaan and M A Syed ldquoDevelopment of a heavy metalsenzymatic-based assay using papainrdquo Analytica Chimica Actavol 566 no 2 pp 283ndash289 2006
[9] M Alina A Azrina A S Mohd Yunus S Mohd Zakiuddin HMohd Izuan Effendi and R Muhammad Rizal ldquoHeavy metals(mercury arsenic cadmium plumbum) in selected marine fishand shellfish along the straits of malaccardquo International FoodResearch Journal vol 19 no 1 pp 135ndash140 2012
[10] M Y Shukor N Masdor N A Baharom et al ldquoAn inhibitivedetermination method for heavy metals using bromelain acysteine proteaserdquo Applied Biochemistry and Biotechnology vol144 no 3 pp 283ndash291 2008
[11] M Y Shukor N A Baharom N A Masdor et al ldquoThedevelopment of an inhibitive determination method for zincusing a serine proteaserdquo Journal of Environmental Biology vol30 no 1 pp 17ndash22 2009
The Scientific World Journal 7
[12] S HanM Zhu Z Yuan and X Li ldquoAmethylene blue-mediatedenzyme electrode for the determination of trace mercury(II)mercury(I) methylmercury and mercury-glutathione com-plexrdquo Biosensors and Bioelectronics vol 16 no 1-2 pp 9ndash162001
[13] K Jung G Bitton and B Koopman ldquoAssessment of urease inhi-bition assays for measuring toxicity of environmental samplesrdquoWater Research vol 29 no 8 pp 1929ndash1933 1995
[14] R M C Dawson D C Elliott W H Elliott and K M JonesData for Biochemical Research Clarendon Press Oxford UK1969
[15] A Bensadoun and D Weinstein ldquoAssay of proteins in thepresence of interfering materialsrdquo Analytical Biochemistry vol70 no 1 pp 241ndash250 1976
[16] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976
[17] G L Ellman K D Courtney V Andres Jr and R MFeatherstone ldquoA new and rapid colorimetric determination ofacetylcholinesterase activityrdquo Biochemical Pharmacology vol 7no 2 pp 88ndash95 1961
[18] H M Farrell Jr E D Wickham and M L Groves ldquoEnvi-ronmental influences on purified casein disulfide interactionsrdquoJournal of Dairy Science vol 81 no 11 pp 2974ndash2984 1998
[19] W Jiang X Zhou Y Zhao and P Liu ldquoIdentification ofa senescence-related protease in coriander leavesrdquo ChineseScience Bulletin vol 47 no 13 pp 1096ndash1099 2002
[20] D A Armbruster M D Tillman and L M Hubbs ldquoLimit ofdetection (LOD)limit of quantitation (LOQ) comparison ofthe empirical and the statistical methods exemplified with GC-MS assays of abused drugsrdquo Clinical Chemistry vol 40 no 7pp 1233ndash1238 1994
[21] DOE Environmental Quality Report Department of Environ-ment Ministry of Science Technology and the EnvironmentMalaysia 2001
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
The Scientific World Journal 3
Table 2 Summary of optimization results for Coriandrum sativum proteases in comparison to other proteases
Optimization parameter Optimum conditions for proteasesCoriandrum sativum Papaina Bromelainb Trypsinc
Enzyme concentration (mgmL)Substrate concentration (mgmL)Temperature (∘C)Incubation time (min)pH
04504253520
8ndash95
010130305ndash7
0102540305-6
010140305ndash7
aShukor et al [8]bShukor et al [10]cShukor et al [11]
Table 3 IC50 values of Coriandrum sativum proteases for Hg and Zn in comparison to other protease assays
IC50 (mgL)Heavymetals
Coriandrum sativumproteases Papaina Bromelainb Trypsinc Immobilized
Ureased 15min Microtoxd 48-hour Daphniamagnad
Hg 3217 024ndash062 013ndash016 1576ndash1704 033 0029ndash005 0005ndash021Zn 0727 211 mdash 48ndash67 146 027ndash29 054ndash51aShukor et al [8]bShukor et al [10]cShukor et al [11]dJung et al [13]
Table 4 LOD and LOQ values for the proteases from Coriandrumsativum
Metals Regression model 1198772 LOD (mgL) LOQ (mgL)
Hg One-phase binding 0966 0241 0802Zn One-phase binding 0984 0228 0650
3 Results and Discussion
31 Screening towards Plant Protease Identification of theplant source that gives the highest enzyme activity is verycrucial in this study since bioassay is only practical forenzyme that shows high enzymatic activity The significantcolor changes of the Bradford reagent are only observable ifthe protease has high activity Table 1 shows the percentageactivity between plant sources Commercialized enzymepapain (Sigma EC 34222 lot no 32K2619 St Louis USA)was used as a positive control and enzyme was replaced withdeionized water for the negative control
Among all plant samples Coriandrum sativum gave thehighest enzyme activity Although high enzyme activity doesnot correlate with the sensitivity of the enzyme towardsheavy metals this plant source was used since the activityis sufficiently high for inhibitive assay The inhibitive assay isonly valid if the difference in absorbance ismore than 02 aftera maximum incubation for 1 h [8] Coriandrum sativum hassenescence-related serinyl protease which is involved in thephysiological role and metabolic activities of the plant [19]
32 Optimized Conditions for Coriandrum sativum Pro-tease Activity The optimum concentration of Coriandrumsativum proteases was at 45120583gmLThe optimum concentra-tion was higher compared to previous optimization studies
on other proteases such as papain bromelain and trypsin asshown in Table 2The optimized concentration would ensurethat degradation was complete before any autodigestion ofproteases takes place Substrate casein gave optimum activityat 425120583gmL Higher concentration of the substrate is notdesired since it would take a longer time for the casein tobe digested The optimized temperature was at 35∘C lowercompared to the proteases such as bromelain and trypsin [1011] Thus it does not require extra heating process since it isalmost similar to the environmental temperature inMalaysiaThe highest enzyme activity was noted at incubation periodof 20min and at the pH range between 8 and 95 in sodiumcarbonate buffer The broad range of pH is favored forassaying environmental samples since it can bear deviationin samplesrsquo pH [11]
33 Heavy Metals Studies The Coriandrum sativum proteaseactivity was inhibited by two heavymetals mercury and zincat 1mgL as shown in Figure 1The other heavymetals did notshow significant inhibition towards the enzyme activity thusthe solution remained brown in color This is because Brad-ford reagent is unable to stain digested casein which is lessthan 2 kDa [10] Mercury and zinc were observed to exhibitdark blue color after the completion of the assay indicatingthat both heavymetals inhibit the activity of the proteaseThemechanism of metal inhibition on enzyme activity especiallyby mercury is through binding of the sulfhydryl groupsin cysteine proteases and through destruction of tertiarystructure in which the disulfide bridges were destroyed andthe casein became unfit to the active site of the enzyme [8]Therefore Bradford reagent stains the undigested casein andthe solution changed to dark blue in color The Coriandrumsativum proteases probably contain a mixture of serine [19]
