Determination of the Binding Parameters between Proteins and

Hindawi Publishing CorporationISRN Analytical ChemistryVolume 2013 Article ID 391053 5 pageshttpdxdoiorg1011552013391053

Research ArticleDetermination of the Binding Parametersbetween Proteins and Luminol by ChemiluminescenceUsing Flow Injection Technique

Jie Guo Donghua Chen and Zhenghua Song

Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of EducationCollege of Chemistry amp Material Science Northwest University Xirsquoan 710069 China

Correspondence should be addressed to Zhenghua Song songzhenghuahotmailcom

Received 8 April 2013 Accepted 16 May 2013

Academic Editors Z Arslan and K Ohyama

Copyright copy 2013 Jie Guo et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The interaction behavior of bovine serum albumin (BSA) lysozyme (LYS) myoglobin (MB) and catalase (CAT) with luminolrespectively was first studied by chemiluminescence (CL) using flow injection (FI) technique based on the fact that the studiedproteins can enhance theCL intensity of luminol A FI-CLmodel of protein-luminol interaction lg[(119868

0minus119868)119868] = 1119899lg[119875]+1119899lg119870

119886+

2lg119899 was constructed and the interaction parameters of BSA LYS MB and CAT with luminol were determined accordingly Thebinding constants 119870

119886are in the descending order of CAT gtMB gt LYS gt BSA at the level of 105 to 107 Lmolminus1 and the number of

binding sites 119899 of luminol to BSA or LYS is around 2 and toMB or CAT is around 1The results of thermodynamic parameters (Δ119867Δ119878 and Δ119866) showed that the binding processes of luminol to the four proteins are spontaneous mainly through the hydrophobicforce

1 Introduction

Proteins possess many biological functions including bind-ing catalysis operating as molecular switches and serving asstructural components of cells and organisms [1 2] and playan important role in the transportation and deposition of var-ious endogenous and exogenous substances [3 4] In recentyears the interaction of protein with small molecules hasbecome a hot spot in the fields of chemistry biology andmedicine [5ndash7] As common model proteins bovine serumalbumin (BSA) [8] lysozyme (LYS) [9] myoglobin (MB)[10] and catalase (CAT) [11] are widely applied to the studyof protein-small molecule interaction Accordingly manymethods have been utilized to investigate this hot topic suchas fluorescence spectroscopy [12] resonance light scattering[13] circular dichroism [14] nuclear magnetic resonancespectroscopy [15] and chemiluminescence (CL) with flowinjection (FI) technique [16] The FI-CL analysis has provento be a very useful analytical method with advantages ofsimple apparatus high sensitivity wide dynamic ranges

reproducibility automatability less reagent consumption [17ndash19] and so forth

Luminol (5-amino-2 3-dihydro-1 4-phthalazinedione) isknown to produce CL with the characteristic wavelength of425 nm under alkaline condition [20 21] making it widelyused around the world as a blood enhancement techniquein forensic science Also this luminescent compound is usedclinically in the treatment of alopecia [22] the promotionof blood clotting [23] and wound healing [24] Due to itshigh quantum yield the CL of luminol has been used inmany laboratory applications including environmental fieldof analyzing trace metals [25 26] food safety field [27]biological field of immunoassays monitoring of metabolicpathways and detection of free radicals [28] Recently theCL of luminol has been employed to study its interactionbehavior with serum albumins [12 29]

In our previous study it was found that BSA LYS MBand CAT can accelerate the electrons transferring rate ofexcited 3-aminophthalate leading to enhancement of CLintensity from luminol [30ndash33] However no comprehensive

2 ISRN Analytical Chemistry

data have been available on the binding parameters of the fourproteins with luminol so far The main aims of the currentpaper are to (a) construct a mathematical model of protein-luminol interaction lg[(119868

0minus 119868)119868] = 1119899lg[119875] + 1119899lg119870

119886+

2lg119899 based on the enhancing effect of protein-to-luminolCL intensity (b) obtain the binding parameters of proteinswith luminol and (c) give the thermodynamic parameters ofprotein-luminol binding process

2 Materials and Methods

21 Reagents All reagents used were of analytical reagentgrade unless otherwise specified and doubly deionized waterwas purified in a Milli-Q system (Millipore Bedford MAUSA) for the preparation of solutions in the whole procedureLuminol (Fluka Switzerland) BSA (Sigma) LYS (Sigma)MB (Sigma) and CAT (Sigma) were used as received withoutfurther purification Luminol (25 times 10minus2mol Lminus1) was pre-pared by dissolving 044 g luminol in 100mL of 01mol Lminus1NaOH solution in a brown calibrated flask BSA LYS MBand CAT (50 times 10minus5mol Lminus1) were prepared in purified waterand stored at 4∘C

