Journal of Luminescence 132 (2012) 2612–2618

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Journal of Luminescence

0022-23

http://d

n Corr

E-m

scbhatt

journal homepage: www.elsevier.com/locate/jlumin

Selective fluorescence resonance energy transfer from serum albumins to abio-active 3-pyrazolyl-2-pyrazoline derivative: A spectroscopic analysis

Arindam Sarkar, Subhash Chandra Bhattacharya n

Department of Chemistry, Jadavpur University, Kolkata 700032, India

a r t i c l e i n f o

Article history:

Received 11 November 2011

Received in revised form

23 April 2012

Accepted 27 April 2012Available online 9 May 2012

Keywords:

Tryptophan

FRET

Emission

Lifetime

Rotamer

13/$ - see front matter & 2012 Elsevier B.V. A

x.doi.org/10.1016/j.jlumin.2012.04.053

esponding author. Tel.: þ91 33 24146223; fa

ail addresses: sbjuchem@yahoo.com,

acharyya@chemistry.jdvu.ac.in (S.C. Bhattach

a b s t r a c t

A novel fluorescent probe and pharmaceutically significant: 3-pyrazolyl-2-pyrazoline derivative (PYZ)

has been selected as an acceptor molecule for fluorescence resonance energy transfer (FRET) interaction

with serum albumins. Steady state and time resolved fluorescence techniques were applied to elucidate

the nature of interaction of PYZ with serum albumins (BSA and HSA). Negligible FRET mediated

emission occurred in the case of HSA but an efficient FRET mediated emission resulted in case of BSA. To

gain further insight into the FRET selectivity of PYZ with the proteins, FRET from L-tryptophan (donor;

native tryptophan) to PYZ (acceptor) was performed with the aim of getting an idea about the steric

restrictions imposed on PYZ by the other groups present in BSA and HSA. The studies revealed that the

surface bound Trp-134 in BSA allows an efficient FRET process with PYZ while the buried Trp-214 in

HSA does not. The unusual selectivity for FRET in case of PYZ and the serum albumins has also been

attributed to the complex structure of PYZ due to the presence of bulkier phenyl moieties in it.

The complex nature of the excited state photophysics of tryptophan (Trp) in proteins also accounts for

this FRET selectivity of PYZ with BSA and HSA.

& 2012 Elsevier B.V. All rights reserved.

1. Introduction

Recently, various fluorescent probes have been developed tostudy biological phenomena in living cells where fluorescenceresonance energy transfer (FRET) has been used in many cases[1,2]. FRET is the radiationless transfer of excited-state energyfrom an initially excited donor to an acceptor [3]. It is a valuabletool for characterizing proximity relationships within andbetween biomolecules [4–14] and a spectroscopic technique formeasuring distances in the 30–80 A range. Excitation energy ofthe donor is transferred to the acceptor via an induced dipole–dipole interaction [15,16]. The FRET technique has been applied toprobe biological systems and also for high-throughput screeningof combinatorial libraries, by means of ratiometric measurementsthat observe the changes in the ratio of the fluorescence inten-sities at two wavelengths. Using ratiometric measurements, it ispossible to reduce the influence of many artifacts due to thechange of the probe concentration and excitation intensity. Thistechnique allows more precise measurements, and with someprobes, quantitative detection is possible.

ll rights reserved.

x: þ91 33 24146584.

arya).