4 The Scientific World Journal
Table 5 Protease inhibitive assay for samples from Prai Industrial Area
Sample GPS location Percentage activity () Concentration of heavy metals in samples (mgL) by ICP-OESZn Hg
1 N05∘20871015840 E100∘246921015840 9359 plusmn 01 004 plusmn 005 nd2 N05∘20871015840 E100∘246921015840 134 plusmn 03 12530 plusmn 033 nd3 N05∘208621015840 E100∘2467410158401015840 9854 plusmn 02 021 plusmn 012 nd4 N05∘208361015840 E100∘251771015840 2254 plusmn 05 763 plusmn 005 minus 9 nd5 N05∘202241015840 E100∘263021015840 9880 plusmn 07 006 plusmn 003 nd6 N05∘219831015840 E100∘240231015840 3880 plusmn 09 089 plusmn 001 nd7 N05∘219671015840 E100∘240441015840 9398 plusmn 01 014 plusmn 002 nd8 N05∘20871015840 E100∘246921015840 946 plusmn 01 1454 plusmn 013 nd9 N05∘20871015840 E100∘246921015840 2521 plusmn 08 397 plusmn 004 nd10 N05∘208621015840 E100∘246741015840 3366 plusmn 02 350 plusmn 002 nd11 N05∘196991015840 E100∘261291015840 1921 plusmn 03 874 plusmn 009 nd12 N05∘196991015840 E100∘261291015840 682 plusmn 03 1200 plusmn 002 152 plusmn 006
13 N05∘196991015840 E100∘261291015840 877 plusmn 08 1580 plusmn 013 nd14 N05∘202631015840 E100∘257741015840 9994 plusmn 09 002 plusmn 001 nd15 N05∘202631015840 E100∘257741015840 9939 plusmn 04 003 plusmn 003 nd16 N05∘201351015840 E100∘269251015840 339 plusmn 03 3865 plusmn 018 374 plusmn 009
17 N05∘211531015840 E100∘260731015840 9481 plusmn 05 002 plusmn 002 nd18 N05∘211531015840 E100∘260731015840 9742 plusmn 07 009 plusmn 001 nd19 N05∘211531015840 E100∘260731015840 9811 plusmn 01 002 plusmn 001 nd20 N05∘200911015840 E100∘252691015840 9746 plusmn 02 006 plusmn 002 nd21 N05∘211321015840 E100∘250811015840 9924 plusmn 02 071 plusmn 001 nd22 N05∘204721015840 E100∘268911015840 1605 plusmn 03 1084 plusmn 006 nd23 N05∘203871015840 E100∘244291015840 495 plusmn 04 005 plusmn 001 nd24 N05∘202631015840 E100∘257741015840 209 plusmn 07 1269 plusmn 003 414 plusmn 015
All data are expressed as mean plusmn SEMnd not detected
Table 6 Protease inhibitive assay for samples from Bukit Tengah Industrial Area
Sample GPS location Percentage activity () Concentration of heavy metals in samples (mgL) by ICP-OESZn Hg
1 N05∘204471015840 E100∘264031015840 639 plusmn 04 1865 plusmn 003 nd2 N05∘206651015840 E100∘263641015840 9684 plusmn 07 nd nd3 N05∘206011015840 E100∘264271015840 387 plusmn 05 1342 plusmn 001 nd4 N05∘206401015840 E100∘264701015840 3247 plusmn 09 nd 236 plusmn 001
5 N05∘189471015840 E100∘263481015840 4870 plusmn 10 nd 157 plusmn 002
All data are expressed as mean plusmn SEMnd not detected
and cysteine proteases Serine proteases are known to beinhibited by zinc [11] while cysteine proteases are stronglyinhibited by mercury [8] Therefore the inhibitive enzymeassay using Coriandrum sativum is a potential candidate forbiomonitoring of Hg and Zn in aquatic environments
34 Comparison of theHalfMaximal Inhibitory Concentration(IC50) Values of Proteases In terms of sensitivity the IC
50
values are often used as a comparison among established
assays [4]The comparison of IC50values of proteases studied
so far with other types of assays is summarized in Table 3 ForHg Coriandrum sativum proteases showed better sensitivitycompared to trypsin with the IC
50value of 3217mgL
(Figure 2) Meanwhile the IC50
value for Zn is 0727mgL(Figure 3) which is more sensitive than other assays likepapain bromelain trypsin and immobilized urease andwithin the range of Microtox andDaphnia magna assaysThelower IC
50value denotes the higher effectiveness of protease
in inhibiting heavy metalsThe advantages of inhibitive assay
The Scientific World Journal 5
Heavy metals
0
10
20
30
40
50
60
70
80
90
100
Enzy
me a
ctiv
ity (
)
Ag As Cd Co Cu Hg Ni Pb Zn ControlCr3+
Figure 1 The effect of heavy metals at 1mgL on Coriandrum sativum proteases activity All data are expressed as mean plusmn SEM
Table 7 Protease inhibitive assay for samples from Endau Rompin
Samples GPS location Percentage activity () Concentration of heavy metals in samples (mgL) by ICP-OESZn Hg
1 N02∘306741015840 E103∘213871015840 9749 plusmn 03 nd nd2 N02∘308021015840 E103∘210861015840 9768 plusmn 02 nd nd3 N02∘307831015840 E103∘211401015840 9593 plusmn 05 nd nd4 N02∘307841015840 E 103∘21021015840 9953 plusmn 07 nd ndAll data are expressed as mean plusmn SEMnd not detected
0
20
40
60
80
100
0 2 4 6 8 10 12 14 16
Enzy
me a
ctiv
ity (
)
Concentration of Hg (mgL)
Figure 2 in which the disulfide bridges were destroyed and thecasein became unfit to the active site of the enzymeInhibitionof proteolytic activity of Coriandrum sativum protease by Hgusing Coomassie brilliant blue assay Data is generated using thenonlinear regression analysis for one-phase bindingmodel using theGraphPad PRISM 5 Software All data are expressed asmeanplusmn SEM
in comparison with Microtox are that it is inexpensive andsuitable for the detection of heavy metals in field trials sinceit is based on colorimetric changes which do not requirephotometer Meanwhile Daphnia magna assays have broadrange of IC
50value and need a longer period of incubation
48 h compared to inhibitive assaywhich only requires 20min
0
20
40
60
80
100
0 05 1 15 2 25 3 35 4
Enzy
me a
ctiv
ity (
)
Concentration of Zn (mgL)
Figure 3 Inhibition of proteolytic activity of Coriandrum sativumprotease by Zn using Coomassie brilliant blue assay Data isgenerated using the nonlinear regression analysis for one-phasebinding model using the GraphPad PRISM 5 Software All data areexpressed as mean plusmn SEM
35 Limits of Detection (LOD) and Limit of Quantitation(LOQ) for Hg and Zn The LOD and LOQ values for Hgand Zn detected by coriander proteases are shown in Table 4LOD is the lowest concentration of heavy metal that caninhibit the enzyme activity which is at least three times thestandard deviation of the blank at 119910-intercept [20] The LODvalues of Hg and Zn are 0241 and 0228mgL respectively
6 The Scientific World Journal
Meanwhile LOQ indicates the minimal concentration ofheavymetal that can produce observable color changes to theBradford assay LOQ is ten times the value of the standarddeviation of themean blank value [20]The LOQvalues ofHgand Zn are 0802 and 0761mgL respectivelyThe LOQ valuefor Hg was lower and about the same for Zn compared tothe trypsin assay 135 and 061mgL respectively [11] Thusproteases from this plant have a promising potential to beused as an inhibitive assay for Hg and Zn
