Research Article Interglobular Diffusion of an Energy …downloads.hindawi.com/journals/jspec/2013/261874.pdf0.050 0.025 0.000 300 325 350 375 400 (nm) 1 2 D (a) 500 600 700 800 400

Hindawi Publishing CorporationJournal of SpectroscopyVolume 2013 Article ID 261874 7 pageshttpdxdoiorg1011552013261874

Research ArticleInterglobular Diffusion of an Energy Donor inTriplet-Triplet Energy Transfer in Proteins

Andrey G Melnikov1 Alexander B Pravdin2 Vyacheslav I Kochubey2

Anna V Kuptsova1 and Gennady V Melnikov1

1 Saratov State Technical University Polytehnicheskaya Street 77 Saratov 410054 Russia2 Saratov State University Astrahanskaya Street 83 Saratov 410012 Russia

Correspondence should be addressed to Vyacheslav I Kochubey saratov gumailru

Received 10 July 2013 Revised 12 September 2013 Accepted 17 September 2013

Academic Editor Ekaterina Borisova

Copyright copy 2013 Andrey G Melnikov 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

The triplet-triplet energy transfer between polarmolecules of luminescent probe (eosin) as an energy donor andnonpolarmoleculesof energy acceptor (anthracene) is studied Both the donor and the acceptor are bound to human serum albumin by noncovalentbonds A dependence of rate constant of triplet-triplet energy transfer on human serum albumin concentration is revealed Arate constant of eosin output from protein globules is determined It is shown that the energy transfer occurs as a result ofinterglobular diffusion of eosinThe obtained results indicate that a protein-luminescent probe based sensor can be used for testinga concentration of polycyclic aromatic hydrocarbons in proteins

1 Introduction

The fundamental process of electron energy transfer formsa basis of light energy conversion in both natural photoac-cepting systems and artificial sensors [1ndash3]There is a numberof studies of energy transfer within the dispersed phaseof microheterogeneous systems this dispersed phase is thesupramolecular structures such as surfactant micelles [4ndash6]and biopolymer molecules in solution [7ndash9]

The energy transfer in microheterogeneous media forexample in a water-micellar media has some features com-pared to homogeneous media The difference is that thedonor and acceptor molecules are not uniformly distributedthroughout a volume of a solution They can be located forexample in a micellar microphase of surfactants solutions orin a protein globule [10] for liquid protein media where theenergy transfer occurs

One type of energy transfer is a triplet-triplet (T-T) oneThis process takes place at a distance between donor and acce-ptor being less or equal to 3ndash10 A [11] This feature allows oneto use the T-T energy transfer between donors and acceptorsbound to a human serum albumin (HSA) to determineminor

structural variations in proteins [12] The phosphorescenceof a probe that is an eosin bound to HSA macromoleculesturned to be sensitive to pH variations in protein solutions[13] and to the presence of heavy metal ions in proteinsQuenching of the eosin phosphorescence in the presence ofheavy metal ions is used thus helping us determine the pre-sence of heavy metals in human serum albumin [14]

Altogether the use of the triplet states of luminescentprobes is a very promising approach for diagnosis of livingconditions of biological objects

In this paper we consider a triplet-triplet energy transferbetween eosin being an energy donor and anthracene as anacceptor both bound to proteins The T-T energy transferbetween eosin and anthracene is studied in detail in [15 16]for homogeneous solutions These results can be extended tothe case of eosin-anthracene interactions in microheteroge-neous media such as protein solution Anthracene is knownto be a sort of polycyclic aromatic hydrocarbons (PAHs)Most of PAHs exhibit carcinogenic andmutagenic properties[17ndash20] and it is very important to develop methods oftheir detection in biological objects In particular elucidatingthe details of interaction of PAHs with transport proteins

2 Journal of Spectroscopy

including HSA is of great interest for medicine because themonitoring of PAH in blood plasma can estimate the risk ofcancer development

Since PAHs effectively interact with blood plasma trans-port proteins [21] we offer the method of detecting PAH inplasma using a sensor composed of a protein (HSA) and aluminescent probe (eosin) The aim of the present work is toprove the feasibility of the method under the conditions oflow PAH and probe concentrations

2 Materials and Methods

Studied solutions of eosin (Eosin Y Sigma) in concentrationof 4sdot10minus6Mwere prepared in phosphate buffer pH 74 For thepreparation of solutions twice-distilled water was used Inthe experiment HSAproduced by ldquoSigmaUSArdquowith contentof 99 of base substance was used In this work we used10 solution of HSA in phosphate buffer pH 74 containing015M Na

2

HPO4

-KH2

PO4

Monitoring of eosin dissolvingwas carried out by measurements of the optical densityof the studied solutions Absorption spectra were recordedon a Lambda 950 spectrophotometer (Perkin Elmer USA)Fluorescence and phosphorescence spectra were obtainedusing a spectrofluorometer Perkin Elmer LS45

Observation of eosin phosphorescence was performed indeoxygenated solutions Removal of oxygen from the testedsolutionswas carried out chemically by awell-knownmethod[22] of using sodium sulfite (manufactured by ldquoReahimrdquoRussia analytically pure grade) whose concentration in thesolution was 001M The anthracene (Fluka) was used as anenergy acceptor

The rate constant of deactivation of triplet states of eosinwas determined as follows At low concentrations of eosinwhen concentration quenching of the luminescence can beneglected the rate of deactivation of electronic excitationenergy of the triplet states [ 3119863] of eosin after pulse photoex-citation is given by [23]

119889 [

3

119863]