2-pyrazoline derivatives constitute a class of fluorescent com-pounds of pharmaceutical importance [17–20]. Recent trend tosynthesize these types of compounds has been augmented for itsanti-inflammatory, antidiabetic, anesthetic, analgesic and gluta-mate transport sensor properties [21–25]. The interest on theinteraction of pyrazoline derivatives with protein using FREToriginates principally from two aspects—the first stems from itsnovel biological applications in pharmaceuticals and the secondone arises due to the presence of electron donors and acceptors inthem. These bioactive fluorescent molecules can effectively serveas reporters of the psychological activities in living systems. Thesolvent dependent radiative transitions and relaxation dynamicsof pyrazole substituted 2-pyrazoline (PZ) from S1 and S2 stateshave been established in our earlier publication [26] In previousstudies from our laboratory, interesting uses of PZ as a sensitivefluorescence probe for exploring the local environments in mem-brane mimetic organized assemblies were demonstrated [27]. Thedual effect of polarity and hydrogen bonding of various solventswas also investigated on our current fluorophore (PYZ) and ityielded interesting results [28]. The interaction between PZ andbiological receptors like serum albumins has also been investi-gated but it produced different results than PYZ [29]. Serumalbumins are the most abundant of all the proteins in bloodplasma, accounting for approximately 60% of the total serumprotein content. The secondary structure of the polypeptidechain consists of about 580 amino acids with approximately

Scheme 1. Two side-on 3D graphic representations of a BSA model structure

based on HSA x-ray crystal structure (PDB ID: 1UOR) [30]. The unbroken circles

show the binding sites [32] which are located in sub-domains IIA and IIIA. The

dashed circles centered at the tryptophan residues show the distance limit for 50%

FRET efficiency.

N

N

Ph

H3C CN

N N

C6H4Br (p)

1

2

3

5

1'2'

3'

4'

5'

PhH

H

Ph

4

Scheme 2. 5-((4S,5R)-1-(4-bromophenyl)-4,5-dihydro-4,5-diphenyl-1H-pyrazol-

3-yl)-3-methyl-1-phenyl-1H-pyrazole-4-carbonitrile [PYZ].

A. Sarkar, S.C. Bhattacharya / Journal of Luminescence 132 (2012) 2612–2618 2613

67% present as alpha-helices without betasheets. The protein iscomposed of three homologous domains (I, II, and III) which aredivided into two sub-domains (A and B). In addition, there are 9loops and 17 disulphide bridges, which make a heart-shaped 3Dstructure of the protein molecule. The principal function of serumalbumin is to transport a wide variety of fatty acids and metabo-lites via the main binding regions located in sub-domains IIA (Site1) and IIIA (Site 2). Human and bovine serum albumins (HSA andBSA, respectively) are probably the most studied serum albuminproteins. They exhibit approximately 76% homology and a repeat-ing pattern of disulphides which are conserved. The main differ-ence between the two proteins is that in HSA there is only onetryptophan amino acid (Trp-214) which is located at position 214(equivalent to Trp-212 for BSA), buried in a hydrophobic pocket atsub-domain IIA; whereas in BSA an additional tryptophan aminoacid Trp-134, which is more exposed to solvent, is found at sub-domain IB [30–32] (Scheme 1). Tryptophan (Trp) residues are theleast common amino acid in proteins and typically dominate thefluorescence of a given protein [33,34]. Hence, they are the mostcommonly used intrinsic fluorophores. The fluorescence emissionfrom Trp residues is extremely sensitive to changes in localenvironment (�30-fold variation in quantum yield and �50 nmchange in emission maxima) [35]. Factors that influence trypto-phan emission are as follows: (i) in nonpolar environment, Trpshows structured blueshifted emission as indole in cyclohexane,(ii) as Trp residues become hydrogen bonded or exposed to water,the emission shifts to longer wavelengths, (iii) resonance energytransfer among the tryptophan (Trp) residues, (iv) quenching byproton transfer from nearby charged amino groups, (v) quenchingby electron acceptors such as protonated carboxyl groups, and (vi)electron transfer quenching by disulfides, amides and by peptidebonds in the protein backbone. As indicated earlier, this sensitiv-ity has been exploited to monitor numerous biological processes;however, the inability to pinpoint the causes of changes in thefluorescence yield limits the informational content of such mea-surements [36–38]. Further complications in interpretation of thechanges in fluorescence at a molecular level are added if there ismore than one Trp in a protein. In the present investigation,an attempt has been made to study the interaction of newlysynthesized bioactive compound 3-pyrazolyl-2-pyrazoline deri-vative (PYZ) with model transport proteins, bovine serum albu-min (BSA) and human serum albumin (HSA) employing steadystate and time resolved fluorescence techniques. It has beenour objective to evaluate the probable in vivo transportationpathways of PYZ and investigate their interactions with human(HSA) and bovine serum albumin (BSA).