36 Field Trials Samples were collected from Prai andBukit Tengah Industrial Areas and Endau Rompin NationalPark The samples were analyzed using the inhibitive assayand the results were validated with ICP-OES Twelve outof 24 samples from Prai Industrial Area gave more than50 inhibition to the protease activity (Table 5) ICP-OESresults showed that the positive samples contain extremelyhigh concentration of Hg and Zn with some exceeding themaximum permissible limit (MPL) for Hg and Zn 0001and 5mgL respectively as outlined by the Department ofEnvironment (DOE) [21] LOQ value for Hg 0802mgL ismuch higher than the MPL for Hg in aquatic environmentas outlined by the DOE of Malaysia 0001mgL Howeverthis assay can be used in monitoring Hg levels in industrialsites since these areasmostly contain very high concentrationof Hg Previous studies carried out in the same area alsoreported that the area is highly polluted with heavy metals[8 10] indicating a continuous trend
Four out of 5 samples from Bukit Tengah Industrial Area(Table 6) gave positive inhibitory result on enzyme activitydue to the presence of Hg and Zn as determined by ICP-OESSince galvanizedmetal factories can be found in this area it issuspected that these types of industries are responsible for theelevatedHg andZn levelThus a fast and inexpensivemethodfor monitoring these heavy metals is urgently needed
Endau Rompin is the one of the largest national park inMalaysia with pristine water and a diverse mixture of floraand fauna Four distinct samples were taken from this areaand the results showed that therewas no significant inhibitoryeffect on enzyme activityThe results obtained from ICP-OESalso proved the absence of heavy metals (Table 7) indicatinga good correlation between inhibitive assay and instrumentalmethod
4 Conclusion
An assay for detectingHg and Zn using proteases from a localplant has been successfully developed The optimizationsof enzyme and substrate concentration pH temperatureand incubation time were performed using the Bradford-protease-casein system In field trials samples obtained frompolluted and nonpolluted sites showed promising resultsThefindings showed that Prai and Juru Industrial Areas are highlypolluted with Hg and Zn The protease has a broad pH rangeand high sensitivity towardsHg andZn suggesting robustnessand suitability for biomonitoring field works For futurestudies it is recommended that the proteases be purifiedto increase the sensitivity towards the heavy metals This
study provides fundamental information for the developmentof rapid sensitive and economic inhibitive assay for thebiomonitoring of heavy metals in the environment
Acknowledgments
The authors would like to thank Shamala Salvamani andZakiuddin Sahlani for their technical support This researchwas funded by The Special Cradle Research DevelopmentFund (CRDF) from the Malaysian Technology DevelopmentCorporation
References
[1] S K Chaerun N P D Pangesti K Toyota andW BWhitmanldquoChanges inmicrobial functional diversity and activity in paddysoils irrigated with industrial wastewaters in Bandung WestJava Province IndonesiardquoWater Air and Soil Pollution vol 217no 1ndash4 pp 491ndash502 2011
[2] R K Sahu S Katiyar J Tiwari and G C Kisku ldquoAssessment ofdrain water receiving effluent from tanneries and its impact onsoil and plants with particular emphasis on bioaccumulation ofheavy metalsrdquo Journal of Environmental Biology vol 28 no 3pp 685ndash690 2007
[3] J K Katnoria S Arora R Bhardwaj andANagpal ldquoEvaluationof genotoxic potential of industrial waste contaminated soilextracts of Amritsar Indiardquo Journal of Environmental Biologyvol 32 no 3 pp 363ndash367 2011
[4] S Knasmuller E GottmannH Steinkellner et al ldquoDetection ofgenotoxic effects of heavy metal contaminated soils with plantbioassaysrdquoMutation Research vol 420 no 1ndash3 pp 37ndash48 1998
[5] V A Kulkarni V S Naidu and T G Jagtap ldquoMarine ecologicalhabitat a case study on projected thermal power plant aroundDharamtar creek Indiardquo Journal of Environmental Biology vol32 no 2 pp 213ndash219 2011
[6] M Oliveira M A Santos and M Pacheco ldquoGlutathioneprotects heavy metal-induced inhibition of hepatic microsomalethoxyresorufin O-deethylase activity in Dicentrarchus labraxLrdquo Ecotoxicology and Environmental Safety vol 58 no 3 pp379ndash385 2004
[7] M F Frasco D Fournier F Carvalho and L GuilherminoldquoImplementation of assay conditions for the use ofAchE activityas a biomarker of metal toxicityrdquo Biomarkers vol 10 no 5 pp360ndash375 2005
[8] Y Shukor N A Baharom F A Rahman M P Abdullah NA Shamaan and M A Syed ldquoDevelopment of a heavy metalsenzymatic-based assay using papainrdquo Analytica Chimica Actavol 566 no 2 pp 283ndash289 2006
[9] M Alina A Azrina A S Mohd Yunus S Mohd Zakiuddin HMohd Izuan Effendi and R Muhammad Rizal ldquoHeavy metals(mercury arsenic cadmium plumbum) in selected marine fishand shellfish along the straits of malaccardquo International FoodResearch Journal vol 19 no 1 pp 135ndash140 2012
[10] M Y Shukor N Masdor N A Baharom et al ldquoAn inhibitivedetermination method for heavy metals using bromelain acysteine proteaserdquo Applied Biochemistry and Biotechnology vol144 no 3 pp 283ndash291 2008
[11] M Y Shukor N A Baharom N A Masdor et al ldquoThedevelopment of an inhibitive determination method for zincusing a serine proteaserdquo Journal of Environmental Biology vol30 no 1 pp 17ndash22 2009
The Scientific World Journal 7
[12] S HanM Zhu Z Yuan and X Li ldquoAmethylene blue-mediatedenzyme electrode for the determination of trace mercury(II)mercury(I) methylmercury and mercury-glutathione com-plexrdquo Biosensors and Bioelectronics vol 16 no 1-2 pp 9ndash162001
[13] K Jung G Bitton and B Koopman ldquoAssessment of urease inhi-bition assays for measuring toxicity of environmental samplesrdquoWater Research vol 29 no 8 pp 1929ndash1933 1995
[14] R M C Dawson D C Elliott W H Elliott and K M JonesData for Biochemical Research Clarendon Press Oxford UK1969
[15] A Bensadoun and D Weinstein ldquoAssay of proteins in thepresence of interfering materialsrdquo Analytical Biochemistry vol70 no 1 pp 241ndash250 1976
[16] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976
[17] G L Ellman K D Courtney V Andres Jr and R MFeatherstone ldquoA new and rapid colorimetric determination ofacetylcholinesterase activityrdquo Biochemical Pharmacology vol 7no 2 pp 88ndash95 1961
[18] H M Farrell Jr E D Wickham and M L Groves ldquoEnvi-ronmental influences on purified casein disulfide interactionsrdquoJournal of Dairy Science vol 81 no 11 pp 2974ndash2984 1998
[19] W Jiang X Zhou Y Zhao and P Liu ldquoIdentification ofa senescence-related protease in coriander leavesrdquo ChineseScience Bulletin vol 47 no 13 pp 1096ndash1099 2002
[20] D A Armbruster M D Tillman and L M Hubbs ldquoLimit ofdetection (LOD)limit of quantitation (LOQ) comparison ofthe empirical and the statistical methods exemplified with GC-MS assays of abused drugsrdquo Clinical Chemistry vol 40 no 7pp 1233ndash1238 1994