22 Apparatus TheFI-CL system used in this workwas illus-trated schematically in Figure 1 The flow system consisted ofthree lines for delivery protein luminolNaOH and carriersolution The IFFM-A Luminescence Analyzer (Xirsquoan RemaxElectronic Science and Technology Co and Ltd XirsquoanChina) contained the sampling system the photomultipliertube (PMT) and the PC with an IFFM-A client system(Remax Xirsquoan China) Polytetrafluoroethylene (PTFE) tube(10mm id) was used to connect all the components of theflow system A six-way valve with a loop of 100120583L was usedfor quantitatively injecting luminol into carrier stream TheCL detector contained a flow cell made by coiling 15 cm ofcolorless glass tube (10mm id) into a spiral disk shape witha diameter of 20 cm and placed close to the PMT Extremeprecautions were taken to ensure that the sample compart-ment and PMT were light tight The CL signal produced inflow cell was detected without wavelength discriminationand the PMT output was recorded by computer with IFFM-Eclient systemThe temperature of the solutionswas controlled(119879plusmn 01∘C) in a water bath

23 General Procedures The carrier (purified water) thesolutions of luminol and proteins were propelled by peri-staltic pumps at a flow rate of 20mLminminus1 on each flow lineThe whole flow system was washed until a stable baselinewas recorded Then 100120583L of luminol solution was injectedinto the carrier stream by the six-way valve and merged withproteins The whole mixed solution was delivered into theflow cell and the emitted CL was collected by the PMTat a voltage of minus700V and recorded by the computer Theincrement of CL intensity (Δ119868 = 119868 minus 119868

0 where 119868 and 119868

0were

CL signals in the presence and in the absence of proteins) wasmeasured

24 Optimization of the Experimental Conditions The effectof luminol concentration on the CL intensity was examined

Carrier

Protein

LuminolNaOHFC

PMT

HV PC

Blank boxP

W

V

M

W

Figure 1 Schematic diagram of the present FI-CL system P pumpM mixing tube V valve FC flow cell W waste PC personalcomputer

over the ranges of 50 times 10minus7 to 50 times 10minus4mol Lminus1 It wasfound that the maximum CL intensity could be obtainedat 25 times 10minus5mol Lminus1 luminol Therefore 25 times 10minus5mol Lminus1luminol was chosen as the optimum concentration and usedin subsequent experiments A series of NaOH solutions withdifferent concentrations ranging from 10times 10minus3 to 01mol Lminus1were tested and 25 times 10minus2mol Lminus1 NaOH was used as theoptimum concentration The flow rate and the length ofmixing tube had a great effect on the CL intensity A flowrate of 20mLminminus1 and the length of mixing tube of 100 cmwere selected in this work with good sensitivity precisionand reproducibility

25 The Operational Stability of the FI-CL System Luminolalkaline solution (100 120583L) was injected into the flow systemin the presence of 25 times 10minus5mol Lminus1 luminol and 50 times10minus8mol Lminus1 proteins and the CL intensity was recorded totest the stability of luminol-protein system This experimentlasted for 3 days and the flow system was regularly used over8 hours per dayThe results showed that the relative standarddeviations were less than 30 so the luminol-protein systemexerted good stability

26 Constructing the Binding Model of Protein with LuminolIt is known that proteins are biomacromolecules with bindingsites for small molecules Assuming that the number ofbinding sites of the small molecule luminol (119871) to protein(119875) is 119899 each site is independent and has the same intrinsicaffinity for the ligand [34 35] The protein-luminol complex(119875119871119899) is formed and the binding process can be expressed as

follows119875 + 119899119871

119870119886

999445999468 119875119871119899

(1)

or1

119899119875 + 119871

119870

999445999468 1198751119899119871 (2)

where119875119871 119899 and119875119871119899represent protein luminol the number

of binding sites and protein-luminol complex respectivelyThe binding constant 119870

119886or 119870 is

119870119886=[119875119871119899]

[119875] [119871]119899

or 119870 =[1198751119899119871]

[119875]1119899[119871] (3)

where [119875119871119899] [119875] and [119871] represent the concentrations of

protein-luminol complex protein and luminol at equilib-rium state respectively and [119875

1119899119871] is the concentration of

product for reaction (2)

ISRN Analytical Chemistry 3

According to chemical reaction equilibrium theory

119870119886= 119870119899 (4)

[1198751119899119871] =1

119899[119875]119887 =1

1198992[119871]119887 (5)

