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Coumarin 6 and 1,6-diphenyl-1,3,5-hexatriene (DPH) as fluorescent probes tomonitor protein aggregation†

Pinakin K. Makwana, Prashant N. Jethva and Ipsita Roy*

Received 27th October 2010, Accepted 25th February 2011

DOI: 10.1039/c0an00829j

We report the use of Coumarin 6 and 1,6-diphenyl-1,3,5-hexatriene (DPH) for the identification of

protein aggregates for the first time. The two dyes can be used at very low (nanomolar) concentrations

and do not interfere with the aggregation process, as is reported for other commonly used fluorescent

protein probes. In the presence of protein aggregates, their quantum yields are significantly high. DPH

is able to recognize both amorphous and fibrillar aggregates but cannot distinguish between them.

Coumarin 6 can distinguish between both types of aggregates. It also exhibits the characteristic

sigmoidal curve of amyloid formation, with higher sensitivity for detection of fibrillation than the

conventionally used Thioflavin T.

Introduction

Misfolded proteins resulting in the formation of aggregates are

encountered in a variety of situations, including downstream

processing of proteins (as in the formation of inclusion bodies

during overexpression of proteins in heterologous systems),

storage stability of biopharmaceuticals (decreasing their

bioavailability and shelf-life) and in numerous neurodegenera-

tive diseases (where they form ‘inclusions’ or ‘plaques’). Under-

standing the nature of these macrostructures is the first step in

devising strategies for inhibition of protein aggregation. As

a biological phenomenon, protein folding has continued to baffle

scientists from different disciplines. Protein misfolding, followed

by its aggregation, is hypothesized to occur via five major routes.1

They are (a) the reversible association mechanism, (b) the

subsequent monomer addition mechanism, (c) the prion aggre-

gation mechanism, (d) quantitative structure activity relationship

model, and (e) the Finke–Watzky two-step minimalistic model.

The protein aggregates formed are of two major classes: amor-

phous and fibrillar. The discovery of the existence of ordered

structure within protein aggregates has focused most of the

attention on the study of fibrillar protein aggregates.

Fluorescence spectroscopy is a technique that has been used

extensively to study proteins and their aggregates. Both intrinsic

fluorescence of proteins and external fluorescence probes have

been used for this purpose. The detection of soluble aggregates of

barstar at acidic pH, which can be transformed to protofibrils, by

two-photon tryptophan fluorescence correlation spectroscopy, is

Department of Biotechnology, National Institute of PharmaceuticalEducation and Research (NIPER), Sector 67, S.A.S. Nagar, Punjab 160062, India. E-mail: ipsita@niper.ac.in; Fax: +91-172-221 4692; Tel:+91-172-229 2061

† Electronic supplementary information (ESI) available. See DOI:10.1039/c0an00829j

This journal is ª The Royal Society of Chemistry 2011

one such example.2 Recently, imaging techniques using a bis-

arsenoid fluorescein analogue dye, viz. FlAsH, that is able to

distinguish between folded and unfolded states of proteins

without interfering with the protein structure3 and JC-1 that is

able to distinguish between monomeric and fibrillar forms of

proteins,4 have been reported. However, fluorescence microscopy

cannot be the method of choice when libraries of compounds

need to be screened as modulators of protein aggregation. In this

case, small molecules that can act as fluorescence probes in

spectroscopic measurement need to be used. Various dyes such as

Thioflavin T,5,6 9-(dicyanovinyl)-julolidine (DCVJ),7 Nile Red,8

bis-ANS,9 8-anilinonaphthalene sulfonate (ANS)10 and Congo

Red5 have generally been used for this purpose. These molecules

can interact with proteins non-covalently via hydrophobic or

electrostatic interactions or can be covalently attached to the

amine or thiol groups of proteins.11 One of the drawbacks of

these dyes is that they either recognize only the fibrillar form of

aggregates or are unable to distinguish between the major types

of protein aggregates.

In this work, we describe the use of Coumarin 6 and 1,6-

diphenyl-1,3,5-hexatriene (DPH) as fluorescent probes to

monitor protein aggregation for the first time. Coumarin 6 has

been reported to be used as a pH sensitive probe, since its fluo-

rescence increases with decrease in pH.12,13 It has also been used

as a fluorescent lipophilic probe for studies of emulsion uptake in

cancer cell lines.14 Since in a majority of cases protein aggrega-

tion is said to result from the interactions between hydrophobic

patches, we decided to examine the suitability of Coumarin 6 as

a fluorescent probe to monitor aggregation of two proteins,

bovine carbonic anhydrase (BCA) and a-synuclein. DPH has

been used extensively as a lipid membrane fluorescent probe and

as a sensor for fatty acyl side chains.15,16 It should thus be able to

detect changes in the hydrophobic milieu of a molecule. We

observed that of the two dyes, DPH is not able to distinguish

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between amorphous and fibrillar classes of aggregates while

Coumarin 6 is able to recognize and discriminate between

amorphous and fibrillar type of protein aggregates.