[21] DOE Environmental Quality Report Department of Environ-ment Ministry of Science Technology and the EnvironmentMalaysia 2001
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
4 The Scientific World Journal
Table 5 Protease inhibitive assay for samples from Prai Industrial Area
Sample GPS location Percentage activity () Concentration of heavy metals in samples (mgL) by ICP-OESZn Hg
1 N05∘20871015840 E100∘246921015840 9359 plusmn 01 004 plusmn 005 nd2 N05∘20871015840 E100∘246921015840 134 plusmn 03 12530 plusmn 033 nd3 N05∘208621015840 E100∘2467410158401015840 9854 plusmn 02 021 plusmn 012 nd4 N05∘208361015840 E100∘251771015840 2254 plusmn 05 763 plusmn 005 minus 9 nd5 N05∘202241015840 E100∘263021015840 9880 plusmn 07 006 plusmn 003 nd6 N05∘219831015840 E100∘240231015840 3880 plusmn 09 089 plusmn 001 nd7 N05∘219671015840 E100∘240441015840 9398 plusmn 01 014 plusmn 002 nd8 N05∘20871015840 E100∘246921015840 946 plusmn 01 1454 plusmn 013 nd9 N05∘20871015840 E100∘246921015840 2521 plusmn 08 397 plusmn 004 nd10 N05∘208621015840 E100∘246741015840 3366 plusmn 02 350 plusmn 002 nd11 N05∘196991015840 E100∘261291015840 1921 plusmn 03 874 plusmn 009 nd12 N05∘196991015840 E100∘261291015840 682 plusmn 03 1200 plusmn 002 152 plusmn 006
13 N05∘196991015840 E100∘261291015840 877 plusmn 08 1580 plusmn 013 nd14 N05∘202631015840 E100∘257741015840 9994 plusmn 09 002 plusmn 001 nd15 N05∘202631015840 E100∘257741015840 9939 plusmn 04 003 plusmn 003 nd16 N05∘201351015840 E100∘269251015840 339 plusmn 03 3865 plusmn 018 374 plusmn 009
17 N05∘211531015840 E100∘260731015840 9481 plusmn 05 002 plusmn 002 nd18 N05∘211531015840 E100∘260731015840 9742 plusmn 07 009 plusmn 001 nd19 N05∘211531015840 E100∘260731015840 9811 plusmn 01 002 plusmn 001 nd20 N05∘200911015840 E100∘252691015840 9746 plusmn 02 006 plusmn 002 nd21 N05∘211321015840 E100∘250811015840 9924 plusmn 02 071 plusmn 001 nd22 N05∘204721015840 E100∘268911015840 1605 plusmn 03 1084 plusmn 006 nd23 N05∘203871015840 E100∘244291015840 495 plusmn 04 005 plusmn 001 nd24 N05∘202631015840 E100∘257741015840 209 plusmn 07 1269 plusmn 003 414 plusmn 015
All data are expressed as mean plusmn SEMnd not detected
Table 6 Protease inhibitive assay for samples from Bukit Tengah Industrial Area
Sample GPS location Percentage activity () Concentration of heavy metals in samples (mgL) by ICP-OESZn Hg
1 N05∘204471015840 E100∘264031015840 639 plusmn 04 1865 plusmn 003 nd2 N05∘206651015840 E100∘263641015840 9684 plusmn 07 nd nd3 N05∘206011015840 E100∘264271015840 387 plusmn 05 1342 plusmn 001 nd4 N05∘206401015840 E100∘264701015840 3247 plusmn 09 nd 236 plusmn 001
5 N05∘189471015840 E100∘263481015840 4870 plusmn 10 nd 157 plusmn 002
All data are expressed as mean plusmn SEMnd not detected
and cysteine proteases Serine proteases are known to beinhibited by zinc [11] while cysteine proteases are stronglyinhibited by mercury [8] Therefore the inhibitive enzymeassay using Coriandrum sativum is a potential candidate forbiomonitoring of Hg and Zn in aquatic environments
34 Comparison of theHalfMaximal Inhibitory Concentration(IC50) Values of Proteases In terms of sensitivity the IC
50
values are often used as a comparison among established
assays [4]The comparison of IC50values of proteases studied
so far with other types of assays is summarized in Table 3 ForHg Coriandrum sativum proteases showed better sensitivitycompared to trypsin with the IC
50value of 3217mgL
(Figure 2) Meanwhile the IC50
value for Zn is 0727mgL(Figure 3) which is more sensitive than other assays likepapain bromelain trypsin and immobilized urease andwithin the range of Microtox andDaphnia magna assaysThelower IC
50value denotes the higher effectiveness of protease
in inhibiting heavy metalsThe advantages of inhibitive assay
The Scientific World Journal 5
Heavy metals
0
10
20
30
40
50
60
70
80
90
100
Enzy
me a
ctiv
ity (
)
Ag As Cd Co Cu Hg Ni Pb Zn ControlCr3+
Figure 1 The effect of heavy metals at 1mgL on Coriandrum sativum proteases activity All data are expressed as mean plusmn SEM
Table 7 Protease inhibitive assay for samples from Endau Rompin
Samples GPS location Percentage activity () Concentration of heavy metals in samples (mgL) by ICP-OESZn Hg
1 N02∘306741015840 E103∘213871015840 9749 plusmn 03 nd nd2 N02∘308021015840 E103∘210861015840 9768 plusmn 02 nd nd3 N02∘307831015840 E103∘211401015840 9593 plusmn 05 nd nd4 N02∘307841015840 E 103∘21021015840 9953 plusmn 07 nd ndAll data are expressed as mean plusmn SEMnd not detected
0
20
40
60
80
100
0 2 4 6 8 10 12 14 16
Enzy
me a
ctiv
ity (
)
Concentration of Hg (mgL)
Figure 2 in which the disulfide bridges were destroyed and thecasein became unfit to the active site of the enzymeInhibitionof proteolytic activity of Coriandrum sativum protease by Hgusing Coomassie brilliant blue assay Data is generated using thenonlinear regression analysis for one-phase bindingmodel using theGraphPad PRISM 5 Software All data are expressed asmeanplusmn SEM
in comparison with Microtox are that it is inexpensive andsuitable for the detection of heavy metals in field trials sinceit is based on colorimetric changes which do not requirephotometer Meanwhile Daphnia magna assays have broadrange of IC
50value and need a longer period of incubation
48 h compared to inhibitive assaywhich only requires 20min
0
20
40
60
80
100
0 05 1 15 2 25 3 35 4
Enzy
me a
ctiv
ity (
)
Concentration of Zn (mgL)
Figure 3 Inhibition of proteolytic activity of Coriandrum sativumprotease by Zn using Coomassie brilliant blue assay Data isgenerated using the nonlinear regression analysis for one-phasebinding model using the GraphPad PRISM 5 Software All data areexpressed as mean plusmn SEM
35 Limits of Detection (LOD) and Limit of Quantitation(LOQ) for Hg and Zn The LOD and LOQ values for Hgand Zn detected by coriander proteases are shown in Table 4LOD is the lowest concentration of heavy metal that caninhibit the enzyme activity which is at least three times thestandard deviation of the blank at 119910-intercept [20] The LODvalues of Hg and Zn are 0241 and 0228mgL respectively
6 The Scientific World Journal
Meanwhile LOQ indicates the minimal concentration ofheavymetal that can produce observable color changes to theBradford assay LOQ is ten times the value of the standarddeviation of themean blank value [20]The LOQvalues ofHgand Zn are 0802 and 0761mgL respectivelyThe LOQ valuefor Hg was lower and about the same for Zn compared tothe trypsin assay 135 and 061mgL respectively [11] Thusproteases from this plant have a promising potential to beused as an inhibitive assay for Hg and Zn