The CL intensity of luminol is proportional to its concen-tration Equations (6) and (7) can be obtained as

1198680prop [119871]119905 (6)

where 1198680is the CL intensity in the absence of protein [119871]

119905is

the total concentration of luminol as

119868 minus 1198680prop [119871]119887 (7)

where 119868 is the CL intensity in the presence of protein [119871]119887is

the concentration of luminol at equilibriumUsing (6) and (7)in (5) leads to

119870 =(11198992) (119868 minus 119868

0)

[119875]11198991198680

(8)

Taking logarithms of both sides

lg119868 minus 1198680

1198680

=1

119899lg [119875] + lg119870 + 2lg119899 (9)

Substituting (4) into (9) the following equation can beobtained

lg119868 minus 1198680

1198680

=1

119899lg [119875] + 1

119899lg119870119886+ 2lg119899 (10)

Equation (10) is the FI-CL model for determination ofbinding parameters of protein with luminol from interceptand slope of lg(119868

0minus 119868)119868 sim lg[119875] curve

3 Results and Discussion

31 Relative CL Intensity-Time Profiles The relative CLintensity-time profiles in different CL systems were presentedin Figure 2 Herein the concentration of luminol was 25 times10minus5mol Lminus1 and the concentrations of BSA LYS MB andCATwere 50times 10minus8mol Lminus1 It can be seen that themaximumCL intensity (119868max) of luminol system (curve 1) is 93 at thetime (119879max) of 39 s the 119868max is 144 152 221 and 410 at the119879max of 37 34 32 and 29 s in the presence of BSA (curve 2)LYS (curve 3) MB (curve 4) and CAT (curve 5) respectivelyIt can be seen that the CL responses for different proteins inluminol system followed the order ofCATgtMBgtLYSgtBSA

32 Binding Parameters of Protein with Luminol Under theoptimum conditions a series of standard solutions of proteinwere analyzed by the FI-CL system The CL intensity incre-ment from luminol obeys the general equation ofΔ119868 = 119860119862

119901+

119861 with the linear equations linear ranges and correlativecoefficients 119877 at different temperatures (288 298 and 308K)listed in Table 1 It can be seen that 119860 varies in the increasingorder of 119860CAT gt 119860MB gt 119860LYS gt 119860BSA indicating that the

Time (s)

1

5

Relat

ive C

L in

tens

ity

0

90

180

270

360

450

0 2 4 6 8 10 12 14 16 18

119879max5 = 29 s119879max4 = 32 s119879max3 = 34 s119879max2 = 37 s119879max1 = 39 s

Figure 2 Relative CL intensity-time profile in different CL systemsat 298K Curve 1 luminol CL system Curve 2 luminol-BSA CLsystem Curve 3 luminol-LYS CL system Curve 4 luminol-MB CLsystem Curve 5 luminol-CAT CL system Concentration luminol25 times 10minus5mol Lminus1 BSA LYS MB and CAT 50 times 10minus8mol Lminus1

sensitivities of determination follow the sequence CAT gtMBgtLYSgtBSAAccording to (10) lg[(119868

0minus119868)119868] = 1119899lg[119875]+

1119899lg119870119886+2lg119899 by plotting lg(119868

0minus119868)119868versus lg[119875] the binding

constants 119870119886and the number of binding sites 119899 of luminol

to CAT MB LYS and BSA were obtained and were given inTable 1 It was clear that the binding constants 119870

119886increase

with the increasing of temperature and the values of119870119886were

at 105 to 107 levels suggesting that there existed a high bindingaffinity of proteinswith luminol in the sequence of CATgtMBgtLYSgtBSAThenumber of binding sites 119899 of luminol to BSAor LYS was about 2 and to MB or CAT was about 1

33TheThermodynamic Parameters of Protein-Luminol Inter-action By the Vanrsquot Hoff equation [36] thermodynamicparameters of BSA LYS MB and CAT with luminol werelisted in Table 2 It can be seen thatΔ119867 gt 0Δ119878 gt 0 andΔ119866 lt0 indicating that the complex formation was a spontaneousand endothermic process with hydrophobic effect as themainbinding force It was also clear that the major contributionfor the Δ119866 comes from Δ119878 rather than Δ119867 suggesting thebinding process was entropy-drivenThe sequence of bindingabilities of different proteins was consistent with the order ofthe sensitivity of determination CAT gt MB gt LYS gt BSAwhich indicated that the binding of CAT with luminol wasmuch easier than MB LYS and BSA

4 ConclusionsThe interaction behavior of BSA LYS MB and CAT withluminol was first studied by FI-CL analysis By the con-structed FI-CL model lg[(119868

