Amino acid-based cationic gemini surfactant–protein interactions

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Amino acid-based cationic gemini surfactant–protein interactions Mafalda A. Branco, Lídia Pinheiro, Célia Faustino ∗ Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal

h i g h l i g h t s

g r a p h i c a l

a b s t r a c t

• (C12 Cys)2 , a new gemini surfactant, forms stable suspensions with and without BSA. • BSA–(C12 Cys)2 represents an oppositely charged biopolymer– surfactant system. • The tensiometric profile is consistent with a cooperative binding process. • (C12 Cys)2 quenches the intrinsic fluorescence of BSA by a static quenching process.

a r t i c l e

i n f o

Article history: Received 30 July 2014 Received in revised form 6 December 2014 Accepted 12 December 2014 Available online xxx Keywords: Amino acid-based surfactant Cationic gemini surfactant Bovine serum albumin Tensiometry Fluorescence spectroscopy

a b s t r a c t A novel cationic amino acid-based gemini surfactant derived from cysteine, (C12 Cys)2 , has been synthesized and both its supramolecular behaviour and its interaction with the model protein bovine serum albumin (BSA) have been characterized under physiological mimetic conditions (PBS, pH 7.4). Surface tension measurements were used to obtain important system parameters, such as critical micelle concentration (CMC), critical aggregation concentration (CAC), protein saturation point (PSP), maximum surface excess concentration ( max ), minimum surface area per molecule (Amin ) at the air/solution interface and the degree of surfactant binding to protein (˛). Formation of a protein–surfactant complex was confirmed by UV–vis and fluorescence spectroscopy. Fluorescence quenching measurements allowed determination of the Stern–Volmer quenching constant (KSV ), surfactant–protein binding constant (Ka ) and number of binding sites (n). UV–vis measurements and the calculated value for the bimolecular quenching constant (kq ) suggest that (C12 Cys)2 quenches BSA intrinsic fluorescence by a static quenching mechanism. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Aggregated proteins are associated with neurodegenerative disorders, such as Alzheimer’s disease (AD), characterized by accumulation of the ␤-amyloid (A␤) peptide and formation of insoluble, extracellular aggregates that lead to neuronal cell death and dementia [1–3]. Cationic gemini surfactants derived from quaternary ammonium salts are known to disassemble and clear mature

∗ Corresponding author. Tel.: +351 217 946 400; fax: +351 217 946 470. E-mail address: [email protected] (C. Faustino).

A␤ fibrils in vitro, forming soluble mixed aggregates, however the cytotoxicity associated with these compounds prevents their clinical use [4,5]. Gemini surfactants are dimeric surfactants formed by two polar head groups and two hydrophobic alkyl chains per molecule linked by a spacer at the level of the head groups. Due to their dimeric structure gemini surfactants usually show lower critical micelle concentration (CMC), higher efficiency in surface tension reduction and better solubilizing capacity when compared to their conventional single chain counterparts [6–8]. Gemini surfactants derived from natural amino acids are biocompatible and biodegradable surfactants that meet the

