Journal of Luminescence 134 (2013) 132–137

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Journal of Luminescence

0022-23

http://d

n Corr

E-m

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

Interaction between viologen-phosphorus dendrimers and a-synuclein

Katarzyna Milowska a,n, Justyna Grochowina a, Nadia Katir b, Abdelkrim El Kadib c, Jean-Pierre Majoral b,Maria Bryszewska a, Teresa Gabryelak a

a Department of General Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, Pomorska 141/143, 90-236 Lodz, Polandb Laboratoire de Chimie de Coordination CNRS, 205 route de Narbonne, 31077 Toulouse, Francec Institute of Nanomaterials and Nanotechnology (INANOTECH)-MAScIR (Moroccan Foundation for Advanced Science, Innovation and Research), ENSET, Avenue de I’Armee Royale,

Madinat El Irfane, 10100 Rabat, Morocco

a r t i c l e i n f o

Article history:

Received 10 July 2012

Accepted 28 August 2012Available online 5 September 2012

Keywords:

Viologen-phosphorus dendrimers

a-Synuclein

Fluorescence quenching

Circular dichroism

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

x.doi.org/10.1016/j.jlumin.2012.08.060

esponding author. Tel./fax: þ48 42 635 44 74

ail address: milowska@biol.uni.lodz.pl (K. Mil

a b s t r a c t

In this study the interaction between viologen-phosphorus dendrimers and a-synuclein (ASN) was

examined. Polycationic viologen-phosphorus dendrimers (two positive charges per viologen unit) are

novel compounds with relatively unknown properties. The influence of these viologen dendrimers on

ASN was tested using fluorimetric and circular dichroism methods. ASN contains four tyrosine residues;

therefore, the influence of dendrimers on protein molecular conformation by measuring the changes in

the ASN fluorescence in the presence of dendrimers was evaluated. The interaction of dendrimers with

free L-tyrosine was also monitored. Results show that viologen-phosphorus dendrimers interact with

ASN; they quenched the fluorescence of ASN as well as free tyrosine by dynamic and static ways.

However, the quenching was not accompanied by modifications in the ASN secondary structure.

& 2012 Elsevier B.V. All rights reserved.

1. Introduction

Disorders in the proteins spatial structure are due to severalfactors including mutations in encoded genes, post-translationalmodification of molecules or cell-damaging agents [1]. Thesechanges may have far-reaching consequences for the functioningof the body.

Improperly (abnormally) folded or damaged proteins canaccumulate and aggregate in the cell forming deposits. Theseinsoluble protein aggregates formed in the cells of the centralnervous system are involved in pathogenesis of many neurode-generative diseases as Alzheimer’s disease (deposits of amyloidb (Ab) and tau protein), Parkinson’s disease (a-synuclein) orCreutzfeldt–Jakob disease (prion protein) [2].

a-Synuclein (ASN) is a 140-amino acid protein that is foundmainly in presynaptic terminals of neurons. In the physiologicalstate, the native a-synuclein is unstructured, highly soluble andthermostable [3]. The primary structure of a-synuclein is char-acterized by the presence of three regions. N-terminal region(amino acids 1–60) is involved in lipid binding. As a result of lipidattachment, the largest part of ASN has a a-helix conformation,which is essential for its proper functioning. Moreover, within theN-terminal domain there are three point mutations associatedwith familial variants of Parkinson’s diseases. The second region

ll rights reserved.

.

owska).

(amino acids 61–95) consists of a highly hydrophobic sequence ofNAC (non-Ab-component of AD amyloid). This domain might beresponsible for the aggregation of a-synuclein. The C-terminalregion (amino acids 96–140) is rich in proline, aspartic acid andglutamic acid. This region plays an important role in preventingaggregation of ASN fibrils, as well as preventing the effects ofoxidative stress and exhibits chaperone activity [3–7].

Despite the well-known a-synuclein structure, its function inthe body has not been fully elucidated. It seems that it is involvedin every step of synaptic vesicle recycling, including trafficking,docking, fusion and recycling after exocytosis, the synthesis andrelease of neurotransmitters, regulation of vesicular transport,and dopamine homeostasis [7–9].

Currently, it is necessary to search for factors contributing tothe inhibition of a-synuclein aggregation, which could havetherapeutic significance in neurodegenerative diseases. Wedecided to focus on dendrimers, because in the previous studiesPAMAM and phosphorus dendrimers showed the properties ofpreventing the ASN fibrillation [4,10]. These dendrimers and PPIalso inhibit the process of aggregation of Ab amyloid and prionPrP 185–208 peptides and show an ability to break up the existingaggregates [11–13].