0minus 119868)119868] = 1119899lg[119875] + 1119899lg119870

119886+

2lg119899 binding constants 119870119886and the number of binding

sites 119899 of protein with luminol were obtained The resultsshowed that the interaction of protein with luminol was


Table 1 The linear equations and binding parameters by (10)

Protein 119879 (K) Linear equation119877Δ119868 = 119860119862

119901+ 119861

a Linear range (nmol Lminus1) lg(1198680minus 119868)119868 versus Lg[119875]

119870119886(L molminus1) 119899

BSA288 Δ119868 = 018119862BSA + 299109977

5ndash250177 times 105 223

298 Δ119868 = 019119862BSA + 324109963 234 times 105 175

308 Δ119868 = 021119862BSA + 355709978 290 times 105 180

LYS288 Δ119868 = 015119862LYS + 214909990

10ndash500193 times 105 195

298 Δ119868 = 021119862LYS + 214309961 426 times 105 175

308 Δ119868 = 048119862LYS + 220209988 559 times 105 196

MB288 Δ119868 = 019119862MB + 326109963

10ndash1000212 times 106 126

298 Δ119868 = 021119862MB + 585509966 240 times 106 135

308 Δ119868 = 029119862MB + 492209961 293 times 106 094

CAT288 Δ119868 = 491119862CAT + 148509962

01ndash100438 times 107 138

298 Δ119868 = 558119862CAT + 157409997 496 times 107 136

308 Δ119868 = 614119862CAT + 133009996 593 times 107 124aEach result is the average of seven separate determinations

Table 2 Thermodynamic parameters of protein-luminol interac-tion determined by FI-CL

Protein 119879 (K) Δ119866 (kJmolminus1) Δ119867 (kJmolminus1) Δ119878 (Jmolminus1 Kminus1)

BSA288 minus2894

1810 16337298 minus3063

308 minus3220

LYS288 minus2914

4068 24303298 minus3211

308 minus3398

MB288 minus3487

1200 16262298 minus3640

308 minus3813

CAT288 minus4213

1119 18507298 minus4390

308 minus4583

an endothermic spontaneous and entropy-driven processmainly via the hydrophobic effect

Conflict of Interests

The authors and commercial identities in this work do nothave any possible conflict of interests

Acknowledgments

The authors gratefully acknowledge the financial supportfrom the National Nature Science Foundation of China (no21275118) the NWU Graduate Innovation and CreativityFund (no 10YZZ29) and the Open Funds from the KeyLaboratory of Synthetic and Natural Functional MoleculeChemistry of Ministry of Education China

References

[1] K Teilum J G Olsen and B B Kragelund ldquoFunctional aspectsof protein flexibilityrdquo Cellular and Molecular Life Sciences vol66 no 14 pp 2231ndash2247 2009

[2] A Dhar K Girdhar D Singh H Gelman S Ebbinghausand M Gruebele ldquoProtein stability and folding kinetics inthe nucleus and endoplasmic reticulum of Eucaryotic CellsrdquoBiophysical Journal vol 101 no 2 pp 421ndash430 2011

[3] G Paramaguru A Kathiravan S Selvaraj P Venuvanalingamand R Renganathan ldquoInteraction of anthraquinone dyes withlysozyme evidences from spectroscopic and docking studiesrdquoJournal of Hazardous Materials vol 175 no 1ndash3 pp 985ndash9912010

[4] R J Pantazes M J Grisewood and C D Maranas ldquoRecentadvances in computational protein designrdquo Current Opinion inStructural Biology vol 21 no 4 pp 467ndash472 2011

[5] H M Zhang J Chen Q H Zhou Y Q Shi and Y Q WangldquoStudy on the interaction between cinnamic acid and lysozymerdquoJournal of Molecular Structure vol 987 no 1ndash3 pp 7ndash12 2011

[6] N Shahabadi and M Mohammadpour ldquoStudy on the interac-tion of sodium morin-5-sulfonate with bovine serum albuminby spectroscopic techniquesrdquo Spectrochimica Acta A vol 86 pp191ndash195 2012

[7] WDu T Teng C C Zhou L Xi and J ZWang ldquoSpectroscopicstudies on the interaction of bovine serum albumin withginkgolic acid binding characteristics and structural analysisrdquoJournal of Luminescence vol 132 no 5 pp 1207ndash1214 2012

[8] GW Zhang N Zhao X Hu and J Tian ldquoInteraction of alpine-tin with bovine serum albumin probing of the mechanism andbinding site by spectroscopic methodsrdquo Spectrochimica Acta Avol 76 no 3-4 pp 410ndash417 2010