2. Experimental Details

PYZ (Empirical Formula: C32H24N5Br) was synthesized asdescribed in Ref. [39] (Scheme 2). BSA (98%, fraction V), HSA(96%), tryptophan and HEPES (N-[2-hydroxyethyl]-piperazine-N’-[2-ethanesulphonicacid]) were obtained from Sigma-Aldrich andwere used as received. 50 mM HEPES buffer solution was pre-pared, and its pH was adjusted to 7.0. The same buffer solutionwas used as medium throughout the experiment. Triply distilledwater was used for the preparation of the experimental solutions.The purified solvents were found to be free from impurities andwere transparent in the spectral region of interest. Absorptionspectra were recorded using a Shimadzu (Japan) UV–vis 1700spectrophotometer with a matched pair of silica cuvettes of pathlength 1 cm. Fluorescence spectra were taken in a F-IIA spectro-fluorimeter (Spex, INC, NJ, USA) with an external slit widthof 1.25 mm. All measurements were done repeatedly and repro-ducible results were obtained. All fluorescence spectra werecorrected for the instrumental response. Fluorescence lifetimeswere determined from time-resolved intensity decay by themethod of time-correlated single-photon counting (TCSPC) usinga pulsed diode Nano LED-295 (295 nm) as light source. Thetypical response time of the system at 295 nm is 10 ps. The datastored in a multichannel analyzer was routinely transferred toIBH DAS-6 decay analysis software. For all the lifetime measure-ments the fluorescence decay curves were analyzed by a biexpo-nential iterative fitting program provided by IBH. Mean (average)lifetimes (t) for biexponential decays of fluorescence were calcu-lated from the decay times and pre-exponential factors usingequation [40]

IðtÞ ¼Xn

i ¼ 1

Aie�t=t

i

where Ai is a pre-exponential factor representing fractionalcontribution to the time resolved decay of the component witha lifetime t. Mean (average) lifetimes (tavg), for biexponentialdecays of fluorescence were calculated from the decay times andpre-exponential factors using the equation

tavg ¼ ða1t1þa2t2Þ=ða1þa2Þ

Geometrical optimization of PYZ was performed using AM1method in Hyperchem 8.0 to calculate the dipole momentand other energy parameters of the molecule (Provided asSupplementary data).

300 350 400 450 500 550

Inte

nsity

wavelength / nm

Serum Albumins emissionPYZ absorbance

Fig. 1. Normalized overlap spectrum of serum albumin emission and PYZ

absorbance.

200 250 300 350 400 450

425

Fluo

resc

ence

Inte

nsity

/ a.

u

wavelength / nm

Inte

nsity

/ a.

u

wavelength / nm

575550525500475450

Fig. 2. Excitation spectra of PYZ. [PYZ]¼8.0�10�6 M and lem¼485 nm. Inset:

emission spectra of PYZ. [PYZ]¼8.0�10�6 M; lex¼378 nm and pH¼7.0.

300 350 400 450 500

300 350 400 450 500

PYZ

BSA

BSA - PYZ

Fluo

resc

ence

Inte

nsity

/ a.

u

wavelength /nm

Fluo

resc

ence

Inte

nsity

/a.u

wavelength / nm

HSA - PYZ

HSA

PYZ

Fig. 3. (a) FRET profile of BSA and PYZ. [BSA]¼4.0�10�6 M; [PYZ]¼0 to

34�10�6 M; lex¼285 nm and pH¼7.0. (b) FRET profile of HSA and PYZ.

[HSA]¼6.0�10�6 M; [PYZ]¼0 to 34�10�6 M; lex¼285 nm and pH¼7.0.