36 Field Trials Samples were collected from Prai andBukit Tengah Industrial Areas and Endau Rompin NationalPark The samples were analyzed using the inhibitive assayand the results were validated with ICP-OES Twelve outof 24 samples from Prai Industrial Area gave more than50 inhibition to the protease activity (Table 5) ICP-OESresults showed that the positive samples contain extremelyhigh concentration of Hg and Zn with some exceeding themaximum permissible limit (MPL) for Hg and Zn 0001and 5mgL respectively as outlined by the Department ofEnvironment (DOE) [21] LOQ value for Hg 0802mgL ismuch higher than the MPL for Hg in aquatic environmentas outlined by the DOE of Malaysia 0001mgL Howeverthis assay can be used in monitoring Hg levels in industrialsites since these areasmostly contain very high concentrationof Hg Previous studies carried out in the same area alsoreported that the area is highly polluted with heavy metals[8 10] indicating a continuous trend
Four out of 5 samples from Bukit Tengah Industrial Area(Table 6) gave positive inhibitory result on enzyme activitydue to the presence of Hg and Zn as determined by ICP-OESSince galvanizedmetal factories can be found in this area it issuspected that these types of industries are responsible for theelevatedHg andZn levelThus a fast and inexpensivemethodfor monitoring these heavy metals is urgently needed
Endau Rompin is the one of the largest national park inMalaysia with pristine water and a diverse mixture of floraand fauna Four distinct samples were taken from this areaand the results showed that therewas no significant inhibitoryeffect on enzyme activityThe results obtained from ICP-OESalso proved the absence of heavy metals (Table 7) indicatinga good correlation between inhibitive assay and instrumentalmethod
4 Conclusion
An assay for detectingHg and Zn using proteases from a localplant has been successfully developed The optimizationsof enzyme and substrate concentration pH temperatureand incubation time were performed using the Bradford-protease-casein system In field trials samples obtained frompolluted and nonpolluted sites showed promising resultsThefindings showed that Prai and Juru Industrial Areas are highlypolluted with Hg and Zn The protease has a broad pH rangeand high sensitivity towardsHg andZn suggesting robustnessand suitability for biomonitoring field works For futurestudies it is recommended that the proteases be purifiedto increase the sensitivity towards the heavy metals This
study provides fundamental information for the developmentof rapid sensitive and economic inhibitive assay for thebiomonitoring of heavy metals in the environment
Acknowledgments
The authors would like to thank Shamala Salvamani andZakiuddin Sahlani for their technical support This researchwas funded by The Special Cradle Research DevelopmentFund (CRDF) from the Malaysian Technology DevelopmentCorporation
References
[1] S K Chaerun N P D Pangesti K Toyota andW BWhitmanldquoChanges inmicrobial functional diversity and activity in paddysoils irrigated with industrial wastewaters in Bandung WestJava Province IndonesiardquoWater Air and Soil Pollution vol 217no 1ndash4 pp 491ndash502 2011
[2] R K Sahu S Katiyar J Tiwari and G C Kisku ldquoAssessment ofdrain water receiving effluent from tanneries and its impact onsoil and plants with particular emphasis on bioaccumulation ofheavy metalsrdquo Journal of Environmental Biology vol 28 no 3pp 685ndash690 2007
[3] J K Katnoria S Arora R Bhardwaj andANagpal ldquoEvaluationof genotoxic potential of industrial waste contaminated soilextracts of Amritsar Indiardquo Journal of Environmental Biologyvol 32 no 3 pp 363ndash367 2011
[4] S Knasmuller E GottmannH Steinkellner et al ldquoDetection ofgenotoxic effects of heavy metal contaminated soils with plantbioassaysrdquoMutation Research vol 420 no 1ndash3 pp 37ndash48 1998
[5] V A Kulkarni V S Naidu and T G Jagtap ldquoMarine ecologicalhabitat a case study on projected thermal power plant aroundDharamtar creek Indiardquo Journal of Environmental Biology vol32 no 2 pp 213ndash219 2011
[6] M Oliveira M A Santos and M Pacheco ldquoGlutathioneprotects heavy metal-induced inhibition of hepatic microsomalethoxyresorufin O-deethylase activity in Dicentrarchus labraxLrdquo Ecotoxicology and Environmental Safety vol 58 no 3 pp379ndash385 2004
[7] M F Frasco D Fournier F Carvalho and L GuilherminoldquoImplementation of assay conditions for the use ofAchE activityas a biomarker of metal toxicityrdquo Biomarkers vol 10 no 5 pp360ndash375 2005
[8] Y Shukor N A Baharom F A Rahman M P Abdullah NA Shamaan and M A Syed ldquoDevelopment of a heavy metalsenzymatic-based assay using papainrdquo Analytica Chimica Actavol 566 no 2 pp 283ndash289 2006
[9] M Alina A Azrina A S Mohd Yunus S Mohd Zakiuddin HMohd Izuan Effendi and R Muhammad Rizal ldquoHeavy metals(mercury arsenic cadmium plumbum) in selected marine fishand shellfish along the straits of malaccardquo International FoodResearch Journal vol 19 no 1 pp 135ndash140 2012
[10] M Y Shukor N Masdor N A Baharom et al ldquoAn inhibitivedetermination method for heavy metals using bromelain acysteine proteaserdquo Applied Biochemistry and Biotechnology vol144 no 3 pp 283ndash291 2008
[11] M Y Shukor N A Baharom N A Masdor et al ldquoThedevelopment of an inhibitive determination method for zincusing a serine proteaserdquo Journal of Environmental Biology vol30 no 1 pp 17ndash22 2009
The Scientific World Journal 7
[12] S HanM Zhu Z Yuan and X Li ldquoAmethylene blue-mediatedenzyme electrode for the determination of trace mercury(II)mercury(I) methylmercury and mercury-glutathione com-plexrdquo Biosensors and Bioelectronics vol 16 no 1-2 pp 9ndash162001
[13] K Jung G Bitton and B Koopman ldquoAssessment of urease inhi-bition assays for measuring toxicity of environmental samplesrdquoWater Research vol 29 no 8 pp 1929ndash1933 1995
[14] R M C Dawson D C Elliott W H Elliott and K M JonesData for Biochemical Research Clarendon Press Oxford UK1969
[15] A Bensadoun and D Weinstein ldquoAssay of proteins in thepresence of interfering materialsrdquo Analytical Biochemistry vol70 no 1 pp 241ndash250 1976
[16] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976
[17] G L Ellman K D Courtney V Andres Jr and R MFeatherstone ldquoA new and rapid colorimetric determination ofacetylcholinesterase activityrdquo Biochemical Pharmacology vol 7no 2 pp 88ndash95 1961
[18] H M Farrell Jr E D Wickham and M L Groves ldquoEnvi-ronmental influences on purified casein disulfide interactionsrdquoJournal of Dairy Science vol 81 no 11 pp 2974ndash2984 1998
[19] W Jiang X Zhou Y Zhao and P Liu ldquoIdentification ofa senescence-related protease in coriander leavesrdquo ChineseScience Bulletin vol 47 no 13 pp 1096ndash1099 2002