In this study we selected two viologen-phosphorus containingdendrimers, new compounds, which have been seldom investi-gated in terms of their biological properties. These types ofcompounds exhibit antimicrobial properties and their behaviordepends on their size, the number of viologen units and thenature of the surface groups [14]. Asaftei and De Clercq [15]

K. Milowska et al. / Journal of Luminescence 134 (2013) 132–137 133

demonstrated antiviral activity of dendrimers containing a violo-gen against the human immunodeficiency virus (HIV), and, toa lesser extent, against other viruses (herpes simplex virus,Reovirus and respiratory syncytial viruses).

In the present work, we investigated whether viologen-phosphorous dendrimers can interact with a-synuclein and affectits conformation. A spectrofluorimetric method based on intrinsictyrosine fluorescence was chosen to study the interactionbetween ASN and viologen-phosphorus dendrimers. Also, theinfluence of these dendrimers on the secondary structure ofASN was studied by circular dichroism.

2. Materials and methods

2.1. Materials

Viologen-phosphorus containing dendrimers of generationzero were synthesized in the Laboratoire de Chimie de Coordina-tion du CNRS. These compounds are characterized by the pre-sence of a hexafunctionalized core (P3N3)(NCH3NQ)6, viologenlinkages as branches and either phosphonate groups or polyethy-lene glycol (PEG) groups on their surface. The chemical structureof viologen-phosphorus containing dendrimers is shown in Fig. 1.

Human ASN was purchased from Sigma-Aldrich (USA). For allexperiments, ASN and dendrimers were dissolved in phosphate-buffered saline (1.9 mM NaH2PO4, 8.1 mM Na2HPO4, pH¼7.4).

All other chemicals were of analytical grade. All solutions weremade with water purified by the Mili-Q system.

2.2. Fluorescence quenching of ASN

Fluorescence measurements were performed using a Perkin–Elmer LS-50B spectrofluorometer with excitation at 280 nm, andemission spectra were recorded in the range of 290–400 nm. ASNat a concentration of 8 mM was contained in 10-mm quartzcuvettes, and it was thermostatted at 37 1C. Next, increasingconcentrations of dendrimers were added to ASN and fluores-cence spectra were recorded.

Before examining fluorescence properties of the protein, thefluorescence spectra of the dendrimers at the used concentrationswere also made.

2.3. Fluorescence quenching of free L-tyrosine

L-tyrosine was dissolved in a phosphate buffer at a concentra-tion of 8 mM. The excitation wavelength of 280 nm was used andthe emission spectra were recorded from 290 to 400 nm using a

Fig. 1. Structure, molecular weight, and the number of cationic end

Perkin–Elmer LS-50B spectrofluorometer. Next, increasing con-centrations of dendrimers were added to tyrosine and fluores-cence spectra were recorded.

2.4. Circular dichroism (CD) spectrometry

The CD spectra (195–260 nm) were recorded for a-synucleinin the presence/absence of dendrimers on a Jasco J-815 CDspectropolarimeter, in 5-mm path length quartz cuvettes, with awavelength step of 1 nm, a response time of 4 s and a scan rate of20 nm/min and thermostatted at 37 1C. Each spectrum was theaverage of three repetitions. ASN was used at a concentration of2 mM. The molar ratio of dendrimers/ASN ranged from 0.5 to 2.Viologen-phosphorus dendrimers alone at the concentrationsused in the experiments did not produce any discernible featuresin their CD spectra and were used as blanks for ASN–dendrimerscomplex spectra.

2.5. Statistical analysis

The results of the fluorescent study are presented asmean7SD from six individual experiments. Each experimentwas repeated three times. The percentage content of the second-ary structure was analyzed after three individual experiments.Statistical evaluation of the difference between the control andthe treated group was performed using Student’s t-test. Po0.05and below was accepted as statistically significant.

3. Results

3.1. Fluorescence quenching of ASN

Fluorescence spectroscopy is a method often used in thestudies on proteins. An analysis of the fluorescence spectra ofthe protein molecules and their dependence on various environ-mental factors (such as pH, temperature, viscosity) can bringseveral interesting conclusions, e.g., on the spatial structure ofproteins or their interaction with other molecules. ASN possessesfour tyrosine residues and no tryptophan. The acidic C-terminaldomain contains three tyrosines at positions 125, 133 and 136;the fourth is in the N-terminal region at the position 39. There-fore, ASN–dendrimer interactions can be evaluated by studyingchanges in tyrosine fluorescence spectra.