119889119905

= minus119896 [

3

119863] minus 120574[

3

119863]

2

(1)

where 119896 is the total rate constant of monomolecular andpseudomonomolecular (quenching by impurities) processesof deactivation of triplet molecules energy and 120574 is thebimolecular rate constant of the deactivation of tripletmolecules (triplet-triplet annihilation of eosin molecules)

If the triplet molecules become deactivated primarily asa result of a first-order reaction that is 119896 ≫ 120574[ 3119863] theequation describing the process of deactivation of tripletenergy of the molecules is simplified

119889 [

3

119863]

119889119905

= minus119896 [

3

119863]

(2)

Solving (2) with respect to [ 3119863] we get

[

3

119863(119905)] = [

3

119863]

0

119890

minus119896119905

(3)

The intensity of the eosin phosphorescence 119868ph is propor-tional to the concentration of its triplet molecules [ 3119863]

119868ph = 119860 [3

119863 (119905)] 119896rad (4)

hence

119868ph = 1198680119890minus119896119905

(5)

where 119868ph is the phosphorescence intensity at time 119905 119860 ishardware function of spectrofluorimeter 119896irr is the rate con-stant of probe phosphorescence [ 3119863]

0

and 1198680

are the concen-tration of triplet molecules and the phosphorescence initialintensity respectively 119896 is the rate constant of deactivation ofthe probe triplet states and 119905 is time

Thus the approximation of experimental data in thecoordinates ln(1198680ph119868ph) versus 119905 allows to determine the totalmonomolecular rate constant of deactivation of triplet mole-cules (119896) and to find a lifetime of the probe triplet states as120591 = 1119896

Statistical processing of the experimental data was per-formed by the method of least squares When measuringthe phosphorescence intensity resulting from the excitationpulse the relative error for seven measurements and forvalidity of 095 was 3The error in the determination of therate constants of the phosphorescence decay was 10

3 Results and Discussion

To study the interaction of proteins with PAHs we obtainedabsorption andfluorescence spectra of anthracene-HSA solu-tions with HSA concentration of 119862HSA = 1mgmL and119862HSA = 10mgmL Figure 1 shows (a) the absorption and (b)the fluorescence spectra of anthracene in solution at differ-ent concentrations of HSA The growth of the absorptionoptical density and the fluorescence of anthracene obse-rved with the increasing of HSA concentrations indicatesa significant increase of the anthracene content in pro-teins with the increase of protein microphase To deter-mine the extinction coefficient of the singlet-singlet absorp-tion of anthracene in HAS we choose HSA concentration119862HSA = 10mgmL According to the obtained linear depen-dence of the optical density of the singlet-singlet absorptionof anthracene on the wavelength of 360 nm (Figure 1(c))boundwith the protein globules versus the anthracene conce-ntration in the protein microphase we define the extinctioncoefficient of the singlet-singlet absorption of anthracene(2355plusmn 132Mminus1 cmminus1) Concentration of anthracene mole-cules in theHSAwas obtained according to119863 = 120576sdot119862sdot119897 where119863 is the optical density of the solution 120576 is the absorptioncoefficient of anthracene and 119897 is the length of the absorbinglayer (1 cm)

Since the anthracene is not soluble in water the obtainedresults indicate that the HSA binds anthracene that is alsoconfirmed by the results of [24]

Our studies show that eosin efficiently binds with HSAIn [25] it is shown that eosin also binds with bovine serumalbumin

Journal of Spectroscopy 3

0150

0125

0100

0075

0050

0025

0000300 325 350 375 400

120582 (nm)

1

2

D

(a)

500

600

700

800

400

300

200

100

0400 450 500 550

Ire

l

120582 (nm)

1

2

(b)

016

012

008

004

2 4 6 8120582 (nm)

D

(c)Figure 1 Spectra of (a) absorption and (b) fluorescence of anthracene in HSA (pH 74) Curves 1 and 2 correspond to HSA concentrations119862HSA = 10mgmL and119862HSA = 1mgmL respectively Wavelength of fluorescence excitation is 380 nm (c)The dependence of optical densityof singlet-singlet absorption of anthracene (120582 = 360 nm) on anthracene concentration in HSA solution (119862HSA = 10mgmL)

Figure 2 represents spectra of delayed fluorescence (DF)(the maximum at 120582max = 550 nm) and room temperaturephosphorescence (RTP) (the maximum at 120582max = 680 nm)of eosin in the HSA at different concentrations of HSA

On the basis of spectral and kinetic studies [13] we foundthat almost all eosin is gathered at protein globuleswhenHSAconcentration in a water solution reaches 119862HSA = 1mgmLBy changing the eosin concentration it was obtained that theoptimal concentration of eosin in HSA for the registration ofprolonged luminescence is 4 sdot 10minus6M

When anthracene is added to a solution of eosin in HASan increase of the rate constant of the eosin phosphorescencedecay is observed This is due to the triplet-triplet energytransfer from eosin to anthracene

When the anthracene concentration lies within 08 sdot10

minus5M and 78 sdot 10minus5M and the HSA concentration is

10mgmL the dependence of the rate constant of the phos-phorescence decay of eosin (119896) on the concentration ofanthracene is found to be almost linear (Figure 3 curve 2)For HSA concentration 119862HSA = 1mgmL a slight deviationfrom the linear dependence is observed (curve 1) Theresulting relationships can be approximated by straight linesin the 119862

119860

values interval from 05 sdot 10minus5M to 3 sdot 10minus5MThese dependencies allow us to determine the effective rateconstant of T-T energy transfer (119896efT-T) in human serumalbumin according to the Stern-Volmer equation