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requirements of both biological and ecological compatibility [9–11] and thus represent a promising class of surfactants to be studied as ␤-amyloid disassembly agents. This class of surfactants offers the additional advantage of being amenable to production by biotechnological procedures (either by fermentation or enzymatic catalysis) as an alternative to chemical synthesis [9,11,12] and have become very attractive for biomedical applications, including drug and gene delivery [13–15]. Ionic surfactants are known to bind to proteins by both electrostatic (specific) and hydrophobic (cooperative) interactions leading to protein unfolding and formation of a surfactant–protein complex [16–23]. This mechanism is on the basis of their employment as denaturing agents in the extraction and purification of proteins from biological matrixes as well as on protein characterization [24,25]. Recent investigations showed that gemini surfactants interact more efficiently with proteins when compared to traditional single-chain surfactants [26–29]. Our report on the interaction between bovine serum albumin (BSA) and anionic amino acid-based gemini surfactants also revealed that surfactant stereochemistry influenced the binding to the protein, as well as external conditions, such as pH, ionic strength and temperature [29]. The biological role of proteins is important for their interaction with surfactant molecules. In mammals, serum albumin is synthesized in the liver and corresponds to the most abundant protein in the plasma, contributing to the osmotic blood pressure and maintaining the blood pH. Serum albumins act as carriers for endogenous ligands such as fatty acids, bilirubin, hematin and steroids [16,17,30]. Their low specificity also allows them to bind to several xenobiotics, including drugs and surfactants, which can make them responsible for termination of the therapeutic action of some drugs. BSA has been extensively used as a model protein to study the interaction between proteins and amphiphilic ligands because of its stability and solubility in aqueous media, its low specificity and its strong resemblance with human serum albumin (HSA) [16,17,30]. The isoelectric point of BSA in 0.15 mol L−1 sodium chloride is about 4.9 [30] so at physiological pH the protein has a net negative charge. In the present study a novel cationic amino acid-based gemini surfactant derived from cysteine, (C12 Cys)2 , has been synthesized from the condensation reaction of l-cystine (the l-cysteine dimer) with dodecylamine, according to the reaction scheme in Fig. 1. Classical synthetic procedures used in amino acid and peptide synthesis [31] have been employed, namely l-cystine protected with tert-butoxycarbonyl (Boc) protecting groups at the amine functions was used and activation of the carboxyl groups of the amino acid was accomplished using O-(benzotriazol1-yl)-N,N,N ,N -tetramethyluronium tetrafluoroborate (TBTU), as described in Section 2. The supramolecular behaviour of the gemini surfactant (C12 Cys)2 has been characterized and its interaction with the model protein bovine serum albumin was studied under physiological mimetic conditions (phosphate-buffered saline, pH 7.4), by means of surface tension, UV–vis spectroscopy and fluorescence quenching methods. The cationic gemini surfactant (C12 Cys)2 forms stable suspensions in PBS (pH 7.4), as well as in the presence of 0.10 g L−1 BSA, after sonication. Suspensions are stable up to a surfactant concentration of 2.0 g L−1 (ca. 3.0 mmol L−1 ), after which a milky suspension forms and sedimentation occurs. Under the experimental conditions employed (PBS, pH 7.4) the pH of BSA is above its isoelectric point so that the overall charge of the protein is negative [30]. Thus, the BSA–(C12 Cys)2 system represents an oppositely charged biopolymer–surfactant system where both electrostatic and hydrophobic interactions between the components are to be expected.