The emission spectra of tyrosine residues in ASN in theabsence and presence of increasing amounts of viologen-phosphorus dendrimers are presented in Fig. 2. A decrease inmaximum fluorescence intensity was observed upon addition of

groups of viologen-phosphorous dendrimers (vpd-1 and vpd-2).

F [a

.u.]

λ [nm]

ASN + vpd-1

ASNASN + D 1 μMASN + D 2 μMASN + D 4 μMASN + D 10 μMASN + D 20 μMD 20 μM

290 310 330 350 370 390 410 430 450 470 490

290 310 330 350 370 390 410 430 450 470 490

F [a

.u.]

λ [nm]

ASN + vpd-2

ASNASN + D 1 μMASN + D 2 μMASN + D 4 μMASN + D 10 μMASN + D 20 μMD 20 μM

0

10

20

30

40

50

60

0

10

20

30

40

50

60

Fig. 2. ASN tyrosine residue fluorescence quenching by dendrimers: (A) vpd-1

and (B) vpd-2.

0

5

10

15

20

25

0 5 10 15 20 25

F 0/ F

Q [μM]

vpd-1vpd-2

Fig. 3. Stern–Volmer plots for intrinsic tyrosine fluorescence quenching by

dendrimers.

Table 1Stern–Volmer constants for quenching of tyrosine fluores-

cence of ASN by dendrimers.

Dendrimer KD (M�1) KS (M�1)

vpd-1 0.355�106 –

vpd-2 0.601�106 0.026�106

λ m

ax [n

m]

c vpd-1 [μM]

315

320

325

330

335

340

315

320

325

330

335

340

0 5 10 15

0 5 10 15 20

λ m

ax [n

m]

c vpd-2 [μM]

20

Fig. 4. Effect of violgen dendrimers: (A) vpd-1 and (B) vpd-2 on the position of the

ASN emission maximum.

K. Milowska et al. / Journal of Luminescence 134 (2013) 132–137134

dendrimers. These results indicate that viologen-phosphorusdendrimers can quench the fluorescence of tyrosine. The effectwas stronger for the vpd-2 dendrimer.

The Stern–Volmer plots for the quenching of ASN with bothused dendrimers (quenchers) are shown in Fig. 3. The Stern–Volmer plot for vpd-1 quenching is linear indicating that thequenching mechanism is dynamic. The Stern–Volmer constantobtained from the slope of this plot was calculated using Stern–Volmer equation:

F0=F ¼ 1þKD½Q �

where F is the fluorescence intensity in the presence of quencher,F0 is its value without the quencher, KD is called the Stern–Volmerquenching constant and [Q] is the quencher concentration.

For the second dendrimer (vpd-2) the Stern–Volmer plotexhibited an upward curvature, suggesting that the staticmechanism also took place. In this case the quenching data can

be analyzed by the following Stern–Volmer equation:

F0=F ¼ ð1þKD½Q �Þð1þKS½Q �Þ ¼ 1þK1½Q �þK2½Q �2

where K1¼(KDþKS) and K2¼(KDKS), and KD and KS are thedynamic and static quenching constants, respectively [16].

The Stern–Volmer constants for the quenching of tyrosinefluorescence by viologen-phosphorus dendrimers are presentedin Table 1.

In the emission spectra a fluorescence maximum shiftingtoward longer wavelengths was noticed. The tyrosine emissionpeaks were observed between 317 and 335 after the addition ofboth used dendrimers (Fig. 3).

In Fig. 3, the emergence of a new peak at 381 nm wavelengthcorrelated with increasing concentrations of vpd-1 can also beseen. The value of the fluorescence intensity was on the samelevel, independent of the dendrimer concentration Fig. 4.

3.2. Fluorescence quenching of free L-tyrosine

In Fig. 5 the fluorescence spectra of L-tyrosine without/withviologen-phosphorus dendrimers are presented. Free L-tyrosineexhibits a maximum emission at 303 nm. After the successiveaddition of dendrimers, decrease in the fluorescence intensity offree L-tyrosine was observed. This quenching indicated the inter-action between dendrimers and tyrosine. During this process, noshift in the fluorescence maximum was observed. For both used

0

20

40

60

80

100

F [a

.u.]