Results and discussion

Bovine carbonic anhydrase (BCA) is a globular protein con-

taining zinc as a prosthetic group. Thermal aggregation of BCA

is a well characterized phenomenon.10 At a certain temperature,

the protein molecule starts to unfold and its hydrophobic

surfaces are exposed. At still higher temperatures, frequencies of

molecular collisions and hydrophobic interactions increase,

leading to the formation of aggregates.

Fig. 1 Characterization of BCA aggregates formed by thermal inacti-

vation of the enzyme. Electrophoresis of aggregated BCA was carried out

on (A) non-denaturing and (B) denaturing polyacrylamide gels. (A)

Native gel electrophoresis of the samples was carried out on 8% cross-

linked gel. Lane 1: native BCA; lane 2: BCA aggregate under non-

reducing conditions; lane 3: BCA aggregate under reducing conditions;

lane 4: BCA aggregate with 6 M urea. (B) SDS (denaturing) gel elec-

trophoresis of the samples was carried out on a 12% cross-linked gel.

Lane 1: native BCA; lane 2: BCA aggregate under non-reducing condi-

tions; lane 3: BCA aggregate under reducing conditions. Gels were

stained by silver staining. (C) Fluorescence of the hydrophobic dye ANS

was measured alone (empty bars) and in the presence of thermally

aggregated BCA (filled bars). (D) Effect of ANS on the aggregation

pattern of BCA. BCA was aggregated in the presence of 10 mM (black)

and 100 mM (grey) of ANS as described in the text. White bar represents

aggregation of BCA in the absence of any dye. Excitation of the samples

was carried out at 350 nm and the emission intensity was recorded at

461 nm. The excitation slit width was kept at 5 nm and the emission slit

width was kept at 7.5 nm. All experiments were carried out in triplicate

and means of individual sets are shown.

Characterization of aggregated BCA

Thermal aggregation of BCA was carried out following the

procedure described in an earlier report.10 Since the results have

already been reported, the data regarding the characterization

will be described in brief. Almost 85% aggregation was observed

when BCA (17.2 mM) was heated at 70 �C for 0.5 h. No change

was observed in the amount of protein aggregated when the

heated solutions were cooled before centrifugation. This indi-

cates that aggregate formation is not reversible and agrees well

with the earlier report.10 The aggregated protein was found to be

enzymatically inactive when it was incubated with p-nitrophenyl

acetate (Fig. S1†). The dye Congo Red has been shown to

interact with amyloid-type aggregates and to exhibit typical

apple-green birefringence under polarized light.17 No birefrin-

gence of Congo Red was seen with BCA aggregates under

polarized light. Thus the BCA aggregates formed on exposure of

the protein to high temperature are not amyloid type. Formation

of non-fibrillar aggregates by BCA upon thermally induced

aggregation is also supported by literature.10

The samples of BCA, before and after aggregation, were

subjected to non-denaturing gel electrophoresis. With cross-

linking higher than 8%, the aggregated protein was unable to

enter the gel. To facilitate the electrophoretic run, the aggregate

was suspended in the gel-loading buffer containing various

additives. It was observed that addition of 6 M urea alone was

able to dissolve the aggregates sufficiently for the protein to enter

the gel (Fig. 1A). Addition of a reducing agent alone was not

enough to disassemble the protein aggregates. Since urea is

known to disrupt non-covalent bonds, the results indicate that at

least some of the bonds responsible for forming aggregates are of

non-covalent nature. When the same samples were run on

a denaturing gel, bands corresponding to higher molecular

weight species were not seen. Instead, bands corresponding to

monomeric protein were observed (Fig. 1B). Thus, amorphous

aggregates of BCA formed at high temperature are SDS-soluble.