[9] W P Wang W A Min J R Chen X H Wu and Z D HuldquoBinding study of diprophylline with lysozyme by spectroscopicmethodsrdquo Journal of Luminescence vol 131 no 4 pp 820ndash8242011

[10] D Ganini M Christoff M Ehrenshaft M B Kadiiska RP Mason and E J H Bechara ldquoMyoglobin-H

2O2catalyzes

the oxidation of 120573-ketoacids to 120572-dicarbonyls mechanism andimplications in ketosisrdquo Free Radical Biology and Medicine vol51 no 3 pp 733ndash743 2011

[11] M I Gonzalez-Sanchez F Garcıa-Carmona H Macia andE Valero ldquoCatalase-like activity of human methemoglobin akinetic and mechanistic studyrdquo Archives of Biochemistry andBiophysics vol 516 no 1 pp 10ndash20 2011


[12] N S Moyon and S Mitra ldquoLuminol fluorescence quenchingin biomimicking environments sequestration of fluorophore inhydrophobic domainrdquo Journal of Physical Chemistry B vol 115no 33 pp 10163ndash10172 2011

[13] J B Xiao J W Chen H Cao and F L Ren ldquoStudy of theinteraction between baicalin and bovine serum albumin bymulti-spectroscopic methodrdquo Journal of Photochemistry andPhotobiology A vol 191 no 2-3 pp 222ndash227 2007

[14] T Banerjee S K Singh and N Kishore ldquoBinding of naproxenand amitriptyline to bovine serum albumin biophysicalaspectsrdquo Journal of Physical Chemistry B vol 110 no 47 pp24147ndash24156 2006

[15] B L Cao S Endsley and N H Andersen ldquo19F NMR studies oftryptophanserum albumin bindingrdquo Bioorganic and MedicinalChemistry vol 11 no 1 pp 69ndash75 2003

[16] Y M Huang Z Z Zhang D J Zhang and J D Lv ldquoFlow-injection analysis chemiluminescence detection combined withmicrodialysis sampling for studying protein binding of drugrdquoTalanta vol 53 no 4 pp 835ndash841 2001

[17] S Kulmala and J Suomi ldquoCurrent status of modern analyticalluminescence methodsrdquo Analytica Chimica Acta vol 500 no1-2 pp 21ndash69 2003

[18] X Wang J M Lin M L Liu and X L Cheng ldquoFlow-basedluminescence-sensing methods for environmental water anal-ysisrdquo Trends in Analytical Chemistry vol 28 no 1 pp 75ndash872009

[19] M C Icardo and J M Calatayud ldquoPhoto-induced lumines-cencerdquo Critical Reviews in Analytical Chemistry vol 38 no 2pp 118ndash130 2008

[20] E H White O Zafiriou H H Kagi and J H M Hill ldquoChemi-luminescence of luminol the chemical reactionrdquo Journal of theAmerican Chemical Society vol 86 no 5 pp 940ndash941 1964

[21] J M Sanders L J Chen L T Burka and H B MatthewsldquoMetabolism and disposition of luminol in the ratrdquoXenobioticavol 30 no 3 pp 263ndash272 2000

[22] S Irie ldquoThe treatment of alopecia areata with 3-aminophthal-hydraziderdquo Current Therapeutic Research Clinical and Experi-mental vol 2 no 3 pp 107ndash110 1960

[23] S Irie ldquoInfluence of 3-aminophthalhydrazide on the prothrom-bin timerdquo Current Therapeutic Research Clinical and Experi-mental vol 2 no 5 pp 153ndash157 1960

[24] S Irie ldquoThe treatment of wounds with 3-aminophthalhydraz-iderdquoThe American surgeon vol 27 pp 642ndash645 1961

[25] H Zhang T Shibata T Krawczyk et al ldquoFacile detectionof proteins on a solid-phase membrane by direct bindingof dextran-based luminol-biotin chemiluminescent polymerrdquoTalanta vol 79 no 3 pp 700ndash705 2009

[26] S Bi H Zhou and S Zhang ldquoMultilayers enzyme-coatedcarbon nanotubes as biolabel for ultrasensitive chemilumi-nescence immunoassay of cancer biomarkerrdquo Biosensors andBioelectronics vol 24 no 10 pp 2961ndash2966 2009

[27] K Mervartova M Polasek and J M Calatayud ldquoRecentapplications of flow-injection and sequential-injection analysistechniques to chemiluminescence determination of pharma-ceuticalsrdquo Journal of Pharmaceutical and Biomedical Analysisvol 45 no 3 pp 367ndash381 2007