[20] D A Armbruster M D Tillman and L M Hubbs ldquoLimit ofdetection (LOD)limit of quantitation (LOQ) comparison ofthe empirical and the statistical methods exemplified with GC-MS assays of abused drugsrdquo Clinical Chemistry vol 40 no 7pp 1233ndash1238 1994
[21] DOE Environmental Quality Report Department of Environ-ment Ministry of Science Technology and the EnvironmentMalaysia 2001
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
The Scientific World Journal 5
Heavy metals
0
10
20
30
40
50
60
70
80
90
100
Enzy
me a
ctiv
ity (
)
Ag As Cd Co Cu Hg Ni Pb Zn ControlCr3+
Figure 1 The effect of heavy metals at 1mgL on Coriandrum sativum proteases activity All data are expressed as mean plusmn SEM
Table 7 Protease inhibitive assay for samples from Endau Rompin
Samples GPS location Percentage activity () Concentration of heavy metals in samples (mgL) by ICP-OESZn Hg
1 N02∘306741015840 E103∘213871015840 9749 plusmn 03 nd nd2 N02∘308021015840 E103∘210861015840 9768 plusmn 02 nd nd3 N02∘307831015840 E103∘211401015840 9593 plusmn 05 nd nd4 N02∘307841015840 E 103∘21021015840 9953 plusmn 07 nd ndAll data are expressed as mean plusmn SEMnd not detected
0
20
40
60
80
100
0 2 4 6 8 10 12 14 16
Enzy
me a
ctiv
ity (
)
Concentration of Hg (mgL)
Figure 2 in which the disulfide bridges were destroyed and thecasein became unfit to the active site of the enzymeInhibitionof proteolytic activity of Coriandrum sativum protease by Hgusing Coomassie brilliant blue assay Data is generated using thenonlinear regression analysis for one-phase bindingmodel using theGraphPad PRISM 5 Software All data are expressed asmeanplusmn SEM
in comparison with Microtox are that it is inexpensive andsuitable for the detection of heavy metals in field trials sinceit is based on colorimetric changes which do not requirephotometer Meanwhile Daphnia magna assays have broadrange of IC
50value and need a longer period of incubation
48 h compared to inhibitive assaywhich only requires 20min
0
20
40
60
80
100
0 05 1 15 2 25 3 35 4
Enzy
me a
ctiv
ity (
)
Concentration of Zn (mgL)
Figure 3 Inhibition of proteolytic activity of Coriandrum sativumprotease by Zn using Coomassie brilliant blue assay Data isgenerated using the nonlinear regression analysis for one-phasebinding model using the GraphPad PRISM 5 Software All data areexpressed as mean plusmn SEM
35 Limits of Detection (LOD) and Limit of Quantitation(LOQ) for Hg and Zn The LOD and LOQ values for Hgand Zn detected by coriander proteases are shown in Table 4LOD is the lowest concentration of heavy metal that caninhibit the enzyme activity which is at least three times thestandard deviation of the blank at 119910-intercept [20] The LODvalues of Hg and Zn are 0241 and 0228mgL respectively
6 The Scientific World Journal
Meanwhile LOQ indicates the minimal concentration ofheavymetal that can produce observable color changes to theBradford assay LOQ is ten times the value of the standarddeviation of themean blank value [20]The LOQvalues ofHgand Zn are 0802 and 0761mgL respectivelyThe LOQ valuefor Hg was lower and about the same for Zn compared tothe trypsin assay 135 and 061mgL respectively [11] Thusproteases from this plant have a promising potential to beused as an inhibitive assay for Hg and Zn
36 Field Trials Samples were collected from Prai andBukit Tengah Industrial Areas and Endau Rompin NationalPark The samples were analyzed using the inhibitive assayand the results were validated with ICP-OES Twelve outof 24 samples from Prai Industrial Area gave more than50 inhibition to the protease activity (Table 5) ICP-OESresults showed that the positive samples contain extremelyhigh concentration of Hg and Zn with some exceeding themaximum permissible limit (MPL) for Hg and Zn 0001and 5mgL respectively as outlined by the Department ofEnvironment (DOE) [21] LOQ value for Hg 0802mgL ismuch higher than the MPL for Hg in aquatic environmentas outlined by the DOE of Malaysia 0001mgL Howeverthis assay can be used in monitoring Hg levels in industrialsites since these areasmostly contain very high concentrationof Hg Previous studies carried out in the same area alsoreported that the area is highly polluted with heavy metals[8 10] indicating a continuous trend
Four out of 5 samples from Bukit Tengah Industrial Area(Table 6) gave positive inhibitory result on enzyme activitydue to the presence of Hg and Zn as determined by ICP-OESSince galvanizedmetal factories can be found in this area it issuspected that these types of industries are responsible for theelevatedHg andZn levelThus a fast and inexpensivemethodfor monitoring these heavy metals is urgently needed
Endau Rompin is the one of the largest national park inMalaysia with pristine water and a diverse mixture of floraand fauna Four distinct samples were taken from this areaand the results showed that therewas no significant inhibitoryeffect on enzyme activityThe results obtained from ICP-OESalso proved the absence of heavy metals (Table 7) indicatinga good correlation between inhibitive assay and instrumentalmethod
4 Conclusion
An assay for detectingHg and Zn using proteases from a localplant has been successfully developed The optimizationsof enzyme and substrate concentration pH temperatureand incubation time were performed using the Bradford-protease-casein system In field trials samples obtained frompolluted and nonpolluted sites showed promising resultsThefindings showed that Prai and Juru Industrial Areas are highlypolluted with Hg and Zn The protease has a broad pH rangeand high sensitivity towardsHg andZn suggesting robustnessand suitability for biomonitoring field works For futurestudies it is recommended that the proteases be purifiedto increase the sensitivity towards the heavy metals This
study provides fundamental information for the developmentof rapid sensitive and economic inhibitive assay for thebiomonitoring of heavy metals in the environment
Acknowledgments
The authors would like to thank Shamala Salvamani andZakiuddin Sahlani for their technical support This researchwas funded by The Special Cradle Research DevelopmentFund (CRDF) from the Malaysian Technology DevelopmentCorporation
References
[1] S K Chaerun N P D Pangesti K Toyota andW BWhitmanldquoChanges inmicrobial functional diversity and activity in paddysoils irrigated with industrial wastewaters in Bandung WestJava Province IndonesiardquoWater Air and Soil Pollution vol 217no 1ndash4 pp 491ndash502 2011
[2] R K Sahu S Katiyar J Tiwari and G C Kisku ldquoAssessment ofdrain water receiving effluent from tanneries and its impact onsoil and plants with particular emphasis on bioaccumulation ofheavy metalsrdquo Journal of Environmental Biology vol 28 no 3pp 685ndash690 2007