2. Materials and methods 2.1. Materials N,N -Di-tert-butoxycarbonyl-l-cystine, O-(benzotriazol-1-yl)N,N,N ,N -tetramethyluronium tetrafluoroborate, dodecylamine and triethylamine of the highest available purity were from Sigma. Concentrated hydrochloric acid (37%), dichloromethane, methanol and acetone were p.a. grade reagents from Merck. BSA (fraction V) with 99% purity was from Sigma and was used without any further purification. 2.2. Synthesis of (C12 Cys)2 The cationic gemini surfactant (C12 Cys)2 was synthesized from the di-tert-butylcarbamate derivative of l-cystine, which was converted into the diamide by condensation reaction with dodecylamine following prior activation of the carboxyl group with TBTU, according to classical peptide synthesis procedures [31]. Removal of the tert-butoxycarbonyl (Boc) protecting groups by a mixture of hydrochloric acid in dichloromethane gave the desired product as a white precipitate which was collected by vacuum filtration and purified from methanol/acetone, according to established literature procedures [32,33]. The synthesis of (C12 Cys)2 and reaction conditions is summarized in Fig. 1. The final product was characterized by 1 H NMR, 13 C NMR and FT-IR spectroscopy. 1 H and 13 C NMR spectra in CDCl3 were obtained in a Bruker Avance ARX-400 spectrometer operating at 400 MHz and 100 MHz, respectively, using the chloroform peak of the solvent as reference. IR spectra, in KBr pellets, were obtained in a Nicolet Impact 400 FT-IR spectrophotometer. (C12 Cys)2 . Yield: 88%. 1 H NMR (CDCl3 ): ı (ppm) = 0.87 (t, 6H, 2 × CH3 ), 1.24 (m, 36H, 2 × (CH2 )9 CH3 ), 1.51 (m, 4H, 2 × CH2 CH2 NHCO), 2.92 (m, 4H, 2 × CH2 NHCO), 3.22 (m, 4H, 2 × CH2 S), 4.77 (d, 2H, 2 × CHNH3 + ), 5.56 (d, 2H, NHC O). 13 C NMR (CDCl3 ): ı (ppm) = 14.26 (CH3 ), 22.85, 27.14, 28.54, 29.45, 29.50, 29.64, 29.74, 29.80, 29.84, 32.12 (CH3 (CH2 )10 ), 39.82 (CH2 S), 47.38 (CH2 (C O)N), 54.95 (CH), 170.24 (C O amide). FT-IR (KBr): max (cm−1 ) = 3485 (NH3 + ), 2920, 2850 (CH2 ), 1665 (C O amide), 1550 (NH3 + bending), 590 (C S). 2.3. Sample preparation BSA stock solutions, with a concentration of 0.10 g L−1 , were prepared by accurately weighting the appropriate amount of protein and dissolving in phosphate-buffered saline (PBS) solution (pH 7.4) under magnetic stirring for at least 12 h prior to use. The concentration of phosphate and sodium chloride in the buffer was 0.020 mol L−1 and 0.150 mol L−1 , respectively. Surfactant solutions, both in the absence and in the presence of 0.10 g L−1 BSA, were prepared by accurately weighting the desired amount of surfactant and dissolving in PBS solution (pH 7.4) or BSA stock solution, with sonication. 2.4. Surface tension measurements Surface tensions of sample solutions were obtained by the Wilhelmy plate technique, using a Krüss K12 tensiometer. Glass vessel was cleaned with chromic mixture, rinsed repeatedly with water and dried, prior to use. The platinum plate was washed with water and acetone and flame-dried before each measurement. Solutions of the desired surfactant concentration were prepared as described in Section 2.3 and aged overnight. The vessel was filled with 10 mL of the sample solution and the equilibrium surface tension was determined from sets of measurements performed until the change in surface tension was less than 0.10 mN m−1 . The equilibrium

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Fig. 1. Synthesis of cationic gemini amino-acid based surfactant (C12 Cys)2 derived from cystine. (a) TBTU (2 equiv.), CH2 Cl2 , rt; (b) CH3 (CH2 )11 NH2 (2 equiv.), Et3 N (2 equiv.), CH2 Cl2 , 24 h, rt; (c) HCl/CH2 Cl2 .

70 60

γ / mN m−1

to the critical aggregation concentration (CAC), representing the onset of cooperative binding, and to the protein saturation point (PSP), respectively. Above PSP, free micelles are formed and thus surface tension reaches a constant value. The maximum surface excess concentration ( max ) and the minimum surface area per molecule (Amin ) at the air/solution interface both in the absence as well as in the presence of BSA can be determined from Eqs. (1) and (2), respectively,

[BSA]/g L−1 0.00 PSP

50

0.10

40 30

CAC

20

CMC

max

10 -2

-1 0 log ([(C12Cys)2] / g L−1)

1 =− 2.303iRT

1

Fig. 2. Surface tension versus logarithm of gemini surfactant concentration in PBS (pH 7.4) at 298.15 K, in the absence and in the presence of 0.10 g L−1 BSA.