119896phos = (119896phos)0

+ 119896

efT-T sdot [119862119860] (6)

where 119896phos and (119896phos)0

are rate constants of eosin phos-phorescence decay in presence of acceptor anthracene and


500 600 700 8000

1

1

2 2

3

Ire

l

120582 (nm)

Figure 2 Luminescence spectra of eosin (119862eosin = 4 sdot 10minus6M) in

HSA Curve 1 119862HSA = 10mgmL curve 2 119862HSA = 1mgmL

800

700

600

500

400

300

2000 2

2

1

4 6 8

CAlowast10minus5 M

k (sminus1)

Figure 3 Dependences of the rate constant of eosin phosphores-cence decay at eosin concentration 4 sdot 10minus6M versus the anthraceneconcentration 119862

119860

both bound to serum albumin HSA concentra-tion is 119862HSA = 1mgmL (curve 1) and 119862HSA = 10mgmL (curve 2)The points represent experimental data the dashed lines are theirleast squares approximations and the solid lines are obtained bycalculations

in its absence respectively and [119862119860

] is the anthracene con-centration in HSA For HSA concentration 119862HSA = 1mgmL(Figure 3 curve 1) the effective rate constant of T-T transferis 14 sdot 106Mminus1sdotsminus1 when the acceptor concentration rangesfrom 08 sdot 10minus5M to 2 sdot 10minus5M When the HSA concentrationis 119862HSA = 10mgmL (Figure 3 curve 2) the rate constantis 30 sdot 106Mminus1sdotsminus1 for the acceptor concentrations rangingfrom 08 sdot 10minus5M to 78 sdot 10minus5M With the increasing of theHSA concentration the phosphorescence decay rate constantdecreases It indicates the decarese of the probability ofencounter of eosin and anthracene molecules as a resultof the distribution of anthracene molecules over a largernumber of HSA globules and the decrease in the numberof anthracene molecules in each protein globule This is due

to the fact that when the HSA concentration is 119862HSA =1mgmL the concentration of protein globules is muchsmaller than at 119862HSA = 10mgmL We hypothesize that asa result eosin and anthracene are more likely localized inthe same protein globule at 119862 = 1mgmL than at 119862 =10mgmL Increased local concentration of donor and accep-tor molecules in a single globule may increase the rateconstant of T-T energy transfer Because of this the detectionsensitivity of PAHs in proteins is increased

Thus these results suggest that the self-assembled systemincluding HSA macromolecule and luminescent probe maybe a sensor for PAHs determination

Since the eosin concentration is significantly lower thanthe concentration of the protein globules for definition of T-T energy transfers it is necessary to consider the interglobulardiffusion of energy donor molecules introduced into a solu-tion of HSA Let us examine the processes of migration ofdonor molecules and deactivation of the energy of the donorand acceptor added into a solution of HAS

(i) Leaving of the donor that is eosin molecules in tripletstate ( 3

119898

119863) from protein globule (119872) to water macrophase

3

119898

119863

119896

minus1

997888997888rarr

3

119863+119872

(7)

where 3119863 is the eosin molecule in triplet state in water phaseof solution and 119896

minus1

is the leaving rate of donor in triplet statefrom the protein globule

(ii) penetration of eosin molecule ( 3119863) from aqueousphase to protein globule (119872) not containing an anthracenemolecule as a result of encounters of eosin molecules withprotein globules

3

119863+119872

(1minus119901

)119896

1

[119872]

0

997888997888997888997888997888997888997888997888997888997888rarr

3

119898

119863

(8)

where 119901 is the probability of the fact that protein globulecontains at least one molecule of the energy acceptor thatis anthracene [119872]

0

is the protein globules concentrationequal toHSA concentration in solution 119896

1

is the rate constantof eosin penetration into protein globule from aqueousmacrophase The value of this rate constant is chosen to beequal to the rate constant of diffusion of eosin molecules inaqueous media 119896

1

= 65 sdot 10

9Mminus1sdotsminus1 [26] We assume thatthe filling of the globules by anthracene molecules obeys aPoisson distribution similar to filling of surfactant micellesby molecules from polycyclic aromatic hydrocarbons row[27 28]

1 minus 119901 = 119890

119901

(9)

where 119901 is the average coefficient of filling of the proteinglobule by luminescent probes For our purpose 119901 =[

1

119898

119860][119872] where [ 1119898

119860] is the concentration of anthracenein HSA globules This is equal to the concentration ofanthracene added to theHSA solution because all anthracenemolecules are bound to protein globules at chosen anthraceneconcentration


(iii) Penetration of the eosin molecules ( 3119863) from anaqueous phase to protein globule (119872) containing anthracenemolecule

3

119863+119872

119901119896

1

[119872]

0

997888997888997888997888997888997888rarr

3

119898

119863

(10)

We assume this is accompanied with a high probability bythe energy transfer between eosin and anthracene that is

immediately after the penetration the process 3119898

119863+

1

119898

119860

119896T-T997888997888997888rarr

1

119898

119863+

3

119898

119860 takes place In this case eosin located in theglobule containing anthracene goes into the ground singletstate Therefore the terms that are responsible for thesetwo processes cancel each other out and do not appearin the equations describing the rate of deactivation energyof the donor molecules ((15) (16)) see below Analogousassumption is as also used in [27] forwater-micellar solutions

(iv) Deactivation of the electronic excitation energy ofdonor bound to a protein globule as a result of monomolecu-lar and pseudomonomolecular processes of energy deactiva-tion with the rate constant 119896