A. Sarkar, S.C. Bhattacharya / Journal of Luminescence 132 (2012) 2612–26182614

3. Results and discussions

3.1. Energy transfer from serum albumins to PYZ: steady state

analysis

Fig. 1 depicts the overlap spectrum of serum albumin emissionand PYZ absorbance. The emission and excitation spectra of PYZhave been given in Fig. 2. There was no change in serum albuminabsorbance on addition of PYZ (figure not shown). Fig. 3a and billustrates the changes in emission of BSA and HSA respectivelyupon addition of PYZ, the fluorescence intensity at short wave-length corresponding to decrease in albumin (donor) emission,whereas that at long wavelength corresponding to increase in PYZ(acceptor) emission. For each spectrum realized in the presence ofserum albumins and PYZ, the corresponding control spectrumwith just PYZ in buffer has been subtracted. Prominent iso-emissive points appeared at 420 nm and 405 nm for BSA andHSA, respectively. The decrease in BSA emission is much promi-nent than that of HSA emission but increase in acceptor emission(PYZ) is quite pronounced in case of both the serum albumins.

This clearly indicates that PYZ behaves differently with BSA andHSA involving different mode of interactions. Here, we havecalculated the FRET efficiencies by measuring the decrease inthe donor fluorescence because it is more reproducible than theFRET efficiencies based on increase in the acceptor fluorescence.The quenching pattern of the tryptophans in serum albumins canbe explained on the basis of the nature and microenvironmentsurrounding the tryptophan groups inside BSA and HSA.

3.2. FRET efficiency

The FRET efficiency (E) is the quantum yield of the energytransfer transition, i.e. the fraction of energy transfer eventoccurring per donor excitation event. The FRET efficiency dependson many parameters that can be grouped as follows: (i) thedistance between the donor and the acceptor, (ii) the spectraloverlap of the donor emission spectrum and the acceptor absorp-tion spectrum, and (iii) the relative orientation of the donoremission dipole moment and the acceptor absorption dipolemoment. E depends on the donor-to-acceptor separation distancer with an inverse 6th power law due to the dipole–dipole coupling

-5.6 -5.5 -5.4 -5.3 -5.2 -5.1 -5.0 -4.9

-0.8

-0.6

-0.4

-0.2

0.0

0.2BSA - PYZ

log[

(F0 /

F) -

1 ]

log[PYZ]

Fig. 5. Binding affinity plot of [log (F0/F)�1] vs log [PYZ] for BSA–PYZ system from

steady state emission data (Fig. 3a). Inset: binding affinity plot of [log (F0/F)�1] vs

log [PYZ] for BSA–PYZ system from fluorescence decay data (Fig. 9a).

A. Sarkar, S.C. Bhattacharya / Journal of Luminescence 132 (2012) 2612–2618 2615

mechanism

E¼1

1þðr=R0Þ6

where R0 is the Forster distance of this pair of donor and acceptor,i.e. the distance at which the energy transfer efficiency is 50% and‘r’ is the distance between donor and acceptor. The Forsterdistance depends on the overlap integral of the donor emissionspectrum with the acceptor absorption spectrum and theirmutual molecular orientation as expressed by the followingequation [41]:

R60 ¼ 8:79� 10�25w2n�4jJ

where j is the fluorescence quantum yield of the donor in theabsence of the acceptor, w2 is the dipole orientation factor, n is therefractive index of the medium and J is the spectral overlapintegral calculated as

J¼

Zf DðlÞeAðlÞl

4dl

Where fD is the normalized donor emission spectrum, and eA isthe acceptor molar extinction coefficient. w2

¼2/3 is oftenassumed. This value is obtained when both fluorophores arefreely rotating and can be considered to be isotropically orientedduring the excited state lifetime. Fluorescent proteins do notreorient on a timescale that is faster than their fluorescencelifetime. In this case, 0rw2r4. The FRET efficiency relates tothe fluorescence lifetime of the donor molecule as follows:

E¼ 12ðt0=tÞ

where t and t0 are the donor lifetimes in the presence andabsence of an acceptor, respectively, or as

E¼ 12ðF0=FÞ

where F0 and F are the donor fluorescence intensities without andwith an acceptor(Q), respectively. Fig. 4 represents the corre-sponding efficiency plot of BSA. The R0 value for BSA is 16 A and ‘r’

is 20 A with a FRET efficiency, E¼20.76%. Analysis of the associa-tion constant (KA) data was also performed via the methodreported by Kang et al. [42]. The number of binding sites (n)was obtained according to the following equation:

log ð½F02F�=F�Þ ¼ log KAþn log ½Q �

0 2 4 6 8 10 12

0.0

0.2

0.4

0.6

0.8

1.0BSA - PYZ

[ 1 -

(F0/

F) ]

[PYZ] x 106 M

Fig. 4. FRET efficiency plot of [1�(F0/F)] vs [PYZ] for BSA–PYZ system from steady

state emission data (Fig. 3a). Inset: FRET efficiency plot of [1�(F0/F)] vs [PYZ] for

BSA–PYZ system from fluorescence decay data (Fig. 9a).

by plotting log ([F0–F]/F) vs log [Q] (Fig. 5). The slope of the doublelogarithm plot obtained from the experimental data gives thenumber of equivalent binding sites (n) (Table 1).

3.3. Fluorescence quenching

To confirm the quenching mechanism, we analyzed the fluor-escence quenching data by the well known Stern–Volmer equa-tion [3] as follows:

ðF0=FÞ ¼ 1þKSV½Q � ¼ 1þkqt0½Q �

where F0 and F denote the steady state fluorescence intensities ofproteins in the absence and in the presence of quencher (PYZ),respectively. KSV is the Stern–Volmer quenching constant, and [Q]is the concentration of quencher; t0 is the average lifetime of thefluorophore in the absence of the quencher. From the plot F0/F vs[Q] (Fig. 6), the slope represents KSV. The linearity of theplot indicates that only one type of quenching occurs in thesystems [3]. In the presence of quencher the absorption spectra ofthe transport protein remain unaltered. This indicates that thestatic quenching does not occur. The Stern–Volmer quenchingconstant has been determined and the values are given in Table 1.

3.4. Interaction of PYZ with L-tryptophan: steady state observation

The intrinsic fluorescence of HSA is attributed mainly to thesole tryptophan residue (Trp-214) present in the hydrophobiccavity of subdomain IIA (Sudlow I). Accordingly, if the probesbinds in Sudlow site I, this may enhance fluorescence quenchingof HSA. No fluorescence quenching mechanism is expected tooccur if the probes bind in the hydrophobic cavity of subdomainIIIA (Sudlow II) due to the far location of the probe from Trp-214.Tryptophan can thus act as an indicator for the interaction ofligands at site I of HSA through efficient energy transfer. Tofurther confirm the site-selective interaction of PYZ we havecarried out FRET experiment from L-tryptophan (donor) to PYZ(acceptor). This study gives an idea about the steric restrictionsimposed on PYZ by the other groups present in BSA and HSA,which affects the energy transfer process between the serumalbumins and PYZ. Fig. 7 represents the quenching of Trp emis-sion on gradual addition of PYZ. Comparing the nature of theemission spectra with that of BSA and HSA, it can be well

0 2 4 6 8 10 12

0.0

0.3

0.6

0.9

1.2

1.5

[(F0 /

F) -

1]

BSA-PYZ

[PYZ] X 106 M

Fig. 6. Stern–Volmer plot of [(F0/F)�1] vs [PYZ] for BSA–PYZ system from steady

state emission data (Fig. 3a). Inset: Stern–Volmer plot of [(F0/F)�1] vs [PYZ] for

Trp–PYZ system from steady state emission data (Fig. 7).