The hydrophobic fluorescent dye, 8-anilinonaphthalene

sulfonate (ANS), has been used as a probe for the identification

of protein aggregates as well as the molten globule state when

used in the micromolar concentration range.10,18,19 It has also

been reported that ANS, when used at a high concentration

relative to protein (500 mM ANS with 3 mM BCA) acts like

a molecular chaperone and prevents thermally induced aggre-

gation of the protein.20 Since studies of ANS with BCA aggregate

have already been reported, we evaluated the response of ANS

2162 | Analyst, 2011, 136, 2161–2167

towards BCA aggregates, comparing this with the new dyes. As

expected, fluorescence of ANS increased in the presence of

protein aggregates in a concentration dependent manner

(Fig. 1C). To confirm whether the dye interferes with protein

aggregation as has been reported,22 the protein was aggregated in

the presence and absence of ANS. It has been reported that ANS

does interfere with aggregation at a concentration of 100 mM,

though the effect, in our case, was found to be marginal

(Fig. 1D). A lower concentration of the dye (nM range) was used

to overcome the effect of ANS on the aggregation of BCA. The

change in the fluorescence intensity of the dye, at this concen-

tration range, in the presence of the aggregated protein was not

significantly different from that of the aggregated protein alone

(Fig. 1C). Hence, we searched for novel fluorophores which

exhibit enhanced fluorescence intensities at lower concentrations

and do not interfere with protein aggregation.

Spectroflourometric analysis of aggregates using Coumarin 6

Comprehensive studies of luminescence properties of coumarins

(structures with fused benzene and pyrone rings) have been

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reported by various authors.21–23 The fluorescence of coumarins

is reported to depend on various parameters of the medium,

including its polarizability, viscosity, pH, polarity, etc. Two main

classes of coumarins with fluorescent properties, based on

substituents at position 7, viz. 7-amino or 7-methoxycoumarins,

have been reported. Coumarin 6 [IUPAC: 3-(1,3-benzothiazol-2-

yl)-7-(diethylamino)chrome-2-one] belongs to the first category.

Unlike the 7-methoxy substituted coumarins, these molecules

exhibit enhanced fluorescence in non-polar media. The forma-

tion of a twisted intramolecular charge-transfer state leads to the

loss of energy by the excited state via non-radiative decay in polar

media.24 The fluorescence of Coumarin 6 has been used to probe

the acidity and heterogeneity of 193 nm photoresist polymer

films.25 The pH sensitivity of Coumarin 6 has been used to

characterize the acidic spots in the internal structure of the

largely non-acidic faujasite zeolite NaY.12 Another important

area where Coumarin 6 has been employed is in probing the

microenvironment of micellar aggregates.26 This has led us to

propose that Coumarin 6 should be able to detect aggregation of

proteins via hydrophobic patches within the protein molecules.

As in the case of ANS, we first checked whether Coumarin 6

interfered with the aggregation of BCA. It was found that under

the conditions used, Coumarin 6 did not have any effect on the

aggregation propensity of the protein (Fig. 2A). When excited at

470 nm, Coumarin 6 alone was found to exhibit an emission

maximum at 515 nm. The fluorescence intensity of Coumarin 6

Fig. 2 (A) Effect of Coumarin 6 on the aggregation pattern of BCA. BCA wa

as described in the text. 100% aggregation refers to the value of the starting pr

(C) and DPH (:) in the presence of aggregated BCA (17.2 mM). Emission in

are shown. Emission intensities of Coumarin 6 (O) and DPH (B) in the prese

slit width was kept at 5 nm and emission slit width was kept at 7.5 nm. The e

comparison. (C) Representative fluorescence emission spectra of 400 nM Cou

aggregates showing blue shift in the latter case. (D) Effect of DPH on the aggre

500 nM of DPH as described in the text. 100% aggregation refers to the valu

carried out in triplicate and means of individual sets are shown.

This journal is ª The Royal Society of Chemistry 2011

was found to increase in the presence of BCA aggregates

(Fig. 2B) and a blue shift of 8 nm was observed in the emission

maximum of the dye (507 nm) (Fig. 2C). This blue shift reflects

the increased energy gap between the ground singlet and excited

states of the dye in the presence of protein aggregates. The dye

did not exhibit any change in its fluorescence intensity/emission

maximum with native BCA used as a control (Fig. 2B). When

Coumarin 6 was heated along with the protein under the above

conditions, the fluorescence intensity of the sample increased

(Fig. S2†) but not to the same extent as that in the case where

Coumarin 6 was added after aggregation of protein was

complete. The emission intensity of the dye alone was found to

decrease when heated at 70 �C. As a general rule, an increase in

temperature is accompanied by a decease in the fluorescence

intensity of the fluorophore. This is attributed to internal

conversion, one of the two mechanisms by which radiationless

decay can occur,27 which results in a change in spin multiplicity.