surface tension was determined from the last ten measurements obeying the former condition. The surface tension equilibrated within minutes or several hours depending on surfactant concentration. The temperature was kept constant at 25.0 ± 0.1 ◦ C by circulating water from a Julabo thermostat. 2.5. Fluorescence measurements The fluorescence spectra were obtained in a Hitachi F2000 fluorescence spectrophotometer using 1.0 cm path length quartz cells. The fluorescence spectra of BSA were scanned in the wavelength range of 295–450 nm at a fixed excitation wavelength of 280 nm, in the absence and in the presence of increasing surfactant concentrations. 2.6. UV–vis absorption measurements UV–vis absorption spectra in the wavelength range 190–400 nm were obtained in a Shimadzu UV-1603 spectrophotometer using 1 cm path length quartz cells. The reference blank was a solution of the same surfactant concentration as the sample solution but without BSA in order to eliminate the disturbance from the absorption of the surfactant. 3. Results and discussion 3.1. Surface tension data The critical micelle concentration (CMC) of (C12 Cys)2 was determined from the breakpoint of the surface tension versus the logarithm of surfactant concentration plot (Fig. 2). In the presence of BSA this plot exhibits two distinct breakpoints, corresponding

Amin =

1 NA max



∂ ∂ log C



(1) T

(2)

where R is the gas constant, NA is the Avogadro number, T is the absolute temperature, (∂/∂ log C)T is the slope below the CMC in the surface tension versus logarithm of surfactant concentration plots and i is the number of species whose concentration at the interface changes with the change in bulk phase concentration of surfactant. The value of i was taken as 1 according to the high ionic strength of the PBS buffer solution used which results in the presence of excess electrolyte. Table 1 summarizes some important aggregation parameters obtained from surface tension measurements, both in the absence and in the presence of BSA, including CMC, CAC, PSP, surface tension values at CMC ( cmc ), CAC ( cac ) and PSP ( psp ),  max and Amin . The novel amino acid-based gemini surfactant, (C12 Cys)2 , has a CMC value of 0.79 mmol L−1 in PBS (pH 7.4) which is similar to the CMC values obtained for quaternary ammonium gemini surfactants of the same alkyl chain length, such as the ones in the alkanediyl␣,␻-bis-(dodecyldimethylammonium bromide) series, designated as 12-s-12, where s is the number of carbon atoms in the spacer chain [34]. The aggregation properties and self-assembly behaviour of these surfactants in aqueous media have been extensively studied [6,7,34–36] and 12-s-12 in water show CMC values of 0.91, 1.00 and 1.12 mmol L−1 for s = 3, 4 and 6, respectively [34]. For 12-s-12 in phosphate buffer (pH 7.0) at 298.15 K, CMC values of 0.40 and 0.56 mmol L−1 have been reported for s = 3 and s = 6, respectively [36]. The ionic strength of the buffer solution is responsible for the lowering of the CMC due to the electrolyte screening effect which reduces the electrostatic repulsions between the head groups in the micelles thus promoting aggregation at lower surfactant concentrations. On the other hand, the correspondent single-chain cationic surfactant, dodecyltrimetylammonium bromide (DTAB) has CMC values of 16.0 mmol L−1 in water [34] and 14.53 mmol L−1 in phosphate buffer (pH 7.0) at 298.15 K [36]. The dimeric structure of gemini surfactants usually results in lower CMC values when compared to their monomeric (single-chain) counterparts due to enhanced hydrophobic interactions [6,7].