λ [nm]

L-Tyr + vpd-1

TyrTyr + D 1 μMTyr + D 2 μMTyr + D 4 μMTyr + D 6 μM

0

20

40

60

80

100

290 310 330 350 370 390 410 430 450 470 490

290 310 330 350 370 390 410 430 450 470 490

F [a

.u.]

λ [nm]

L-Tyr+ vpd-2

TyrTyr + D 1 μMTyr + D 2 μMTyr + D 4 μMTyr + D 6 μM

Fig. 5. Free L-tyrosine fluorescence quenching by dendrimers: (A) vpd-1 and

(B) vpd-2.

0

5

10

15

20

25

30

35

0 2 4 6 8 10

F 0/ F

Q [μM]

vpd-1vpd-2

Fig. 6. Stern–Volmer plots for free L-tyrosine fluorescence quenching by dendrimers.

Table 2Stern–Volmer constants for quenching of free L-

tyrosine fluorescence by dendrimers.

Dendrimer KD (M�1) KS (M�1)

vpd-1 0.407�106 0.277�106

vpd-2 0.615�106 0.318�106

-12

-10

-8

-6

-4

-2

0

2

CD

[mde

g]

[nm]

ASN + vpd-1

ASNASN+ D 1:0.5ASN + D 1:1ASN + D 1:1.5ASN + D 1:2

-12

-10

-8

-6

-4

-2

0

2

190 200 210 220 230 240 250 260

190 200 210 220 230 240 250 260C

D [m

deg]

[nm]

ASN + vpd-2

ASNASN + D 1:0.5ASN + D 1:1ASN + D 1:1.5ASN + D 1:2

Fig. 7. CD spectra of a-synuclein (c¼2 mM) alone and in the presence of dendrimers:

(A) vpd-1 and (B) vpd-2.

K. Milowska et al. / Journal of Luminescence 134 (2013) 132–137 135

dendrimers quenching occurred by both dynamic and staticmechanisms. The Stern–Volmer plots are shown in Fig. 6 andthe Stern–Volmer constants are given in Table 2.

3.3. Circular dichroism (CD) spectrometry

In this work we also examined the effect of dendrimers on thesecondary structure of ASN using the circular dichroism method.

Fig. 7 presents the ASN CD spectra alone and in the presence ofdendrimers. The CD spectra of non-incubated ASN were typical ofa substantially unfolded polypeptide chain; a minimum negativepeak typical of random coil was observed at 200 nm and a weakerbroad minimum was observed at E225 nm. The addition ofvpd-1 dendrimers at the lowest used concentrations and vpd-2dendrimers with the exception of their largest concentrations, did

not cause significant changes in the spectra shape. In other cases,only slight changes in the intensity of the negative peak wereobserved.

4. Discussion

The present work was aimed at investigating whether twoviologen-phosphorus dendrimers interact with a-synuclein andchange its conformation and structure. There was one importantreason for choosing this protein: ASN is a protein whose struc-tural changes and aggregation cause neurodegenerative diseases.On the other hand, the chosen dendrimers are new compounds,whose properties are not well known. They are incorporatingwithin their structure both neutral cyclic phosphorus nitrogenmoieties and polycationic cyclic nitrogen units. Moreover one ofthese dendrimers is decorated with phosphonate end groups onits surface which bring fascinating properties in different fieldsincluding NK cells amplification [17], anti-inflammatory proper-ties [18], etc. The presence of charges within the cascade structuremight be also of interest since polycationic phosphorus dendri-mers can be used as transporters of genetic material and drugs[19,20], exhibit anti-HIV activity [21], can prevent aggregation ofproteins associated with prion [22] and Alzheimer’s and Parkin-son’s diseases [10,12,23]. Lastly the grafting of PEG groups on thesurface of the other one may also bring properties in the field of

K. Milowska et al. / Journal of Luminescence 134 (2013) 132–137136

medicinal chemistry, decreasing toxicity and enhancing watersolubility.

Asaftei and De Clerq’s studies [15] performed using monomersand dendrimer derivatives of viologens showed their antiviralproperties, particularly against the HIV virus.