[3] J K Katnoria S Arora R Bhardwaj andANagpal ldquoEvaluationof genotoxic potential of industrial waste contaminated soilextracts of Amritsar Indiardquo Journal of Environmental Biologyvol 32 no 3 pp 363ndash367 2011
[4] S Knasmuller E GottmannH Steinkellner et al ldquoDetection ofgenotoxic effects of heavy metal contaminated soils with plantbioassaysrdquoMutation Research vol 420 no 1ndash3 pp 37ndash48 1998
[5] V A Kulkarni V S Naidu and T G Jagtap ldquoMarine ecologicalhabitat a case study on projected thermal power plant aroundDharamtar creek Indiardquo Journal of Environmental Biology vol32 no 2 pp 213ndash219 2011
[6] M Oliveira M A Santos and M Pacheco ldquoGlutathioneprotects heavy metal-induced inhibition of hepatic microsomalethoxyresorufin O-deethylase activity in Dicentrarchus labraxLrdquo Ecotoxicology and Environmental Safety vol 58 no 3 pp379ndash385 2004
[7] M F Frasco D Fournier F Carvalho and L GuilherminoldquoImplementation of assay conditions for the use ofAchE activityas a biomarker of metal toxicityrdquo Biomarkers vol 10 no 5 pp360ndash375 2005
[8] Y Shukor N A Baharom F A Rahman M P Abdullah NA Shamaan and M A Syed ldquoDevelopment of a heavy metalsenzymatic-based assay using papainrdquo Analytica Chimica Actavol 566 no 2 pp 283ndash289 2006
[9] M Alina A Azrina A S Mohd Yunus S Mohd Zakiuddin HMohd Izuan Effendi and R Muhammad Rizal ldquoHeavy metals(mercury arsenic cadmium plumbum) in selected marine fishand shellfish along the straits of malaccardquo International FoodResearch Journal vol 19 no 1 pp 135ndash140 2012
[10] M Y Shukor N Masdor N A Baharom et al ldquoAn inhibitivedetermination method for heavy metals using bromelain acysteine proteaserdquo Applied Biochemistry and Biotechnology vol144 no 3 pp 283ndash291 2008
[11] M Y Shukor N A Baharom N A Masdor et al ldquoThedevelopment of an inhibitive determination method for zincusing a serine proteaserdquo Journal of Environmental Biology vol30 no 1 pp 17ndash22 2009
The Scientific World Journal 7
[12] S HanM Zhu Z Yuan and X Li ldquoAmethylene blue-mediatedenzyme electrode for the determination of trace mercury(II)mercury(I) methylmercury and mercury-glutathione com-plexrdquo Biosensors and Bioelectronics vol 16 no 1-2 pp 9ndash162001
[13] K Jung G Bitton and B Koopman ldquoAssessment of urease inhi-bition assays for measuring toxicity of environmental samplesrdquoWater Research vol 29 no 8 pp 1929ndash1933 1995
[14] R M C Dawson D C Elliott W H Elliott and K M JonesData for Biochemical Research Clarendon Press Oxford UK1969
[15] A Bensadoun and D Weinstein ldquoAssay of proteins in thepresence of interfering materialsrdquo Analytical Biochemistry vol70 no 1 pp 241ndash250 1976
[16] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976
[17] G L Ellman K D Courtney V Andres Jr and R MFeatherstone ldquoA new and rapid colorimetric determination ofacetylcholinesterase activityrdquo Biochemical Pharmacology vol 7no 2 pp 88ndash95 1961
[18] H M Farrell Jr E D Wickham and M L Groves ldquoEnvi-ronmental influences on purified casein disulfide interactionsrdquoJournal of Dairy Science vol 81 no 11 pp 2974ndash2984 1998
[19] W Jiang X Zhou Y Zhao and P Liu ldquoIdentification ofa senescence-related protease in coriander leavesrdquo ChineseScience Bulletin vol 47 no 13 pp 1096ndash1099 2002
[20] D A Armbruster M D Tillman and L M Hubbs ldquoLimit ofdetection (LOD)limit of quantitation (LOQ) comparison ofthe empirical and the statistical methods exemplified with GC-MS assays of abused drugsrdquo Clinical Chemistry vol 40 no 7pp 1233ndash1238 1994
[21] DOE Environmental Quality Report Department of Environ-ment Ministry of Science Technology and the EnvironmentMalaysia 2001
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
6 The Scientific World Journal
Meanwhile LOQ indicates the minimal concentration ofheavymetal that can produce observable color changes to theBradford assay LOQ is ten times the value of the standarddeviation of themean blank value [20]The LOQvalues ofHgand Zn are 0802 and 0761mgL respectivelyThe LOQ valuefor Hg was lower and about the same for Zn compared tothe trypsin assay 135 and 061mgL respectively [11] Thusproteases from this plant have a promising potential to beused as an inhibitive assay for Hg and Zn
36 Field Trials Samples were collected from Prai andBukit Tengah Industrial Areas and Endau Rompin NationalPark The samples were analyzed using the inhibitive assayand the results were validated with ICP-OES Twelve outof 24 samples from Prai Industrial Area gave more than50 inhibition to the protease activity (Table 5) ICP-OESresults showed that the positive samples contain extremelyhigh concentration of Hg and Zn with some exceeding themaximum permissible limit (MPL) for Hg and Zn 0001and 5mgL respectively as outlined by the Department ofEnvironment (DOE) [21] LOQ value for Hg 0802mgL ismuch higher than the MPL for Hg in aquatic environmentas outlined by the DOE of Malaysia 0001mgL Howeverthis assay can be used in monitoring Hg levels in industrialsites since these areasmostly contain very high concentrationof Hg Previous studies carried out in the same area alsoreported that the area is highly polluted with heavy metals[8 10] indicating a continuous trend
Four out of 5 samples from Bukit Tengah Industrial Area(Table 6) gave positive inhibitory result on enzyme activitydue to the presence of Hg and Zn as determined by ICP-OESSince galvanizedmetal factories can be found in this area it issuspected that these types of industries are responsible for theelevatedHg andZn levelThus a fast and inexpensivemethodfor monitoring these heavy metals is urgently needed
Endau Rompin is the one of the largest national park inMalaysia with pristine water and a diverse mixture of floraand fauna Four distinct samples were taken from this areaand the results showed that therewas no significant inhibitoryeffect on enzyme activityThe results obtained from ICP-OESalso proved the absence of heavy metals (Table 7) indicatinga good correlation between inhibitive assay and instrumentalmethod
4 Conclusion
An assay for detectingHg and Zn using proteases from a localplant has been successfully developed The optimizationsof enzyme and substrate concentration pH temperatureand incubation time were performed using the Bradford-protease-casein system In field trials samples obtained frompolluted and nonpolluted sites showed promising resultsThefindings showed that Prai and Juru Industrial Areas are highlypolluted with Hg and Zn The protease has a broad pH rangeand high sensitivity towardsHg andZn suggesting robustnessand suitability for biomonitoring field works For futurestudies it is recommended that the proteases be purifiedto increase the sensitivity towards the heavy metals This