[28] A R Bowie M G Sanders and P J Worsfold ldquoAnalyticalapplications of liquid phase chemiluminescence reactionsmdashareviewrdquo Journal of Bioluminescence and Chemiluminescence vol11 no 2 pp 61ndash90 1996

[29] N S Moyon and S Mitra ldquoOn the interaction of luminol withhuman serum albumin nature and thermodynamics of ligandbindingrdquoChemical Physics Letters vol 498 no 1ndash3 pp 178ndash1832010

[30] X J Tan Z H Song D H Chen and Z M Wang ldquoStudy onthe chemiluminescence behavior of bovine serum albuminwithluminol and its analytical applicationrdquo Spectrochimica Acta Avol 79 no 1 pp 232ndash235 2011

[31] D H Chen and Z H Song ldquoIn vitro monitoring of picogramlevels of risperidone in human urine via luminollysozyme flowinjection chemiluminescencerdquo Microchimica Acta vol 171 no3-4 pp 437ndash440 2010

[32] ZMWang D H Chen X Gao and Z H Song ldquoSubpicogramdetermination of melamine in milk products using a luminol-myoglobin chemiluminescence systemrdquo Journal of Agriculturaland Food Chemistry vol 57 no 9 pp 3464ndash3469 2009

[33] D H Chen Z M Wang Y Zhang X Y Xiong and ZH Song ldquoStudy on the interaction behavior of catalase withcephalosporins by chemiluminescence with flow injection anal-ysisrdquo Analytical Methods vol 4 no 6 pp 1485ndash1487 2012

[34] G Scatchard ldquoThe attraction of proteins for small moleculesand ionsrdquo Annals of the New York Academy of Sciences vol 51pp 660ndash672 1949

[35] A A Spector J E Fletcher and J D Ashbrook ldquoAnalysis oflong-chain free fatty acid binding to bovine serum albumin bydetermination of stepwise equilibrium constantsrdquoBiochemistryvol 10 no 17 pp 3229ndash3232 1971

[36] P D Ross and S Subramanian ldquoThermodynamics of proteinassociation reactions forces contributing to stabilityrdquo Biochem-istry vol 20 no 11 pp 3096ndash3102 1981

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


Journal of

Chemistry


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


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy



Analytical ChemistryVolume 2014

Journal of


Quantum Chemistry


Organic Chemistry International


CatalystsJournal of

ElectrochemistryInternational Journal of



data have been available on the binding parameters of the fourproteins with luminol so far The main aims of the currentpaper are to (a) construct a mathematical model of protein-luminol interaction lg[(119868

0minus 119868)119868] = 1119899lg[119875] + 1119899lg119870

119886+

2lg119899 based on the enhancing effect of protein-to-luminolCL intensity (b) obtain the binding parameters of proteinswith luminol and (c) give the thermodynamic parameters ofprotein-luminol binding process

2 Materials and Methods

21 Reagents All reagents used were of analytical reagentgrade unless otherwise specified and doubly deionized waterwas purified in a Milli-Q system (Millipore Bedford MAUSA) for the preparation of solutions in the whole procedureLuminol (Fluka Switzerland) BSA (Sigma) LYS (Sigma)MB (Sigma) and CAT (Sigma) were used as received withoutfurther purification Luminol (25 times 10minus2mol Lminus1) was pre-pared by dissolving 044 g luminol in 100mL of 01mol Lminus1NaOH solution in a brown calibrated flask BSA LYS MBand CAT (50 times 10minus5mol Lminus1) were prepared in purified waterand stored at 4∘C

22 Apparatus TheFI-CL system used in this workwas illus-trated schematically in Figure 1 The flow system consisted ofthree lines for delivery protein luminolNaOH and carriersolution The IFFM-A Luminescence Analyzer (Xirsquoan RemaxElectronic Science and Technology Co and Ltd XirsquoanChina) contained the sampling system the photomultipliertube (PMT) and the PC with an IFFM-A client system(Remax Xirsquoan China) Polytetrafluoroethylene (PTFE) tube(10mm id) was used to connect all the components of theflow system A six-way valve with a loop of 100120583L was usedfor quantitatively injecting luminol into carrier stream TheCL detector contained a flow cell made by coiling 15 cm ofcolorless glass tube (10mm id) into a spiral disk shape witha diameter of 20 cm and placed close to the PMT Extremeprecautions were taken to ensure that the sample compart-ment and PMT were light tight The CL signal produced inflow cell was detected without wavelength discriminationand the PMT output was recorded by computer with IFFM-Eclient systemThe temperature of the solutionswas controlled(119879plusmn 01∘C) in a water bath