The stronger the inverse relationship between temperature and

fluorescence, the higher is the contribution of internal conversion

to radiationless decay. At higher temperature, the probability of

molecular collision is high. The kinetics of non-radiative decay

processes also increase. Both of these lead to a decay of the

fluorescence signal. In the case of Coumarin 6, the fluorescence

intensity of the dye is higher in the presence of protein aggregates

at lower (room) temperature, i.e. when the dye is added to the

protein after aggregation is over, than when the dye and the

s aggregated in the presence of 250, 500, 750 and 1000 nM of Coumarin 6

otein in the absence of any dye. (B) Fluorescence intensity of Coumarin 6

tensities of Coumarin 6 (,) and DPH (>) in the presence of native BCA

nce of 3.4 mMof BCA aggregates are also shown. In both cases, excitation

mission intensities of Coumarin 6 (�) and DPH (*) alone are shown for

marin 6 in the absence (spectrum I) and presence (spectrum II) of BCA

gation pattern of BCA. BCA was aggregated in the presence of 5, 100 and

e of the starting protein in the absence of any dye. All experiments were

Analyst, 2011, 136, 2161–2167 | 2163

Table 1 Quantum yields of Coumarin 6 and DPH in the absence andpresence of aggregates. The quantum yield of ANS is shown forcomparison

Quantum Quantum

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protein are heated simultaneously. Thus, internal conversion is

probably the major route of radiationless decay of the singlet

excited state. In all the cases described here, Coumarin 6 has been

added to the protein sample after aggregation.

Dye yield (alone) yield of dye (in the presence of aggregate)

Coumarin 6 0.37 0.70DPH 0.014 0.77ANS 0.00450 0.46 (with BSA)51

Spectroflourometric analysis of aggregates using DPH

Because of its negligible fluorescence in aqueous medium and

high quantum yield in non-polar milieu, 1,6-diphenyl-1,3,5-

hexatriene (DPH) has found applications in biomembrane/lipid

research.28–30 The fluorescence polarization of DPH has been

reported to change with variation in lipid composition of two

autologous human melanoma cell lines from a superficial mela-

noma and the lymph node of the same patient.31 Recently, DPH

has also been entrapped in silica nanoparticles and the fluores-

cence of the probe has been used to monitor particle–protein

interactions.32

It was observed that DPH did not interfere with the aggrega-

tion of BCA up to a concentration of 500 nM when the protein

solution was incubated at 70 �C for 0.5 h. Fluorescence

measurements of DPH in the presence of BCA aggregates were

carried out as described in the Methods section. In contrast to

ANS and Coumarin 6, DPH alone exhibited negligible fluores-

cence even at a very high concentration (5 mM, Fig. S3†) when

excited at 360 nm. In the presence of the protein aggregate,

a significant increase in the fluorescence intensity of the dye was

observed (Fig. 2B) at 430 nm. Thus it appears as if the aggre-

gation of protein affects the immediate environment of the dye

sufficiently to induce a change in fluorescence intensity of the

latter. DPH also works in a nanomolar concentration range and

its advantage over Coumarin 6 is that the fluorescence of the dye

is significantly higher in the presence of the aggregates than in its

absence as compared to Coumarin 6 even at a very low

concentration of protein (3.4 mM) (Fig. 2B). In this case also, we

first checked whether DPH interfered with the aggregation of

BCA. It was found that under the conditions used, DPH did not

have any effect on the aggregation propensity of the protein

(Fig. 2D). No increase in fluorescence intensity of DPH was

observed in the presence of native BCA which confirms that the

increase in fluorescence intensity in the presence of aggregates is

because of the modulation of the local environment of the dye by

the aggregated protein. Unlike Coumarin 6, DPH does not

exhibit any decrease in its emission intensity on being heated in

the presence of protein aggregates. However, at higher concen-

trations ($5 mM), the fluorescence intensity of the dye alone is

quenched (Fig. S4†). This quenching is not observed in the

presence of the protein aggregates. Compared to ANS, the

advantage with these two dyes is the much lower concentration

(nM) at which they work, which ensures that they do not inter-

fere with aggregation of the protein.

Calculation of quantum yields

Quantum yields for Coumarin 6 and DPH were calculated using

fluorescein and quinine sulfate as the respective standards. The

values of quantum yield in the presence and absence of protein

aggregate are shown in Table 1. It is clear that the fluorescence

properties of Coumarin 6 and DPH increase significantly in the

presence of amorphous aggregates of BCA. Fluorophores with

2164 | Analyst, 2011, 136, 2161–2167

blue-shifted fluorescence spectra (as in the case with Coumarin 6

in the presence of BCA aggregates) are expected to have high

quantum yields.33 The higher values of quantum yield of these

dyes in the presence of BCA aggregates also point to these being

more sensitive probes than conventional dyes.