2

3

119898

119863

119896

2

997888rarr

1

119898

119863+ℎ]119898phos (11)

where 1119898

119863 is the eosin molecule in ground state and ℎ]119898phosis the quantum of phosphorescence emitted by eosin in thetriplet state bound to protein globule The rate constant ofmonomolecular processes of deactivation of eosin energy(1198962

) can be represented by the following equation

119896

2

= 119896phos + 119896119902 [119876] (12)

where [119876] is the concentration of the quencher (usually thisis a residual molecular oxygen) 119896phos is the rate constantof the eosin phosphorescence and 119896

119902

is the rate constantfor quenching of the eosin triplet states by admixtures [119876]At a constant concentration of quencher [119876] and at thechosen chemical method of solution deoxygenation theconcentration of the residual oxygen reaches 119862 = 1 nM [29]and the summand 119896

119902

[119876] is the pseudomonomolecular rateconstant of the deactivation of triplet states of eosin as a resultof encounters with the quencher molecules According toFigure 3 the rate constants of donor triplet states deactivationare 340 sminus1 and 260 sminus1 for HSA concentration 1mgmL and10mgmL respectively These values were defined by eosinphosphorescence decay after pulse photoexcitation (119896

2

) in theabsence of acceptor molecules Reducing of the rate constantof the deactivation in the HSA addition may occur due toan increase of the eosin residence time in globular proteinphase This leads to the reducing of the probability of theencounter of eosin molecules with molecules of the residualoxygen contained in the water

(v) Monomolecular processes of donor triplet statesdeactivation in water macrophase (119896

3

)

3

119863

119896

3

997888rarr

1

119863+ℎ]119908phos (13)

where ℎ]119908phos is the quantum energy of light irradiated byeosin in water macrophase In water phase the rate constantof eosin triplet states deactivation reaches 1300 sminus1

(vi) Triplet-triplet transfer of the electronic excitationenergy between eosin penetrating from water macrophase toprotein globule and anthracene bound to protein globule

3

119863+

1

119898

119860

119901sdot119896

1

sdot[119872]

0

997888997888997888997888997888997888997888rarr

1

119863+

3

119898

119860

(14)

The concentration of proteins containing anthracene119901[119872]

0

is chosen approximately equal to the molecular con-centration of anthracene [ 1

119898

119860] since anthracene is practicallyinsoluble in water

On the base of the adduced processes the equation oftriplet states deactivations of eosin molecules can be writtenas follows

(a) for the donormolecules bound to the protein globule

119889 [

3

119898

119863]

119889119905

= 119896

1

[119872] [

3

119863] minus 119896

minus1

[

3

119898

119863] minus 119896

2

[

3

119898

119863]

(15)

(b) for the donor molecules in the water macrophase

119889 [

3

119863]

119889119905

= minus119896

1

[119872] [

3

119863] minus 119896

1

[

1

119898

119860] [

3

119863] + 119896

minus1

[

3

119898

119863] minus 119896

3

[

3

119863]

(16)

Here [119872] = (1 minus 119901)[119872]0

is the concentration of theanthracene-free protein globules The first term of (15)describes the entry of the excited eosin molecules into theanthracene-free globulesThe second term takes into accountthe exit of eosin in the excited state from the protein glob-ules and the third one corresponds to the monomolecularprocesses of deactivation of the excited eosin The first termof (16) takes into account the transition of the excited eosinfrom the water macrophase to the protein globules withoutanthracene The second term is responsible for the entranceof the excited eosin to the protein globule with anthracene Infact the second term describes the eosin deactivation by theT-T transfer at the entrance of the excited eosin to the proteinglobule with anthracene The third term takes into accountthe monomolecular processes of the eosin deactivation inwater macrophase

The solution of linear differential equations (15) and (16)is a sum of two exponentials whose exponents are the rootsof the characteristic equation of the system Both of themare negative and have magnitudes 105 and 102 respectivelyHence the first exponential term decays much faster than thesecond one and neglecting it one can write the solution as[

3

119863] [ 3119898

119863] sim exp(minus119896119905) Here 119896 is the smallest by magnitudecharacteristic number which plays the role of the decay rateof the eosin phosphorescence

We are interested in the dependence of 119896 on the con-centration of anthracene 119862

119860

and on the concentration ofHSA Graphs of this relationship are shown in Figure 3 bysolid lines The best agreement between experimental andtheoretical data is achieved with the following parameters119896

1

= 65 sdot 10

9Mminus1 sdot sminus1 1198963

= 1300 sminus1 for HSA concentration


of 119862HSA = 10mgmL 119896minus1

= 750 sminus1 1198962

= 260 sminus1 for theconcentration of HAS 119862HSA = 1mgmL 119896

minus1

= 500 sminus1119896

2

= 320 sminus1The values were quite large which justifies the accounting

of the process of the exit of eosin molecules from a proteinglobule when considering the processes of deactivation ofthe eosin triplet states in protein microphase Under theseexperimental conditions we did not observe an absorptionand fluorescence spectra of eosin in the water macrophaseThis can be explained by the fact that the rate constantfor eosin leaving from the protein globule is less than therate constant of the eosin entry into the protein globuleConsequently eosin is predominantly localized in the proteinmicrophase Its concentration in the aqueous macrophase ismuch less than in protein microphase This makes it difficultto record absorption and fluorescence spectra of eosin inwater macrophase