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Table 1 Aggregation parameters for (C12 Cys)2 in the absence and in the presence of BSA obtained from surface tension measurements in PBS (pH 7.4) at 298.15 K. Values in brackets are in mmol L−1 . [BSA]/g L−1

CAC/g L−1

 cac /mN m−1

CMC or PSP/g L−1

 cmc or  psp /mN m−1

106  max /mol m−2

1018 Amin /m2

0.00 0.10

– 0.32 (0.50)

– 42.1

0.51 (0.79) 0.70 (1.09)

31.6 35.6

2.34 1.67

0.71 0.99

The CMC value of (C12 Cys)2 is also similar to those obtained from conductivity measurements for cationic gemini surfactants derived from the amino acid arginine, namely N˛ ,Nω bis(N˛ -dodecylarginine)-␣,␻-alkyldiamide dihydrochloride salts, Cn (C12 Arg)2 , which are 0.5 and 0.4 mmol L−1 for n = 3 and n = 6, respectively [37]. Solutions of Cn (C12 Arg)2 with n = 9, 12 appear as translucent and viscous dispersions [37,38], similarly to solutions of (C12 Cys)2 whose turbidity increases with increasing surfactant concentration. The change in turbidity of a surfactant solution has been attributed to the change in the amount and/or size of the surfactant aggregates [37–39]. Moreover, for the cationic gemini surfactants with dodecyl chains derived from cystine and cystamine, bis(dodecyldimethyl glycine) cystine dimethylester dihydrochloride (DABC) and bis(dodecyldimethylglycine)cystamine dihydrochloride (DABK), the CMC obtained from surface tension measurements with the Wilhelmy plate yielded values of 0.065 and 0.120 mmol L−1 [40], respectively, which are lower than the CMC of (C12 Cys)2 . The presence of the quaternary ammonium groups and the position of the amide bond in the surfactant structure may be responsible for the fact as DABC and DABK were prepared by condensation reaction of dodecyldimethylaminobetaine with cystine dimethylester or cystamine [41], while (C12 Cys)2 was obtained from the condensation reaction of dodecylamine with cystine. Thus, (C12 Cys)2 , a novel cationic gemini surfactant derived from cystine, resembles more the gemini surfactants derived from arginine, Cn (C12 Arg)2 than the cationic gemini surfactants of the same alkyl chain length that also containing a disulfide bond derived from cystine (DABC) and cystamine (DABK). The peculiar properties of the Cn (C12 Arg)2 surfactants have been attributed to the strong hydrogen-bonding of the amide bond located between the polar amino acid residue and the hydrophobic alkyl chain of the surfactant molecules [11,37,39], a characteristic structural feature that is also present in (C12 Cys)2 . An important property of amino acid-based surfactants is their pH sensitivity. Cationic lipids derived from N-oleyl cysteine linked through a tripeptide are known to exhibit pH-sensitive hydrolysis when pH decreased from 7.5 to 5.5, which is the pH characteristic of the endosomal/lysosomal compartment, thus being potential transfection agents in gene therapy [42]. The cationic charge of amino acid-based surfactants such as (C12 Cys)2 is due to protonated amino groups of the cysteine residues (pKa 10.70) and the behaviour of the surfactant is pH-dependent, which is not the case of DABC and DABK that contain quaternary ammonium groups [11,40,41]. Thus, (C12 Cys)2 is expected to be essentially in its protonated form in PBS (pH 7.4) medium, although significant pKa shifts have been reported for amino acid-based gemini surfactants upon micellization as aggregation can induce changes in surfactant protonation [37,43–46]. For arginine-derived surfactants like Cn (C12 Arg)2 , the guanidinium group (pKa 12.48) of the amino acid residues is positively charged in acidic, neutral and most basic media. However, strong negative pKa shift of the guanidinium groups has been observed for arginine diacyl surfactant derivatives [37,43], the surfactants being significantly more acidic then the amino acid from which they were prepared. Charge reduction of the surfactant is accompanied by a decrease in monomer solubility leading to low CMC values and formation of non-globular aggregates, (for which the charge has a stronger influence compared to spherical aggregate formation) [37,43].