Aggregation of a-synuclein

a-Synuclein is a heat-resistant, natively unfolded, acidic protein

composed of 140 amino acids residues, with a molecular weight

of about 16 kDa.34 It can assume a variety of conformations,

from unfolded in solution to a-helical in the presence of lipids to

a b-pleated sheet in protein aggregates.35 Aggregation of a-syn-

uclein is nucleation dependent which means that the initial

interaction between monomers acts as a seed for the subsequent

molecules to interact. The monomeric protein molecule, which is

soluble in the cytoplasm, forms oligomers and becomes part of

a larger aggregate as polymerization proceeds.36 Factors that

shift the kinetic equilibrium towards partial unfolding can

facilitate fibril formation. Fibrillation of a-synuclein is moni-

tored using thioflavin T (ThT), a cationic benzothiazole dye that

has been used to identify amyloid aggregates since it was first

demonstrated that its fluorescence increases upon binding to

amyloid fibrils.37 Upon binding with amyloid, ThT experiences

changes in both fluorescence emission and excitation spectra.5,6

a-Synuclein was incubated at a concentration of 7 mgml�1 at 37�C. The kinetics of fibrillation were examined by monitoring the

change in fluorescence intensity of thioflavin T. Aliquots (10 ml)

were withdrawn at regular intervals and added to 1.0 ml of 10 mM

thioflavin T in 20 mM Tris HCl buffer, pH 7.8. The fluorescence

intensity of thioflavin T was detected at 482 nm. Fig. 3 (inset)

shows the time-dependent change in thioflavin T fluorescence

during fibrillation of a-synuclein. A characteristic sigmoidal curve

is seen, with distinct lag, growth and saturation phases, which are

characteristic of a nucleation-dependent fibrillation process. The

lag time of nucleation was calculated to be 74.9 h.

The same process was repeated and aliquots were now added

to Coumarin 6 and DPH solutions. In each case, aliquots (10 ml)

were withdrawn at regular intervals and added to 1.0 ml of 50 nM

dye in 20 mM Tris HCl buffer, pH 7.8. The fluorescence inten-

sities of Coumarin 6 and DPH were detected at 508 and 423 nm,

respectively. In case of Coumarin 6, a characteristic sigmoidal

curve with discrete nucleation, fibrillation (exponential growth)

and saturation phases, could be seen (Fig. 3). The initial lag

phase corresponds to the stage where the critical nucleus for

fibrillation is formed. The lag phase was calculated to be 58 h.

Interestingly, at least in the case of a-synuclein, Coumarin 6 is

able to detect the shift from nucleation to exponential growth

phase earlier than thioflavin T. The concentration of Courmarin

This journal is ª The Royal Society of Chemistry 2011

Fig. 3 Fluorescence intensities of Coumarin 6 (curve I) and DPH (curve

II) in the presence of aggregated a-synuclein. The emission intensities of

the two dyes were monitored at 507 nm and 423 nm, respectively, after

exciting the samples at 450 nm and 360 nm. Inset shows the change in

fluorescence intensity of thioflavin T with time in the presence of a-syn-

uclein at 482 nm. Excitation and emission slit widths were 5 nm and 10

nm, respectively.

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6 required for detection of fibrillation is also much lower than

thioflavin T. DPH, on the other hand, exhibited a different trend.

In this case also, the fluorescence intensity of the dye increased

with increase in time of aggregation of a-synuclein (Fig. 3).

However, the characteristic sigmoidal curve of fibrillation was

not observed. To find out if the presence of the dye interferes with

the folding/unfolding equilibrium, altering the fraction of

monomers which could be responsible for difference in lag times,

the samples were centrifuged after addition of the dyes and the

amount of protein in the supernatant was estimated.38 The

amount of protein in the supernatant (containing the soluble

monomers) did not vary in any case (Fig. S5†), showing that the

presence of the dye does not interfere with the folding/unfolding

equilibrium of a-synuclein. It can be speculated that DPH

interacts non-specifically with hydrophobic clusters in protein

aggregates, resulting in an increase in fluorescence intensity while

Coumarin 6 interacts more specifically with crossed b-sheet

structure that is characteristic of amyloid fibrils.

A novel use of two fluorophores has been reported for the

detection of protein aggregates. Both Coumarin 6 and DPH were

able to detect fibrillar, and amorphous protein aggregates. The

fluorescence intensity of DPH was enhanced in the presence of

fibrillar aggregates but it did not exhibit the sigmoidal curve that

is typical of nucleation-dependent fibrillogenesis. Coumarin 6, on

the other hand, exhibited a nucleation-dependent increase in

fluorescence intensity with three distinct stages of lag, growth

and saturation phases. The lag time observed with Coumarin 6

was less than that seen with thioflavin T, indicating that

Coumarin 6 is able to detect the transition from nucleation to

fibrillation stage earlier than thioflavin T. In addition, since the

two dyes work at much lower concentrations than the conven-

tionally used fluorophores, viz. ANS and thioflavin T, there is no

interference in the protein aggregation process, as has been

reported in the case of ANS.