23 General Procedures The carrier (purified water) thesolutions of luminol and proteins were propelled by peri-staltic pumps at a flow rate of 20mLminminus1 on each flow lineThe whole flow system was washed until a stable baselinewas recorded Then 100120583L of luminol solution was injectedinto the carrier stream by the six-way valve and merged withproteins The whole mixed solution was delivered into theflow cell and the emitted CL was collected by the PMTat a voltage of minus700V and recorded by the computer Theincrement of CL intensity (Δ119868 = 119868 minus 119868

0 where 119868 and 119868

0were

CL signals in the presence and in the absence of proteins) wasmeasured

24 Optimization of the Experimental Conditions The effectof luminol concentration on the CL intensity was examined

Carrier

Protein

LuminolNaOHFC

PMT

HV PC

Blank boxP

W

V

M

W

Figure 1 Schematic diagram of the present FI-CL system P pumpM mixing tube V valve FC flow cell W waste PC personalcomputer

over the ranges of 50 times 10minus7 to 50 times 10minus4mol Lminus1 It wasfound that the maximum CL intensity could be obtainedat 25 times 10minus5mol Lminus1 luminol Therefore 25 times 10minus5mol Lminus1luminol was chosen as the optimum concentration and usedin subsequent experiments A series of NaOH solutions withdifferent concentrations ranging from 10times 10minus3 to 01mol Lminus1were tested and 25 times 10minus2mol Lminus1 NaOH was used as theoptimum concentration The flow rate and the length ofmixing tube had a great effect on the CL intensity A flowrate of 20mLminminus1 and the length of mixing tube of 100 cmwere selected in this work with good sensitivity precisionand reproducibility

25 The Operational Stability of the FI-CL System Luminolalkaline solution (100 120583L) was injected into the flow systemin the presence of 25 times 10minus5mol Lminus1 luminol and 50 times10minus8mol Lminus1 proteins and the CL intensity was recorded totest the stability of luminol-protein system This experimentlasted for 3 days and the flow system was regularly used over8 hours per dayThe results showed that the relative standarddeviations were less than 30 so the luminol-protein systemexerted good stability

26 Constructing the Binding Model of Protein with LuminolIt is known that proteins are biomacromolecules with bindingsites for small molecules Assuming that the number ofbinding sites of the small molecule luminol (119871) to protein(119875) is 119899 each site is independent and has the same intrinsicaffinity for the ligand [34 35] The protein-luminol complex(119875119871119899) is formed and the binding process can be expressed as

follows119875 + 119899119871

119870119886

999445999468 119875119871119899

(1)

or1

119899119875 + 119871

119870

999445999468 1198751119899119871 (2)

where119875119871 119899 and119875119871119899represent protein luminol the number

of binding sites and protein-luminol complex respectivelyThe binding constant 119870

119886or 119870 is

119870119886=[119875119871119899]

[119875] [119871]119899

or 119870 =[1198751119899119871]

[119875]1119899[119871] (3)

where [119875119871119899] [119875] and [119871] represent the concentrations of

protein-luminol complex protein and luminol at equilib-rium state respectively and [119875

1119899119871] is the concentration of

product for reaction (2)



119870119886= 119870119899 (4)

[1198751119899119871] =1

119899[119875]119887 =1

1198992[119871]119887 (5)


1198680prop [119871]119905 (6)


119905is


119868 minus 1198680prop [119871]119887 (7)



119870 =(11198992) (119868 minus 119868

0)

[119875]11198991198680

(8)


lg119868 minus 1198680

1198680

=1

119899lg [119875] + lg119870 + 2lg119899 (9)


lg119868 minus 1198680

1198680

=1

119899lg [119875] + 1

119899lg119870119886+ 2lg119899 (10)






119901+


Time (s)