4 Conclusion

Thus T-T transfer between luminescent probe andPAH bothbound to protein occurring when the probe concentration ismuch lower than the concentration of protein globules maybe sustained by interglobular diffusion of hydrophilic polardonor (eosin)

It is shown that the self-assemble system consisting of aHSAmacromolecule and a fluorescent probemay be a sensorfor PAHs determination To achieve the optimal sensitivitythe appropriate ratios between components of the sensornamely HSA eosin and anthracene were suggested

Self-assembled luminescent sensor based onHSAmacro-molecule and probes noncovalently bound to the proteinallows the detection of the carcinogenic PAH molecules inproteins by means of the registration of the change in thekinetics of the eosin probe phosphorescence decay Sensitivityof determination of PAHs by the suggested sensor increasesin the decreasing of the content of proteins in the solution to119862HSA = 1mgmL

Acknowledgments

This study was supported in part by RF GovernmentalContracts 14B37110563 and by Russian Foundation for BasicResearch Grant N12-02-31196

References

[1] H V Hsieh D B Sherman S A Andaluz T J Amiss and J BPitner ldquoFluorescence resonance energy transfer glucose sensorfrom site-specific dual labeling of glucosegalactose bindingprotein using ligand protectionrdquo Journal of Diabetes Science andTechnology vol 6 no 6 pp 1286ndash1295 2012

[2] I L Medintz E R Goldman M E Lassman and J M MauroldquoA fluorescence resonance energy transfer sensor based onmaltose binding proteinrdquo Bioconjugate Chemistry vol 14 no 5pp 909ndash918 2003

[3] H M Green and J Alberola-Ila ldquoDevelopment of ERK activitysensor an in vitro FRET-based sensor of extracellular regulatedkinase activityrdquo BMC Chemical Biology vol 5 2005

[4] J R Escabi-Perez F Nome and J H Fendler ldquoEnergy transferin micellar systems Steady state and time resolved lumines-cence of aqueous micelle solubilized naphthalene and terbiumchloriderdquo Journal of the American Chemical Society vol 99 no24 pp 7749ndash7754 1977

[5] J R Darwent W Dong C D Flint and N W SharpeldquoIntermolecular energy transfer between phenanthrene andlanthanide ions in aqueous micellar solutionrdquo Journal of theChemical Society Faraday Transactions vol 89 no 6 pp 873ndash880 1993

[6] J P Chauvet M Bazin and R Santus ldquoOn the triplet-tripletenergy transfer from chlorophyll to carotene in triton x 100micellesrdquo Photochemistry and Photobiology vol 41 pp 83ndash901985

[7] K M Sanchez D E Schlamadinger J E Gable and J E KimldquoForster resonance energy transfer and conformational stabilityof proteins an advanced biophysical module for physicalchemistry studentsrdquo Journal of Chemical Education vol 85 no9 pp 1253ndash1256 2008

[8] H Zemel andBMHoffman ldquoLong-range triplet-triplet energytransfer within metal-substituted hemoglobinsrdquo Journal of theAmerican Chemical Society vol 103 no 5 pp 1192ndash1201 1981

[9] J Tian X Liu Y Zhao and S Zhao ldquoStudies on the interactionbetween tetraphenylporphyrin compounds and bovine serumalbuminrdquo Luminescence vol 22 no 5 pp 446ndash454 2007

[10] A Husain J Ovadia and L I Grossweiner ldquoPulse radiolysis ofthe eosin-human serum albumin complexrdquo Transactions of theFaraday Society vol 66 pp 1472ndash1484 1970

[11] V L Ermolaev E G Sveshnikova and T A Shakhverdov ldquoEne-rgy transfer between organic molecules and transition metalionsrdquo Russian Chemical Reviews vol 44 no 1 pp 26ndash40 1975

[12] A G MelrsquoNikov A M Saletskii V I Kochubey A B PravdinI S Kurchatov and G V MelrsquoNikov ldquoTriplet-triplet energytransfer between luminescent probes bound to albuminsrdquoOptics and Spectroscopy vol 109 no 2 pp 188ndash192 2010

[13] A M Saletskii A G Melrsquonikov A B Pravdin V I Kochubeiand G V Melrsquonikov ldquoStructural changes in human serum albu-min according to the data on the phosphorescence kinetics of aluminescent probemdasheosinrdquo Journal of Applied Spectroscopy vol72 no 5 pp 723ndash727 2005

[14] E V Naumova A G Melnikov and G V Melnikov ldquoLumi-nescence quenching by heavy metal ions of probes basedon anthracene pyrene and eosin in human serum albuminrdquoJournal of Applied Spectroscopy vol 80 no 2 pp 159ndash163 2013

[15] K Kikuchi H Kokubun and M Koizumi ldquoStudies of tripletenergy transfer by means of an emission-absorption flashtechnique i reversible energy transfer between xanthene dyesand anthracenesrdquo Bulletin of the Chemical Society of Japan vol43 pp 2732ndash2739 1970

[16] G A Ketsle L V Levshin G V Melnikov and B F MinaevldquoMechanism of the effect of a magnetic field on mixed-typetriplet-triplet annihilationrdquo Optics and Spectroscopy vol 51 no4 pp 367ndash369 1981

[17] P B Farmer O Sepai R Lawrence et al ldquoBiomonitoringhuman exposure to environmental carcinogenic chemicalsrdquoMutagenesis vol 11 no 4 pp 363ndash381 1996

[18] M McKenzie T McLemore and P Rankin ldquoA human plasmacomponent that binds benzo(a)pyrenerdquo Cancer vol 42 no 6pp 2733ndash2737 1978