Experimental

Materials

Carbonic anhydrase from bovine erythrocytes (BCA), 8-anilino-

1-naphthalene sulfonic acid ammonium salt, 95% (ANS),

This journal is ª The Royal Society of Chemistry 2011

1,6-diphenyl-1,3,5-hexatriene (DPH), Coumarin 6, 98%, Thio-

flavin T, LB broth, ampicillin, phenylmethanesulfonyl fluoride

(PMSF) and isopropyl-1-thio-b-D-galactopyranoside (IPTG)

were purchased from Sigma-Aldrich Chemicals Pvt. Ltd., Ban-

galore, India. Fluorescein sodium and quinine hemisulfate

monohydrate were purchased from Fluka, Bangalore, India.

Plasmid pRSETB (a-synuclein) was received as a gift from Dr

Roberto Cappai, Department of Pathology, University of Mel-

bourne, Australia.

Methods

Aggregation of BCA. BCA solutions (17.2 mM) were prepared

in 0.05 M phosphate buffer, pH 7.6 and aggregation was carried

out with a slight modification of the protocol reported earlier.10

The protein solution was heated at 70 �C for 0.5 h. The amount

of protein precipitated was calculated by subtracting the amount

of protein present in the supernatant obtained after centrifuga-

tion from the initially added protein, which was assumed to be

100%. The amount of protein was estimated by the dye-binding

method38 using bovine serum albumin (BSA) as a standard

protein.

Characterization of BCA aggregates

Electrophoresis. Native (8%) and denaturing (12%) PAGE

were carried out for BCA and its aggregates using a miniVE

electrophoresis unit (GE Healthcare, Hong Kong), under

conditions of constant voltage. Protein bands were visualized

using the silver staining method.39

Congo Red birefringence study. The procedure for sample

preparation has already been described in the literature.5,40

Samples were viewed at 500� magnification with a polarized

light microscope (Leica Microsystems, Germany) under crossed

polarized and visible light.

Spectroflourometric analysis of aggregates using ANS. Stock

solution of ANS (10 mM) was prepared in 100% methanol.10

BCA aggregates were prepared as described earlier. For spec-

troflourometric experiments, desired concentrations of ANS

were taken and the total volume was made up to 1 ml with

0.05 M phosphate buffer, pH 7.6, containing BCA aggregates.

Samples were excited at 350 nm and the emission intensity was

recorded at 461 nm.

Purification of a-synuclein. Escherichia coli BL21 cells were

transformed with the human a-synuclein cDNA sequence cloned

in pRSETB vector following standard protocol.41 The expression

and purification of a-synuclein were carried out as per reported

protocol.42 The purification of the protein was confirmed by

SDS-PAGE and immunoblotting using anti-a-synuclein mono-

clonal antibody as the primary antibody (Fig. S6†).

Aggregation of a-synuclein. Lyophilized a-synuclein was dis-

solved in 20 mM Tris HCl buffer, pH 7.8 buffer and subjected to

ultracentrifugation (100 000 g for 1 h) to remove preformed

aggregates. The final concentration of the purified protein was

adjusted to 7 mg ml�1 in the presence of 0.02% sodium azide.43

Samples were incubated at 37 �C. At predefined time intervals,

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aliquots were withdrawn and added to solutions of thioflavin T

(final concentration of 2 mM). The fluorescence emission of the

samples was monitored at 470–560 nm after exciting them at

450 nm. For plotting the fibrillation kinetics, emission intensity

values at 482 nm (lmax) were considered.

The aggregation kinetics were followed by fitting the data

using the formula44

y ¼ yi þmxi þ yf þmxf

1þ ex� x0

s

where yi + mxi is the initial line, yf + mxf is the final line and x0 is

the midpoint of maximum signal. The apparent rate constant

(kapp) is 1/s and the lag time is calculated to be x0 � 2s.

Spectroflourometric analysis of aggregates using Coumarin 6.

Coumarin 6 was dissolved inN,N-dimethylformamide (DMF) to

prepare a stock solution of 10 mM.45 The concentration of

Coumarin 6 was varied from 50 nM to 500 nM in a total volume

of 1 ml solution (in 0.05 M phosphate buffer, pH 7.6) containing

BCA aggregates. In case of aggregated a-synuclein, analysis was

carried out only with 50 nMCoumarin 6. Samples were excited at

470 nm and the emission spectra were recorded between 500 and

600 nm.

Spectroflourometric analysis of aggregates using DPH. A stock

solution of DPH (5 mM) was prepared in 100% acetonitrile. For

analysis of aggregates, various concentrations were taken (50

nM–5 mM). In each case, DPH was taken in a fixed volume of

acetonitrile (10 ml) that was added to a total volume of 1 ml

solution (in 0.05 M phosphate buffer, pH 7.6) containing BCA

aggregates. In case of aggregated a-synuclein, analysis was carried

out only with 50 nM DPH. Samples were excited at 360 nm and

the emission spectra were recorded between 400 and 520 nm.