study provides fundamental information for the developmentof rapid sensitive and economic inhibitive assay for thebiomonitoring of heavy metals in the environment
Acknowledgments
The authors would like to thank Shamala Salvamani andZakiuddin Sahlani for their technical support This researchwas funded by The Special Cradle Research DevelopmentFund (CRDF) from the Malaysian Technology DevelopmentCorporation
References
[1] S K Chaerun N P D Pangesti K Toyota andW BWhitmanldquoChanges inmicrobial functional diversity and activity in paddysoils irrigated with industrial wastewaters in Bandung WestJava Province IndonesiardquoWater Air and Soil Pollution vol 217no 1ndash4 pp 491ndash502 2011
[2] R K Sahu S Katiyar J Tiwari and G C Kisku ldquoAssessment ofdrain water receiving effluent from tanneries and its impact onsoil and plants with particular emphasis on bioaccumulation ofheavy metalsrdquo Journal of Environmental Biology vol 28 no 3pp 685ndash690 2007
[3] J K Katnoria S Arora R Bhardwaj andANagpal ldquoEvaluationof genotoxic potential of industrial waste contaminated soilextracts of Amritsar Indiardquo Journal of Environmental Biologyvol 32 no 3 pp 363ndash367 2011
[4] S Knasmuller E GottmannH Steinkellner et al ldquoDetection ofgenotoxic effects of heavy metal contaminated soils with plantbioassaysrdquoMutation Research vol 420 no 1ndash3 pp 37ndash48 1998
[5] V A Kulkarni V S Naidu and T G Jagtap ldquoMarine ecologicalhabitat a case study on projected thermal power plant aroundDharamtar creek Indiardquo Journal of Environmental Biology vol32 no 2 pp 213ndash219 2011
[6] M Oliveira M A Santos and M Pacheco ldquoGlutathioneprotects heavy metal-induced inhibition of hepatic microsomalethoxyresorufin O-deethylase activity in Dicentrarchus labraxLrdquo Ecotoxicology and Environmental Safety vol 58 no 3 pp379ndash385 2004
[7] M F Frasco D Fournier F Carvalho and L GuilherminoldquoImplementation of assay conditions for the use ofAchE activityas a biomarker of metal toxicityrdquo Biomarkers vol 10 no 5 pp360ndash375 2005
[8] Y Shukor N A Baharom F A Rahman M P Abdullah NA Shamaan and M A Syed ldquoDevelopment of a heavy metalsenzymatic-based assay using papainrdquo Analytica Chimica Actavol 566 no 2 pp 283ndash289 2006
[9] M Alina A Azrina A S Mohd Yunus S Mohd Zakiuddin HMohd Izuan Effendi and R Muhammad Rizal ldquoHeavy metals(mercury arsenic cadmium plumbum) in selected marine fishand shellfish along the straits of malaccardquo International FoodResearch Journal vol 19 no 1 pp 135ndash140 2012
[10] M Y Shukor N Masdor N A Baharom et al ldquoAn inhibitivedetermination method for heavy metals using bromelain acysteine proteaserdquo Applied Biochemistry and Biotechnology vol144 no 3 pp 283ndash291 2008
[11] M Y Shukor N A Baharom N A Masdor et al ldquoThedevelopment of an inhibitive determination method for zincusing a serine proteaserdquo Journal of Environmental Biology vol30 no 1 pp 17ndash22 2009
The Scientific World Journal 7
[12] S HanM Zhu Z Yuan and X Li ldquoAmethylene blue-mediatedenzyme electrode for the determination of trace mercury(II)mercury(I) methylmercury and mercury-glutathione com-plexrdquo Biosensors and Bioelectronics vol 16 no 1-2 pp 9ndash162001
[13] K Jung G Bitton and B Koopman ldquoAssessment of urease inhi-bition assays for measuring toxicity of environmental samplesrdquoWater Research vol 29 no 8 pp 1929ndash1933 1995
[14] R M C Dawson D C Elliott W H Elliott and K M JonesData for Biochemical Research Clarendon Press Oxford UK1969
[15] A Bensadoun and D Weinstein ldquoAssay of proteins in thepresence of interfering materialsrdquo Analytical Biochemistry vol70 no 1 pp 241ndash250 1976
[16] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976
[17] G L Ellman K D Courtney V Andres Jr and R MFeatherstone ldquoA new and rapid colorimetric determination ofacetylcholinesterase activityrdquo Biochemical Pharmacology vol 7no 2 pp 88ndash95 1961
[18] H M Farrell Jr E D Wickham and M L Groves ldquoEnvi-ronmental influences on purified casein disulfide interactionsrdquoJournal of Dairy Science vol 81 no 11 pp 2974ndash2984 1998
[19] W Jiang X Zhou Y Zhao and P Liu ldquoIdentification ofa senescence-related protease in coriander leavesrdquo ChineseScience Bulletin vol 47 no 13 pp 1096ndash1099 2002
[20] D A Armbruster M D Tillman and L M Hubbs ldquoLimit ofdetection (LOD)limit of quantitation (LOQ) comparison ofthe empirical and the statistical methods exemplified with GC-MS assays of abused drugsrdquo Clinical Chemistry vol 40 no 7pp 1233ndash1238 1994
[21] DOE Environmental Quality Report Department of Environ-ment Ministry of Science Technology and the EnvironmentMalaysia 2001
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
The Scientific World Journal 7
[12] S HanM Zhu Z Yuan and X Li ldquoAmethylene blue-mediatedenzyme electrode for the determination of trace mercury(II)mercury(I) methylmercury and mercury-glutathione com-plexrdquo Biosensors and Bioelectronics vol 16 no 1-2 pp 9ndash162001
[13] K Jung G Bitton and B Koopman ldquoAssessment of urease inhi-bition assays for measuring toxicity of environmental samplesrdquoWater Research vol 29 no 8 pp 1929ndash1933 1995
[14] R M C Dawson D C Elliott W H Elliott and K M JonesData for Biochemical Research Clarendon Press Oxford UK1969
[15] A Bensadoun and D Weinstein ldquoAssay of proteins in thepresence of interfering materialsrdquo Analytical Biochemistry vol70 no 1 pp 241ndash250 1976
[16] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976
[17] G L Ellman K D Courtney V Andres Jr and R MFeatherstone ldquoA new and rapid colorimetric determination ofacetylcholinesterase activityrdquo Biochemical Pharmacology vol 7no 2 pp 88ndash95 1961
[18] H M Farrell Jr E D Wickham and M L Groves ldquoEnvi-ronmental influences on purified casein disulfide interactionsrdquoJournal of Dairy Science vol 81 no 11 pp 2974ndash2984 1998
[19] W Jiang X Zhou Y Zhao and P Liu ldquoIdentification ofa senescence-related protease in coriander leavesrdquo ChineseScience Bulletin vol 47 no 13 pp 1096ndash1099 2002
[20] D A Armbruster M D Tillman and L M Hubbs ldquoLimit ofdetection (LOD)limit of quantitation (LOQ) comparison ofthe empirical and the statistical methods exemplified with GC-MS assays of abused drugsrdquo Clinical Chemistry vol 40 no 7pp 1233ndash1238 1994
[21] DOE Environmental Quality Report Department of Environ-ment Ministry of Science Technology and the EnvironmentMalaysia 2001
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
International Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
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