350 400 450 500

Fluo

resc

ence

Inte

nsity

(a.u

)

wavelength / nm

Trp - PYZ

PYZTrp

Fig. 7. FRET profile of L-Trp and PYZ. [L-Trp]¼8.0�10�6 M; [PYZ]¼0 to

28�10�6 M; lex¼285 nm and pH¼7.0.

0 5 10 15 20 25

0.0

0.1

0.2

0.3

0.4

0.5

[1 -

(F0 /

F)]

[PYZ] X 106 M

Trp - PYZ

Fig. 8. FRET efficiency plot [1�(F0/F)] vs [PYZ] for Trp–PYZ system from steady

state emission data (Fig. 7). Inset: binding affinity plot of [log (F0/F)�1] vs

log [PYZ] for Trp–PYZ system from steady state emission data (Fig. 7).

Table 1Binding and quenching parameters of PYZ.

Medium Ka /mol�1 dm3 (from steady

state data)

n (from steady

state data)

Ka/mol�1 dm3 (from

lifetime data)

n (from

lifetime data)

Ksv /mol�1 dm3 (from

steady state data)

BSA 4.47�106 1.33 2.29�107 1.76 1.05�105

Tryptophan 7.94�103 0.92 1.38�102 0.46 1.76�104

HSA – – 1.44�102 0.53 –

A. Sarkar, S.C. Bhattacharya / Journal of Luminescence 132 (2012) 2612–26182616

interpreted that PYZ mainly interacts with the surface bound Trpin BSA (Trp-134) while it experiences steric restriction in case ofthe buried Trp in HSA (Trp-214). In case of L-tryptophan there isno hindrance created by any other groups, so PYZ interacts withL-Trp promptly and as a result of which there is an efficientenergy transfer between L-Trp and PYZ. There is an appreciabledecrease and increase in donor and acceptor emission spectrarespectively in cases of BSA and L-tryptophan. In HSA, decrease indonor emission is very negligible compared to the increase inacceptor emission (PYZ). The big fluorescence intensity drop andblueshift (about 8 nm) of the Trp fluorescence emission in BSAis clear evidence that the Trp-134 is more exposed to an

environment that allows PYZ to quench its fluorescence. Thisspecificity in FRET may be due to the nature of the bulky PYZmolecule whose complex structure (AM1 calculations given asSupplementary data) prevents it from interacting with the buriedTrp-214 in HSA. Relatively, the surface bound Trp-134 in BSAfinds it easier to transfer energy to PYZ as it is more exposed tothe environment and to the direction in which PYZ approachesBSA. The efficiency plot for L-Trp–PYZ has been given in Fig. 8.

3.5. Time resolved studies

The decay profiles of BSA and HSA are presented in Fig. 9aand b respectively and the parameters obtained from lifetimestudies of BSA, HSA and L-tryptophan (Trp) in presence of PYZare given in Tables 2–4 respectively. BSA and HSA exhibitedbi-exponential fitting curves. The distribution width varies butthere are no appreciable changes in the average lifetime values ofBSA and HSA. In contrast L-Trp displayed a single exponentialfitting pattern as it is free from environments experienced by BSAand HSA. Moreover, the extent of decrease in average lifetimevalues of HSA is more (4.98–3.46 ns) than BSA (5.88–5.52 ns) andL-Trp (2.58–2.50 ns). This ambiguity from steady state resultsindicate a variation of microenvironments experienced by theL-Trp and BSA but without a change in local polarity whichindicates that there is not enough solvent access to the two Trp’sin BSA. As a result the extent of decrease in lifetime values ofL-Trp and BSA is very much less than that of HSA. A confoundingproblem associated with Trp fluorescence is that often the life-time decay of even single Trp containing proteins (like in HSA)exhibits complex patterns [37]. Several models have beenproposed to describe the lifetime heterogeneity observed for