The method of tryptophan or tyrosine residues fluorescencequenching has found wide applications in the studies on mole-cular dynamics and structure of proteins. The conformationalchanges of ASN were evaluated by the measurements of ASNtyrosine residue fluorescence intensity before and after theaddition of dendrimers. The emission properties of tyrosinedepend on a combination of specific and non specific interactionsbetween the chromophore and its environment. The obtainedresults indicate a statistically significant quenching of freeL-tyrosine and ASN intrinsic tyrosine fluorescence by both useddendrimers. These data suggest that viologen-phosphorus den-drimers act as effective quenchers of tyrosine. The data for vpd-1dendrimer (phosphonate end groups) show only the dynamicmechanism of intrinsic tyrosine fluorescence quenching, becausethe equation assumes a linear plot of F0/F versus [Q] and the slopeis equal to KSV. The Stern–Volmer constants express quenchingefficiency and thus sensitivity of the sensor [24]. For the seconddendrimer, the Stern–Volmer plot is curved upwards, whichmeans that there are both dynamic (collisional) and staticmechanisms of fluorescence quenching. The static quenching isoften observed for efficient quenchers and is likely to be accom-panied by the conformational changes of the molecules [25].

The quenching of intrinsic tyrosine fluorescence by cationicphosphorus dendrimers was observed in our previous work [10].This research showed that the interaction between ASN anddendrimers depends on their size (generation) and the fluores-cence is quenched by a dynamic mechanism.

The mechanism of fluorescence quenching depends on thechemical structure of the quencher and in many cases has notbeen completely clear. Thus, fluorescence can be quenched by: anelectron spin exchange process, leading to a rapid move of anelectron from the excited singlet state to the triplet state, electrontransfer from the chromophore singlet state, spin–orbit couplingbetween the chromophore molecules in the excited state and thequencher, and resonance energy transfer, proton transfer, forma-tion of non fluorescence complexes and other processes [26–29].

The effect of dendrimers on free L-tyrosine was studied toeliminate the possibility that the observed influence of dendri-mers on ASN was only a consequence of simple diffusionprocesses of the dendrimers in a solution. Both dendrimersproved to be strong quenchers of free tyrosine, and the Stern–Volmer plots exhibited strong upward curvature. Thus, in bothcases the quenching mechanisms are dynamic and static. In thedynamic quenching the excited fluorophore molecules collidewith a molecule of a quencher. Static quenching involves animmediate acquisition of fluorophore excitation energy by thequencher, or formation of a non fluorescent complex, before themoment of excitation.

Free L-tyrosine exhibits maximum fluorescence at 303 nm.The addition of dendrimers did not change the position of themaximum, so the quenching was not accompanied by the fluores-cence maximum shift.

In the case of ASN intrinsic tyrosine fluorescence a maximumred-shift was observed after the addition of dendrimers. Tyrosineemission peak was observed at wavelengths between 317 and335 nm for both used dendrimers. The fluorescent properties oftyrosine are solvent dependent. The shift in the position of themaximum corresponds with the changes of the polarity aroundthe chromophore molecules [30]. The emission is red-shifted in amore polar solvent. In a basic aqueous solution, spectra showlarge red-shift with a decrease in fluorescence quantum yield, due

to deprotonation of the aromatic hydroxyl group [31]. In thespectra of ASN, apart from the fluorescence quenching andmaximum shift, a new peak was observed at 381. In the spectrumof vpd-1 dendrimer, a very small peak was noticed at 381 nm.When the dendrimer was added to ASN, the peak was higher.These results suggest the energy transfer from ASN to thedendrimer.

Circular dichroism (CD) spectroscopy was used to examine thestructural changes in ASN in the presence of viologen-phosphorusdendrimers. A similar shape of CD spectra in the presence andabsence of dendrimers indicates that the structure of ASN stayspredominantly unfolded. Although the fluorescence methodshowed conformational changes, those changes do not affect thesecondary structure of the protein.

The photo-physical and structural interaction betweenviologen-phosphorus dendrimers and human serum albuminhave also been tested previously [25]. The results showed thatthe used dendrimers strongly quenched HSA fluorescence, but thedendrimer with PEG groups on the surface, did not quenchfluorescence of free L-tryptophan. It did not influence the albuminCD spectra either [25].

In conclusion, the used viologen-phosphorus dendrimersquenched the fluorescence of ASN as well as free tyrosine, butdid not change the secondary structure of ASN. Both dendrimerscaused a red-shift in the maximum of fluorescence and theobtained results suggest transfer of energy from ASN to thedendrimer.

Acknowledgment

The project was supported by the COST Action TD0802.

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