The surface tension at the CMC is a measure of the effectiveness of the surfactant, thus (C12 Cys)2 , with a  cmc of 31.6 mN m−1 , is more efficient in surface tension reduction than 12-s-12, with  cmc values in the range [35–43] mN m−1 [34] or DABC ( cmc 35 mN m−1 ) [41]. DABK and Cn (C12 Arg)2 have  cmc values around 30 mN m−1 [41,47] similar to (C12 Cys)2 . The value of Amin for pure gemini surfactant (C12 Cys)2 is lower than the usual Amin values reported for 12-s-12 gemini surfactants, which are usually around 1–2 nm2 [34]. DABC and DABK have Amin values of 1.02 and 1.19 nm2 , respectively, [41] while values between 0.8 and 1.3 nm2 have been reported for Cn (C12 Arg)2 [47]. The lower value obtained for (C12 Cys)2 may be due to the high ionic strength of the phosphate buffer used, as dense ionic atmosphere is known to have a screening effect in the electrostatic repulsions between the surfactant head groups, contributing to stabilization of the aggregates either in the bulk or at interface [30]. The same effect has been reported for cationic gemini surfactants of different spacer-chain lengths derived from cetyltrimethylammonium bromide (CTAB) in 60 mmol L−1 phosphate buffer solution [28] and for Cn (C12 Arg)2 in NaCl 0.01 mol L−1 [47]. The latter showed Amin values around 0.5–0.6 nm2 , lower than the ones observed in water at the same temperature. Discrepancies may also be related to the value of i in the Gibbs adsorption isotherm (Eq. (2)) as for gemini surfactants in pure water a value of 3 is used instead of 1. In the presence of BSA, the initial binding of (C12 Cys)2 to the protein is the result of specific electrostatic interactions of the cationic head groups with opposite charged amino acid residues of the macromolecule along with binding of the alkyl chains of the gemini surfactant to hydrophobic regions of the biopolymer in the vicinity of the binding sites [15,16,23]. Thus the CAC value is lower than the CMC of the surfactant in the absence of protein, a behaviour that has been described in the literature for ionic surfactants and globular proteins [23,28,48,49]. Formation of the surfactant–protein complex hinders the micellization process and thus the surfactant self-assembles to form free micelles in solution at higher concentrations than the CMC of pure surfactant solution. As a consequence, interfacial saturation of the interaction profile, corresponding to CMC, occurs at a higher surfactant concentration compared to the dilution profile, an indication of effective interaction between (C12 Cys)2 and BSA [23,28,48,49]. The ratio CAC/CMC measures the strength of the interaction between surfactant and BSA. The lower the CAC, the stronger is the binding between surfactant and protein. For the (C12 Cys)2 –BSA system the CAC/CMC ratio is 0.63 indicating a relatively weak interaction [17,29]. The difference between the CMC values in the presence and in the absence of protein can be used to determine the degree of binding of surfactant to protein, ˛, according to equation [17,50,51]: ˛=

[CMC] [PSP] − [CMC] = [BSA] [BSA]

(3)

A ˛ value of 1.9 has been obtained for the (C12 Cys)2 –BSA system, which means that 1.9 g of gemini surfactant bounds to 1 g of protein, in saturation conditions. A value of 1.50 has been referred for cationic surfactant DDAB [50]. The surfactant saturation binding is an important parameter that has been extensively studied [23,49,50,52–54]. From dialysis studies Reynolds and Tanford [52] concluded that different proteins, with different molecular weights,