1

5

Relat

ive C

L in

tens

ity

0

90

180

270

360

450

0 2 4 6 8 10 12 14 16 18




0minus119868)119868] = 1119899lg[119875]+





119886increase





0minus 119868)119868] = 1119899lg[119875] + 1119899lg119870

119886+






119901+ 119861


119870119886(L molminus1) 119899

BSA288 Δ119868 = 018119862BSA + 299109977

5ndash250177 times 105 223

298 Δ119868 = 019119862BSA + 324109963 234 times 105 175

308 Δ119868 = 021119862BSA + 355709978 290 times 105 180

LYS288 Δ119868 = 015119862LYS + 214909990

10ndash500193 times 105 195

298 Δ119868 = 021119862LYS + 214309961 426 times 105 175

308 Δ119868 = 048119862LYS + 220209988 559 times 105 196

MB288 Δ119868 = 019119862MB + 326109963

10ndash1000212 times 106 126

298 Δ119868 = 021119862MB + 585509966 240 times 106 135

308 Δ119868 = 029119862MB + 492209961 293 times 106 094

CAT288 Δ119868 = 491119862CAT + 148509962

01ndash100438 times 107 138

298 Δ119868 = 558119862CAT + 157409997 496 times 107 136




BSA288 minus2894

1810 16337298 minus3063

308 minus3220

LYS288 minus2914

4068 24303298 minus3211

308 minus3398

MB288 minus3487

1200 16262298 minus3640

308 minus3813

CAT288 minus4213

1119 18507298 minus4390

308 minus4583




Acknowledgments


References











2O2catalyzes






































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy




Journal of


Quantum Chemistry




CatalystsJournal of





119870119886= 119870119899 (4)

[1198751119899119871] =1

119899[119875]119887 =1

1198992[119871]119887 (5)


1198680prop [119871]119905 (6)


119905is


119868 minus 1198680prop [119871]119887 (7)



119870 =(11198992) (119868 minus 119868

0)

[119875]11198991198680

(8)


lg119868 minus 1198680

1198680

=1

119899lg [119875] + lg119870 + 2lg119899 (9)


lg119868 minus 1198680

1198680

=1

119899lg [119875] + 1

119899lg119870119886+ 2lg119899 (10)






119901+


Time (s)

1

5

Relat

ive C

L in

tens

ity

0

90

180

270

360

450

0 2 4 6 8 10 12 14 16 18




0minus119868)119868] = 1119899lg[119875]+





119886increase





0minus 119868)119868] = 1119899lg[119875] + 1119899lg119870

119886+






119901+ 119861


119870119886(L molminus1) 119899

BSA288 Δ119868 = 018119862BSA + 299109977

5ndash250177 times 105 223

298 Δ119868 = 019119862BSA + 324109963 234 times 105 175

308 Δ119868 = 021119862BSA + 355709978 290 times 105 180

LYS288 Δ119868 = 015119862LYS + 214909990

10ndash500193 times 105 195

298 Δ119868 = 021119862LYS + 214309961 426 times 105 175

308 Δ119868 = 048119862LYS + 220209988 559 times 105 196

MB288 Δ119868 = 019119862MB + 326109963

10ndash1000212 times 106 126

298 Δ119868 = 021119862MB + 585509966 240 times 106 135

308 Δ119868 = 029119862MB + 492209961 293 times 106 094

CAT288 Δ119868 = 491119862CAT + 148509962

01ndash100438 times 107 138

298 Δ119868 = 558119862CAT + 157409997 496 times 107 136




BSA288 minus2894

1810 16337298 minus3063

308 minus3220

LYS288 minus2914

4068 24303298 minus3211

308 minus3398

MB288 minus3487

1200 16262298 minus3640

308 minus3813

CAT288 minus4213

1119 18507298 minus4390

308 minus4583




Acknowledgments


References











2O2catalyzes






































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy




Journal of


Quantum Chemistry




CatalystsJournal of






119901+ 119861


119870119886(L molminus1) 119899

BSA288 Δ119868 = 018119862BSA + 299109977

5ndash250177 times 105 223

298 Δ119868 = 019119862BSA + 324109963 234 times 105 175

308 Δ119868 = 021119862BSA + 355709978 290 times 105 180

LYS288 Δ119868 = 015119862LYS + 214909990

10ndash500193 times 105 195

298 Δ119868 = 021119862LYS + 214309961 426 times 105 175

308 Δ119868 = 048119862LYS + 220209988 559 times 105 196

MB288 Δ119868 = 019119862MB + 326109963

10ndash1000212 times 106 126

298 Δ119868 = 021119862MB + 585509966 240 times 106 135

308 Δ119868 = 029119862MB + 492209961 293 times 106 094

CAT288 Δ119868 = 491119862CAT + 148509962

01ndash100438 times 107 138

298 Δ119868 = 558119862CAT + 157409997 496 times 107 136




BSA288 minus2894

1810 16337298 minus3063

308 minus3220

LYS288 minus2914

4068 24303298 minus3211

308 minus3398

MB288 minus3487

1200 16262298 minus3640

308 minus3813

CAT288 minus4213

1119 18507298 minus4390

308 minus4583




Acknowledgments


References











2O2catalyzes






































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy




Journal of


Quantum Chemistry




CatalystsJournal of






































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy




Journal of


Quantum Chemistry




CatalystsJournal of



Determination of the Binding Parameters between Proteins and

Documents

Transcript of Determination of the Binding Parameters between Proteins and