0 10 20 30 40 50

10

100

1000

prompt BSA 0.002mM PYZ 0.004mM PYZ 0.006mM PYZ 0.008mM PYZ 0.010mM PYZ 0.012mM PYZ 0.014mM PYZ

log

(cou

nts)

time / ns

PYZ - BSA

0 10 20 30 40 50

10

100

1000

prompt HSA 0.002mM PYZ 0.004mM PYZ 0.006mM PYZ 0.008mM PYZ 0.010mM PYZ 0.012mM PYZ 0.014mM PYZ

log

(cou

nts)

time / ns

PYZ - HSA

Fig. 9. (a) Fluorescence decay profile of BSA–PYZ. [BSA]¼2.5�10�6 M;

lex¼295 nm; lem¼348 nm and pH¼7.0. (b) Fluorescence decay profile of HSA–

PYZ. [HSA]¼4.0�10�6 M; lex¼295 nm; lem¼340 nm and pH¼7.0.

Table 2

Fluorescence decay parameters of BSA (2.5�10�6 M) in PYZ [lem¼348 nm].

[PYZ]�106 M B1 B2 t1 (ns) t2 (ns) tavg (ns) w2

0 0.55 0.45 4.61 7.43 5.88 1.02

2 0.60 0.40 4.68 7.66 5.87 1.08

4 0.53 0.47 4.47 7.38 5.84 1.21

6 0.51 0.49 4.28 7.38 5.80 1.16

8 0.40 0.60 3.65 7.02 5.67 1.14

10 0.41 0.59 3.66 7.06 5.66 1.12

12 0.45 0.55 3.76 7.12 5.61 1.04

14 0.34 0.66 3.11 6.76 5.52 1.09

Table 3

Fluorescence decay parameters of HSA (4.0�10�6 M) in PYZ [lem¼340 nm].

[PYZ]�106 M B1 B2 t1 (ns) t2 (ns) tavg (ns) w2

0 0.42 0.58 2.49 6.80 4.98 1.08

2 0.58 0.42 2.40 6.75 4.23 1.11

4 0.56 0.44 2.28 6.66 4.20 1.21

6 0.62 0.38 2.41 6.65 4.02 1.23

8 0.64 0.36 2.43 6.76 3.98 1.17

10 0.60 0.40 2.18 6.55 3.92 1.17

12 0.61 0.39 2.32 6.53 3.60 1.16

14 0.65 0.35 2.32 6.57 3.46 1.22

Table 4Fluorescence decay parameters of L-Trp (4.0�10�6 M) in

PYZ [lem¼360 nm].