[19] S L Tannheimer S L Barton S P Ethier and S W Bur-chiel ldquoCarcinogenic polycyclic aromatic hydrocarbons increase


intracellular Ca2+ and cell proliferation in primary humanmammary epithelial cellsrdquo Carcinogenesis vol 18 no 6 pp1177ndash1182 1997

[20] D YuM G Kazanietz R G Harvey and TM Penning ldquoPoly-cyclic aromatic hydrocarbon o-Quinones inhibit the activity ofthe catalytic fragment of protein kinase CrdquoBiochemistry vol 41no 39 pp 11888ndash11894 2002

[21] K Skupinska M Zylm I Misiewicz and T Kasprzycka-Gut-tman ldquoInteraction of anthracene and its oxidative derivativeswith human serum albuminrdquo Acta Biochimica Polonica vol 53no 1 pp 101ndash112 2006

[22] V M Mazhul and D G Scherbin ldquoTryptophan phosphores-cence as a monitor of flexibility of membrane proteins in cellsrdquoin Advances in Fluorescence Sensing Technology III Proceedingsof SPIE pp 507ndash514 February 1997

[23] C A Parker Photoluminescence of Solutions with Applicationsto Photochemistry and Analytical Chemistry Elsevier 1968

[24] A M Saletskii A G MelrsquoNikov A B Pravdin V I Kochubeiand G V Melnrsquoikov ldquoComplexation of pyrene and anthracenewith human blood plasmardquo Journal of Applied Spectroscopy vol75 no 3 pp 402ndash406 2008

[25] A A Waheed K S Rao and P D Gupta ldquoMechanism of dyebinding in the protein assay using eosin dyesrdquo Analytical Bio-chemistry vol 287 no 1 pp 73ndash79 2000

[26] V L Ermolaev E N Bodunov E B Sveshnikova and TA Shakhverdov Nonradiative Transfer of Electronic ExcitationEnergy Nauka Leningrad Russia 1977 (Russian)

[27] K Glasle U K A Klein and M Hauser ldquoIntermicellar excha-nge dynamics of solubilized reactantsrdquo Journal of MolecularStructure vol 84 no 3-4 pp 353ndash360 1982

[28] G Rothenberger P P Infelta and M Gratzel ldquoDynamics andstatistics of triplet-triplet annihilation in micellar assembliesrdquoJournal of Physical Chemistry vol 85 no 13 pp 1850ndash1856 1981

[29] S Z Kananovich and V M Mazhulrsquo ldquoFluorimetric analysisof the structural-dynamic state of alkaline phosphatase ofEscherichia colirdquo Journal of Applied Spectroscopy vol 70 no 5pp 765ndash769 2003

Submit your manuscripts athttpwwwhindawicom

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including HSA is of great interest for medicine because themonitoring of PAH in blood plasma can estimate the risk ofcancer development

Since PAHs effectively interact with blood plasma trans-port proteins [21] we offer the method of detecting PAH inplasma using a sensor composed of a protein (HSA) and aluminescent probe (eosin) The aim of the present work is toprove the feasibility of the method under the conditions oflow PAH and probe concentrations

2 Materials and Methods

Studied solutions of eosin (Eosin Y Sigma) in concentrationof 4sdot10minus6Mwere prepared in phosphate buffer pH 74 For thepreparation of solutions twice-distilled water was used Inthe experiment HSAproduced by ldquoSigmaUSArdquowith contentof 99 of base substance was used In this work we used10 solution of HSA in phosphate buffer pH 74 containing015M Na

2

HPO4

-KH2

PO4

Monitoring of eosin dissolvingwas carried out by measurements of the optical densityof the studied solutions Absorption spectra were recordedon a Lambda 950 spectrophotometer (Perkin Elmer USA)Fluorescence and phosphorescence spectra were obtainedusing a spectrofluorometer Perkin Elmer LS45

Observation of eosin phosphorescence was performed indeoxygenated solutions Removal of oxygen from the testedsolutionswas carried out chemically by awell-knownmethod[22] of using sodium sulfite (manufactured by ldquoReahimrdquoRussia analytically pure grade) whose concentration in thesolution was 001M The anthracene (Fluka) was used as anenergy acceptor

The rate constant of deactivation of triplet states of eosinwas determined as follows At low concentrations of eosinwhen concentration quenching of the luminescence can beneglected the rate of deactivation of electronic excitationenergy of the triplet states [ 3119863] of eosin after pulse photoex-citation is given by [23]

119889 [

3

119863]

119889119905

= minus119896 [

3

119863] minus 120574[

3

119863]

2

(1)

where 119896 is the total rate constant of monomolecular andpseudomonomolecular (quenching by impurities) processesof deactivation of triplet molecules energy and 120574 is thebimolecular rate constant of the deactivation of tripletmolecules (triplet-triplet annihilation of eosin molecules)

If the triplet molecules become deactivated primarily asa result of a first-order reaction that is 119896 ≫ 120574[ 3119863] theequation describing the process of deactivation of tripletenergy of the molecules is simplified

119889 [

3

119863]

119889119905

= minus119896 [

3

119863]

(2)

Solving (2) with respect to [ 3119863] we get

[

3

119863(119905)] = [

3

119863]

0

119890

minus119896119905

(3)

The intensity of the eosin phosphorescence 119868ph is propor-tional to the concentration of its triplet molecules [ 3119863]