Quantum yields of Coumarin 6 and DPH. For calculating

quantum yields of Coumarin 6 and DPH, two dyes, viz. fluo-

rescein and quinine sulfate, were chosen as the respective stan-

dards. Quantum yields of dyes were estimated as per the

procedure described,46,47 using the following equation:

Fun ¼ Fstd [Gradun/Gradstd][hun2/hstd

2]

where Fun ¼ quantum yield of unknown dye, Fstd ¼ quantum

yield of standard dye, Gradun ¼ slope from the plot of integrated

fluorescence intensity vs. absorbance of unknown dye, Gradstd ¼slope from the plot of integrated fluorescence intensity vs.

absorbance of standard dye, hun ¼ refractive index of the solvent

of unknown dye, and hstd ¼ refractive index of the solvent of

standard dye.

When calculating quantum yields, quantum yield values of

fluorescein and quinine sulfate were taken to be 0.79,48 and

0.54,49 respectively.

Acknowledgements

We thank Prof. A. K. Bansal (NIPER) for allowing us to use

polarized microscope. We are indebted to Dr I. P. Singh and Dr

S. Jachak (NIPER) for allowing us to use the Abbe refrac-

trometer. We are also grateful to Department of Biotechnology,

2166 | Analyst, 2011, 136, 2161–2167

Govt. of India and Third World Academy of Science (TWAS,

Trieste, Italy) for financial support.

References

1 A. M. Morris, M. A. Watzky and R. G. Finke, Biochim. Biophys.Acta, 2009, 1794, 375–397.

2 B. Sahoo, J. Balaji, S. Nag, S. K. Kaushalya and S. Maiti, J. Chem.Phys., 2008, 129, 075103.

3 Z. Ignatova and L. M. Gierasch, Methods Cell Biol., 2008, 89, 59–70.4 J. H. Lee, I. H. Lee, Y. J. Choe, S. Kang, Y. H. Kim, W. P. Gai,J. S. Hahn and S. R. Paik, Biochem. J., 2009, 418, 311–323.

5 M. R. Nilsson, Methods, 2004, 34, 151–160.6 N. Motamedi-Shad, E. Monsellier, S. Torrassa, A. Relini andF. Chiti, J. Biol. Chem., 2009, 284, 29921–29934.

7 C. E. Kung and J. K. Reed, Biochemistry, 1989, 28, 6678–6686.8 B.Demeule,R.Gurny andT.Arvinte, Int. J. Pharm., 2007, 329, 37–45.9 A. Hawe, W. Friess, M. Sutter and W. Jiskoot, Anal. Biochem., 2008,378, 115–122.

10 B. Kundu and P. Guptasarma, Biochem. Biophys. Res. Commun.,2002, 293, 572–577.

11 A. Hawe, M. Sutter and W. Jiskoot, Pharm. Res., 2008, 25, 1487–1499.

12 S. Corrent, P. Hahn, G. Pohlers, T. J. Connolly, J. C. Scaiano,V. Fornes and H. Garc�ıa, J. Phys. Chem., 1998, 102, 5852–5858.

13 M. Frenette, G. I. Marius and J. C. Scaiano, Can. J. Chem., 2005, 83,869–874.

14 D. Goldstein, T. Nassar, G. Lambert, J. Kadouche and S. Benita,J. Controlled Release, 2005, 108, 418–432.

15 L. A.Munishkina andA. L. Fink, Biochim. Biophys. Acta, 2007, 1768,1862–1885.

16 R. J. Spiegel, I. T. Magarth and J. A. Shutta, Cancer Res., 1981, 41,452–458.

17 P. Frid, S. V. Anisimov and N. Popovic, Brain Res. Rev., 2007, 53,135–160.

18 A. Rana, T. P. Gupta, S. Bansal and B. Kundu, Biochim. Biophys.Acta, 2008, 1784, 930–935.

19 G. V. Semisotnov, N. A. Rodionova, O. I. Razgulyaev,V. N. Uversky, A. F. Gripas and R. I. Gilmanshin, Biopolymers,1991, 31, 119–128.