[PYZ]�106 M t (ns) w2

0 2.58 1.21

4 2.55 1.10

8 2.55 1.18

16 2.52 1.23

24 2.52 1.16

40 2.50 1.18

A. Sarkar, S.C. Bhattacharya / Journal of Luminescence 132 (2012) 2612–2618 2617

individual Trp residues within proteins. According to the ‘‘classical

rotamer model’’, different ground state conformers of Trp, whichexist on a longer timescale than the lifetime of the excited state,have different interactions within the protein matrix, thus produ-cing distinct lifetimes [43,44]. Tryptophan fluorescence is stronglyinfluenced by the proximity of other residues (i.e. nearby proto-nated groups such as Asp or Glu). Also, energy transfer betweentryptophan and the other fluorescent amino acids is possible,which would affect the lifetime analysis. In human serumalbumin (HSA), with only one tryptophan residue, the biexpo-nential decay can be attributed to a single electronic transition oftryptophan, which may exist as different conformational isomersin the protein. The presence of rotamers, however, does not fullyexplain the multiexponential decay in single tryptophan proteins.As the indole ring assumes different positions in a polypeptidechain, slightly different position of neighboring quenching groupsmay also result in multiexponential decay. Alternatively, Trplifetimes can be analyzed as continuous distributions rather thanas discrete emitting species, potentially providing information onlocal conformation and dynamics [45–47]. The complex nature ofthe decay of BSA and HSA alone makes it difficult to determinewhich mechanism of quenching is involved, and whether it is theonly process responsible for the observed changes. Indeed, PYZinduced perturbations of the tryptophan neighborhood are likelyto cause changes in fluorescence decays of the rotamers. It seemsthat tryptophan in free BSA and HSA is more flexible than it canbe assumed from its decay fitting to a multi-exponential function.This qualitative change in the lifetime distribution profile ismainly caused by PYZ-induced structural modification of thetryptophan local environment stabilizing the range of possibletryptophan orientations, rather than by quenching, which, how-ever, can also contribute here. It seems that both perturbations inthe tryptophan environment and FRET can result in the observedbehavior of the decay function. In the case of environmentperturbations, the distribution of rotamers can gradually varywidely. On the other hand, FRET between the rotamers and PYZcan be more effective due to distance or orientation-relatedfactors. As lifetime analysis cannot distinguish between theseeffects, other experiments might be helpful in confirming thesuggested mechanisms. More recently, Rolinski et al. [48] havepresented a Langevin-equation based refinement of the rotamermodel to account for observed non-exponentiality of Trp life-times. The ‘‘solvent/spectral relaxation model’’ suggests that theheterogeneity is due to the increase in the dipole moment ofthe excited state that produces short-lived ‘‘blue’’ and long-lived ‘‘red’’ species that combine to produce the steady stateemission [49]. The lifetime distribution profiles show a broaddistribution of Trp conformations in the PYZ bound state whichlikely produce alternate quenching/ FRET patterns among thedifferent conformers. Such a distribution implies that some statesare more efficient in energy transfer and/or alternative quenchingprocesses taking place [50] whereas other conformers wouldproduce less efficient quenching from FRET or other processes.

A. Sarkar, S.C. Bhattacharya / Journal of Luminescence 132 (2012) 2612–26182618

4. Conclusion

We observed negligible FRET mediated emission in the case ofHSA and an efficient FRET-mediated emission in case of BSA andTrp. The unusual selectivity of PYZ for the serum albumins can beattributed to the steric factors due to the presence of bulkier phenylmoieties in PYZ. The surface bound Trp-134 in BSA allows anefficient FRET process with PYZ while the buried Trp-214 in HSAdoes not. The steady state emission data and lifetime parameterscharacterize the nature of Trp–PYZ interactions, but they cannot beinterpreted in terms of a simple model. The emission behavior oftryptophan alone is a subject of a long standing problem, and the‘‘rotamer model’’ seems to be only a good approximation of the realprocess. In our system, photophysics of tryptophan is comparedwith serum albumin–PYZ interactions, thus the precise interpreta-tion of the resulting fluorescence responses has to be supported byadditional information. In our opinion, further progress in thisimportant aspect of probe-protein research can be stimulated bymolecular dynamics, which may be able to find possible locationsand orientations of fluorophore attached to proteins, and thus helpin building improved models to explain the fluorescence behavior.It would clearly be informative if a FRET analysis can furtheraccount for specific donor–acceptor locations as demonstrated inour present study. Since the study on the selective FRET process of afluorophore with serum albumins is very rare, the selective inter-actions of novel functional molecule PYZ with BSA and HSA canopen up new avenues in the wide field of probe-protein studies. Theselective FRET sensing of PYZ with the proteins (BSA and HSA) canbe efficiently applied in other bio-assemblies containing proteinswhere PYZ can act as a selective FRET receptor molecule dependingon the structure and nature of the protein molecules and theposition of tryptophan groups inside them.

Acknowledgment

Authors are indebted to Prof. K.K. Mahalanabis andDr. A. Mukherjee of Jadavpur University for their co-operationwith the synthesis of the compound. Author A.S. thanks CSIR forfunding the work. [CSIR Project no: 01(2429)/10/EMR-II].

Appendix A. Supplementary Information

Supplementary data associated with this article can be foundin the online version at http://dx.doi.org/10.1016/j.jlumin.2012.04.055.

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