119868ph = 119860 [3

119863 (119905)] 119896rad (4)

hence

119868ph = 1198680119890minus119896119905

(5)

where 119868ph is the phosphorescence intensity at time 119905 119860 ishardware function of spectrofluorimeter 119896irr is the rate con-stant of probe phosphorescence [ 3119863]

0

and 1198680

are the concen-tration of triplet molecules and the phosphorescence initialintensity respectively 119896 is the rate constant of deactivation ofthe probe triplet states and 119905 is time

Thus the approximation of experimental data in thecoordinates ln(1198680ph119868ph) versus 119905 allows to determine the totalmonomolecular rate constant of deactivation of triplet mole-cules (119896) and to find a lifetime of the probe triplet states as120591 = 1119896

Statistical processing of the experimental data was per-formed by the method of least squares When measuringthe phosphorescence intensity resulting from the excitationpulse the relative error for seven measurements and forvalidity of 095 was 3The error in the determination of therate constants of the phosphorescence decay was 10

3 Results and Discussion

To study the interaction of proteins with PAHs we obtainedabsorption andfluorescence spectra of anthracene-HSA solu-tions with HSA concentration of 119862HSA = 1mgmL and119862HSA = 10mgmL Figure 1 shows (a) the absorption and (b)the fluorescence spectra of anthracene in solution at differ-ent concentrations of HSA The growth of the absorptionoptical density and the fluorescence of anthracene obse-rved with the increasing of HSA concentrations indicatesa significant increase of the anthracene content in pro-teins with the increase of protein microphase To deter-mine the extinction coefficient of the singlet-singlet absorp-tion of anthracene in HAS we choose HSA concentration119862HSA = 10mgmL According to the obtained linear depen-dence of the optical density of the singlet-singlet absorptionof anthracene on the wavelength of 360 nm (Figure 1(c))boundwith the protein globules versus the anthracene conce-ntration in the protein microphase we define the extinctioncoefficient of the singlet-singlet absorption of anthracene(2355plusmn 132Mminus1 cmminus1) Concentration of anthracene mole-cules in theHSAwas obtained according to119863 = 120576sdot119862sdot119897 where119863 is the optical density of the solution 120576 is the absorptioncoefficient of anthracene and 119897 is the length of the absorbinglayer (1 cm)

Since the anthracene is not soluble in water the obtainedresults indicate that the HSA binds anthracene that is alsoconfirmed by the results of [24]

Our studies show that eosin efficiently binds with HSAIn [25] it is shown that eosin also binds with bovine serumalbumin


0150

0125

0100

0075

0050

0025

0000300 325 350 375 400

120582 (nm)

1

2

D

(a)

500

600

700

800

400

300

200

100

0400 450 500 550

Ire

l

120582 (nm)

1

2

(b)

016

012

008

004

2 4 6 8120582 (nm)

D








119860


119896phos = (119896phos)0

+ 119896

efT-T sdot [119862119860] (6)




500 600 700 8000

1

1

2 2

3

Ire

l

120582 (nm)



800

700

600

500

400

300

2000 2

2

1

4 6 8

CAlowast10minus5 M

k (sminus1)


119860








119898


3

119898

119863

119896

minus1

997888997888rarr

3

119863+119872

(7)


minus1



3

119863+119872

(1minus119901

)119896

1

[119872]

0

997888997888997888997888997888997888997888997888997888997888rarr

3

119898

119863

(8)


0


1


1

= 65 sdot 10


1 minus 119901 = 119890

119901

(9)


1

119898

119860][119872] where [ 1119898




3

119863+119872

119901119896

1

[119872]

0

997888997888997888997888997888997888rarr

3

119898

119863

(10)



119863+

1

119898

119860

119896T-T997888997888997888rarr

1

119898

119863+

3

119898



2

3

119898

119863

119896

2

997888rarr

1

119898

119863+ℎ]119898phos (11)

where 1119898



119896

2

= 119896phos + 119896119902 [119876] (12)


119902


119902


2



3

)

3

119863

119896

3

997888rarr

1

119863+ℎ]119908phos (13)



3

119863+

1

119898

119860

119901sdot119896

1

sdot[119872]

0

997888997888997888997888997888997888997888rarr

1

119863+

3

119898

119860

(14)


0


119898




119889 [

3

119898

119863]

119889119905

= 119896

1

[119872] [

3

119863] minus 119896

minus1

[

3

119898

119863] minus 119896

2

[

3

119898

119863]

(15)


119889 [

3

119863]

119889119905

= minus119896

1

[119872] [

3

119863] minus 119896

1

[

1

119898

119860] [

3

119863] + 119896

minus1

[

3

119898

119863] minus 119896

3

[

3

119863]

(16)

Here [119872] = (1 minus 119901)[119872]0



3

119863] [ 3119898



119860


1

= 65 sdot 10





= 750 sminus1 1198962


minus1

= 500 sminus1119896

2



4 Conclusion




Acknowledgments


References









































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


0150

0125

0100

0075

0050

0025

0000300 325 350 375 400

120582 (nm)

1

2

D

(a)

500

600

700

800

400

300

200

100

0400 450 500 550

Ire

l

120582 (nm)

1

2

(b)

016

012

008

004

2 4 6 8120582 (nm)

D








119860


119896phos = (119896phos)0

+ 119896

efT-T sdot [119862119860] (6)




500 600 700 8000

1

1

2 2

3

Ire

l

120582 (nm)



800

700

600

500

400

300

2000 2

2

1

4 6 8

CAlowast10minus5 M

k (sminus1)


119860








119898


3

119898

119863

119896

minus1

997888997888rarr

3

119863+119872

(7)


minus1



3

119863+119872

(1minus119901

)119896

1

[119872]