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3.2. Fluorescence emission and UV–vis absorption spectra Fluorescence of BSA is usually dominated by the contribution of its aromatic amino acid residues, namely tyrosine (Tyr), tryptophan (Trp) and phenylalanine (Phe), the emission of the latter one being negligible in most cases [26–28,50,57]. The variations in the fluorescence spectra obtained by exciting the protein at 295 nm have been attributed to the presence of tryptophan residues while the changes that result from protein excitation at 280 nm are associated with both tryptophan and tyrosine residues [26,28]. As the fluorescence intensity and the wavelength of emission maximum ( max ) are sensitive to protein conformation, these parameters become important tools in probing protein folding and/or unfolding processes [20–23,50,57]. BSA has 19 Tyr residues in different domains and two Trp residues, namely Trp 134, present at the protein surface in domain I, and Trp 213, present in the hydrophobic binding pocket of the protein in domain II, that act as intrinsic fluorophores [58,59]. At the excitation wavelength of 280 nm, the quenching in BSA mainly occurs from binding of the surfactant ion in the vicinity of the forementioned Trp residues [60]. The fluorescence spectra of pure BSA and of BSA in the presence of increasing surfactant concentrations recorded at the excitation wavelength of 280 nm are shown in Fig. 3. BSA shows a strong fluorescence emission peak at 340 nm whose intensity gradually decreases upon addition of increasing surfactant concentrations, being accompanied by a blue shift of the maximum emission wavelength from 340 nm to 333 nm. The Trp 134 present at the surface of the protein in domain I is surrounded by a number of negatively charged aspartic (Asp) and glutamic (Glu) acid residues to which the cationic gemini (C12 Cys)2 can bind by electrostatic

200

1

8 6 F0/F

2 3

Fluorescence intensity

bind identical amounts of surfactant, and reported a binding of 1.4 g of surfactant per gram of protein for the sodium dodecylsulphate (SDS)–BSA system in phosphate buffer solution (pH 6.7) and low ionic strength ( = 0.005). However this ratio was found to be dependent on the method used [53] and on the ionic strength [49,54], with the amount of surfactant bound to protein increasing from 1.0 to 2.2 g/g protein when buffer concentration increased from 10 to 220 mmol L−1 [54]. The initial surface tension values for the interaction profile are lower than that of the dilution profile reflecting the surface activity of BSA. In PBS medium (pH 7.4) BSA has an overall negative charge so the interaction between the protein and the cationic surfactant at the interface is dominated by electrostatic interactions. With the increase in surfactant concentration, the available charges in the protein backbone are compensated by the oppositely charged surfactant ions leading to formation of electrically neutral complexes of increased hydrophobicity and enhanced surface activity [28,55,56]. Unfolding of the protein exposes more hydrophobic binding sites and hydrophobic interactions between (C12 Cys)2 and protein predominate with further increase in surfactant concentration. The BSA species at the interface can then be gradually displaced by gemini surfactant molecules due to competitive adsorption [55,56], leading to a higher surface tension at the CMC in the presence of protein, i.e.,  psp >  cmc . The value of surface tension therefore depends on the interplay between the self-assembly of surfactant at the interface and the binding process between surfactant and protein both in the bulk and at the interface [28,55,56]. The lower  max values and hence enhanced Amin values obtained for dilution of the surfactant in BSA compared to that in buffer indicate a less compact monolayer in the presence of the protein. The relaxed monolayer can be attributed to the flexibility of unfolded protein and suggests the presence of BSA–surfactant complexes at the air/solution interface [28,30].

5

150

4 2 0

0

1

2

[Q] / g L−1

4

100 −1

[(C12Cys)2]/g L 5

1 2 3 4 5 6 7

50 6 7

0

300

320

340

360

380

0.00 0.02 0.05 0.10 0.50 0.70 1.00

400

λ / nm Fig. 3. Fluorescence emission spectra of BSA (0.10 g L−1 ) in the presence of increasing surfactant concentrations, obtained at the excitation wavelength of 280 nm. The insert represents the Stern–Volmer plot (see Section 3.3).