20 B. Kundu and P. Guptasarma, Proteins, 1999, 37, 321–324.21 P.-S. Song and W. H. Gordon III, J. Phys. Chem., 1970, 74, 4234–

4240.22 H. E. Katerinopoulos, Curr. Pharm. Des., 2004, 10, 3835–3852.23 S. R. Trenor, A. R. Shultz, B. J. Love and T. E. Long, Chem. Rev.,

2004, 104, 3059–3077.24 G. Jones II and J. A. Jimenez, J. Photochem. Photobiol., B, 2001, 65,

5–12.25 M. D. Mason, K. Ray, G. Pohlers, J. F. Cameron and R. D. Grober,

J. Phys. Chem. B, 2003, 107, 14219–14224.26 J. A. Molina-Bol�ıvar, J. Aguiar and C. C. Ruiz,Mol. Phys., 2001, 99,

1729–1741.27 D. M. Herculeus, Fluorescence and phosphorescence analysis:

Principles and application, Interscience, New York, 1966.28 F. J€ahnig, Proc. Natl. Acad. Sci. U. S. A., 1979, 76, 6361–6365.29 S. Shrivastava, Y. D. Paila, A. Dutta and A. Chattopadhyay,

Biochemistry, 2008, 47, 5668–5677.30 H. G. Franquelim, L. M. S. Loura, N. C. Santos and

M. A. R. B. Castanho, J. Am. Chem. Soc., 2008, 130, 6215–6223.31 A. Le Bivic, H. Sari, M. Reynier, S. Lebec and F. Bardin, J. Natl.

Cancer Inst., 1987, 79, 1181–1188.32 L. Latterini and M. Amelia, Langmuir, 2009, 25, 4767–4773.33 J. R. Lakowicz, Principles of fluorescence spectroscopy, Springer, New

York, 3rd edn, 2006.34 L. Narhi, S. J. Wood, S. Steavenson, Y. Jiang, G. M. Wu, D. Anafi,

S. A. Kaufman, F. Martin, K. Sitney, P. Denis, J. C. Louis,J. Wypych, A. L. Biere and M. Citron, J. Biol. Chem., 1999, 274,9843–9846.

35 B. I. Giasson, I. V. J. Murray, J. Q. Trojanowski and V. M. Y. Lee,J. Biol. Chem., 2001, 276, 2380–2386.

36 A. Abeliovich, Y. Schmitz, I. Fari~nas, D. Choi-Lundberg, W. H. Ho,P. E. Castillo, N. Shinsky, J. M. Verdugo, M. Armanini, A. Ryan,M. Hynes, H. Phillips, D. Sulzer and A. Rosenthal, Neuron, 2000,25, 239–252.

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View Article Online

37 H. Naiki, K. Higuchi, M. Hosokawa and T. Takeda, Anal. Biochem.,1989, 177, 244–249.

38 M. M. Bradford, Anal. Biochem., 1976, 72, 248–254.39 R. C. Switzer, C. R. Merril and S. Shifrin, Anal. Biochem., 1979, 98,

231–237.40 N. K. Jain and I. Roy, Eur. J. Pharm. Biopharm., 2008, 69, 824–

834.41 J. Sambrook and D. Russell,Molecular cloning: A laboratory manual,

Cold Spring Harbor Laboratory Press, New York, 2001.42 R. Cappai, S. L. Leck, D. J. Tew, N. A. Williamson, D. P. Smith,

D. Galatis, R. A. Sharples, C. C. Curtain, F. E. Ali, R. A. Cherny,J. G. Culvenor, S. P. Bottomley, C. L. Masters, K. J. Barnham andA. F. Hill, FASEB J., 2005, 19, 1377–1379.

43 S. J. Wood, J. Wypych, S. Steavenson, J.-C. Louis, M. Citron andA. L. Biere, J. Biol. Chem., 1999, 274, 19509–19512.

This journal is ª The Royal Society of Chemistry 2011

44 V. N. Uversky, J. Li and A. L. Fink, J. Biol. Chem., 2001, 276, 10737–10744.

45 G. He, L. L. Ma, J. Pan and S. Venkatraman, Int. J. Pharm., 2007,334, 48–55.

46 A. T. R. Williams, S. A. Winfield and J. N. Miller, Analyst, 1983, 108,1067–1071.

47 S. Dhami, A. J. D. Mello, G. Rumbles, S. M. Bishop, D. Phillips andA. Beeby, Photochem. Photobiol., 1995, 61, 341–346.

48 J. Q. Umberger and V. K. Lamer, J. Am. Chem. Soc., 1945, 67, 1099–1109.

49 W. H. Melhuish, J. Phys. Chem., 1961, 65, 229–235.50 V. N. Uversky, S. Winter and G. L€oberc, Biochim. Biophys. Acta,

1998, 1388, 133–142.51 K. Takehara, Y. Morinaga, S. Nakashima, S. Matsuoka, H. Kamaya

and I. Ueda, Anal. Sci., 2006, 22, 1571–1575.

Analyst, 2011, 136, 2161–2167 | 2167