0

997888997888997888997888997888997888997888997888997888997888rarr

3

119898

119863

(8)


0


1


1

= 65 sdot 10


1 minus 119901 = 119890

119901

(9)


1

119898

119860][119872] where [ 1119898




3

119863+119872

119901119896

1

[119872]

0

997888997888997888997888997888997888rarr

3

119898

119863

(10)



119863+

1

119898

119860

119896T-T997888997888997888rarr

1

119898

119863+

3

119898



2

3

119898

119863

119896

2

997888rarr

1

119898

119863+ℎ]119898phos (11)

where 1119898



119896

2

= 119896phos + 119896119902 [119876] (12)


119902


119902


2



3

)

3

119863

119896

3

997888rarr

1

119863+ℎ]119908phos (13)



3

119863+

1

119898

119860

119901sdot119896

1

sdot[119872]

0

997888997888997888997888997888997888997888rarr

1

119863+

3

119898

119860

(14)


0


119898




119889 [

3

119898

119863]

119889119905

= 119896

1

[119872] [

3

119863] minus 119896

minus1

[

3

119898

119863] minus 119896

2

[

3

119898

119863]

(15)


119889 [

3

119863]

119889119905

= minus119896

1

[119872] [

3

119863] minus 119896

1

[

1

119898

119860] [

3

119863] + 119896

minus1

[

3

119898

119863] minus 119896

3

[

3

119863]

(16)

Here [119872] = (1 minus 119901)[119872]0



3

119863] [ 3119898



119860


1

= 65 sdot 10





= 750 sminus1 1198962


minus1

= 500 sminus1119896

2



4 Conclusion




Acknowledgments


References









































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


500 600 700 8000

1

1

2 2

3

Ire

l

120582 (nm)



800

700

600

500

400

300

2000 2

2

1

4 6 8

CAlowast10minus5 M

k (sminus1)


119860








119898


3

119898

119863

119896

minus1

997888997888rarr

3

119863+119872

(7)


minus1



3

119863+119872

(1minus119901

)119896

1

[119872]

0

997888997888997888997888997888997888997888997888997888997888rarr

3

119898

119863

(8)


0


1


1

= 65 sdot 10


1 minus 119901 = 119890

119901

(9)


1

119898

119860][119872] where [ 1119898




3

119863+119872

119901119896

1

[119872]

0

997888997888997888997888997888997888rarr

3

119898

119863

(10)



119863+

1

119898

119860

119896T-T997888997888997888rarr

1

119898

119863+

3

119898



2

3

119898

119863

119896

2

997888rarr

1

119898

119863+ℎ]119898phos (11)

where 1119898



119896

2

= 119896phos + 119896119902 [119876] (12)


119902


119902


2



3

)

3

119863

119896

3

997888rarr

1

119863+ℎ]119908phos (13)



3

119863+

1

119898

119860

119901sdot119896

1

sdot[119872]

0

997888997888997888997888997888997888997888rarr

1

119863+

3

119898

119860

(14)


0


119898




119889 [

3

119898

119863]

119889119905

= 119896

1

[119872] [

3

119863] minus 119896

minus1

[

3

119898

119863] minus 119896

2

[

3

119898

119863]

(15)


119889 [

3

119863]

119889119905

= minus119896

1

[119872] [

3

119863] minus 119896

1

[

1

119898

119860] [

3

119863] + 119896

minus1

[

3

119898

119863] minus 119896

3

[

3

119863]

(16)

Here [119872] = (1 minus 119901)[119872]0



3

119863] [ 3119898



119860


1

= 65 sdot 10





= 750 sminus1 1198962


minus1

= 500 sminus1119896

2



4 Conclusion




Acknowledgments


References









































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



3

119863+119872

119901119896

1

[119872]

0

997888997888997888997888997888997888rarr

3

119898

119863

(10)



119863+

1

119898

119860

119896T-T997888997888997888rarr

1

119898

119863+

3

119898



2

3

119898

119863

119896

2

997888rarr

1

119898

119863+ℎ]119898phos (11)

where 1119898



119896

2

= 119896phos + 119896119902 [119876] (12)


119902


119902


2



3

)

3

119863

119896

3

997888rarr

1

119863+ℎ]119908phos (13)



3

119863+

1

119898

119860

119901sdot119896

1

sdot[119872]

0

997888997888997888997888997888997888997888rarr

1

119863+

3

119898

119860

(14)


0


119898




119889 [

3

119898

119863]

119889119905

= 119896

1

[119872] [

3

119863] minus 119896

minus1

[

3

119898

119863] minus 119896

2

[

3

119898

119863]

(15)


119889 [

3

119863]

119889119905

= minus119896

1

[119872] [

3

119863] minus 119896

1

[

1

119898

119860] [

3

119863] + 119896

minus1

[

3

119898

119863] minus 119896

3

[

3

119863]

(16)

Here [119872] = (1 minus 119901)[119872]0



3

119863] [ 3119898



119860


1

= 65 sdot 10





= 750 sminus1 1198962


minus1

= 500 sminus1119896

2



4 Conclusion




Acknowledgments


References









































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



= 750 sminus1 1198962


minus1

= 500 sminus1119896

2



4 Conclusion




Acknowledgments


References









































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










Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

Documents

Research Article Interglobular Diffusion of an Energy …downloads.hindawi.com/journals/jspec/2013/261874.pdf0.050 0.025 0.000 300 325 350 375 400 (nm) 1 2 D (a) 500 600 700 800 400