interactions thus perturbing the native conformation of BSA [60]. The observed changes suggest a shift of the fluorophores to a more hydrophobic environment that can be attributed to internalization of the fluorophore towards the protein hydrophobic core due to protein refolding (which is possible when protein gets stabilized [59,61]) or to hydrophobic interactions between the dodecyl chains of (C12 Cys)2 and hydrophobic amino acid residues around Trp 134, such as leucine (Leu), Tyr and Phe [60], leading to a decrease in intrinsic fluorescence intensity [26,28,57,62,63]. For surfactant concentrations above the CAC (0.30 g L−1 ) the blue shift vanishes and the intensity of the emission reaches a constant value at a surfactant concentration of 0.70 g L−1 . Therefore, no further change of the microenvironment of the fluorophore residues occurs above this concentration, which corresponds to the protein saturation point and formation of free micelles, according to the tensiometric profile. Fluorescence quenching may occur as a result of collision between the fluorophore and the quencher in the excited state or from ground-state complex formation, thus involving dynamic or static quenching mechanisms, respectively [23,26–28,50,57]. There are several ways to distinguish static from dynamic quenching, which include determination of the bimolecular quenching rate constant value, comparison of the values of the Stern–Volmer quenching constant obtained at different temperatures, and comparison of protein absorption spectra in the presence and in the absence of surfactant (quencher) [23,26–28,50,57,64,65]. Higher temperature results in faster diffusion thus enhancing dynamic quenching, and the Stern–Volmer quenching constant increases, while for a static quenching process the Stern–Volmer constant decreases with increasing temperature due to reduced stability of the complex [23,26–28,50,57,64]. The UV–vis absorption spectra can help distinguish both processes because collisional quenching only affects the excited states of the fluorophores, which do not perturb the UV absorption spectra, a situation that is in marked contrast to ground-state complex formation [26,27,50,66]. The UV–vis absorption spectra of BSA (Fig. 4) are perturbed by addition of increasing concentration of surfactant, resulting in a significant red shift ( max = + 26 nm) of the maximum absorption peak at 210 attributed to the – * transition of the peptide bond in BSA [27,58,66,67], while absorption at 280 nm is due to the n– * transition of the aromatic amino acid residues [58,67]. Hence, fluorescence quenching is likely to occur by a static quenching mechanism initiated by ground-state complex formation.

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Table 2 Stern–Volmer quenching constant (KSV ), bimolecular quenching constant (kq ), binding constant (Ka ), binding sites (n) and Gibbs energy (Gb ) for the binding of gemini surfactant (C12 Cys)2 with BSA in PBS (pH 7.4) at 298.15 K. [BSA]/g L−1

10−4 KSV /L mol−1

10−12 kq /L mol−1 s−1

10−4 Ka /L mol−1

n

Gb /kJ mol−1

0.10

1.53

1.50

1.68

1.0 ± 0.1

−24.12

3 −1

Absorbance

[(C12Cys)2]/g L

2

1 2 3 4 5 6 7 8 9

1 ↓ 9

1

0 200

250

300

1.00 0.70 0.50 0.30 0.20 0.10 0.05 0.02 0.00

350

λ / nm Fig. 4. UV–vis absorption spectra for 0.10 g L−1 BSA in PBS (pH 7.4), in the absence and in the presence of increasing concentrations of (C12 Cys)2 .

3.3. Fluorescence quenching data The fluorescence quenching process can be described by the Stern–Volmer equation [26,27,50,58,61,66]: F0 = 1 + KSV [Q ] = 1 + kq 0 [Q ] F

(4)

where F0 and F are the steady-state fluorescence intensities in the absence and in the presence of quencher (surfactant), KSV is the Stern–Volmer quenching constant, [Q] is quencher concentration, kq is the bimolecular quenching constant and  0 is the average lifetime of fluorescence in the absence of quencher, its value being in the order of 10−8 s for biopolymers [68]. Therefore, KSV was determined from the slope of the plot of F0 /F against [Q] (Fig. 3, insert) and values are summarized in Table 2. The calculated kq (=KSV / 0 ) is two orders of magnitude larger than the largest dynamic bimolecular quenching constant in aqueous medium, which is 2 × 1010 L mol−1 s−1 [69], an indication that the fluorescence quenching of BSA in the presence of (C12 Cys)2 is initiated through a static quenching mechanism [64,65]. For a static quenching process, when the ligand binds independently to a set of equivalent sites on the protein, the binding constant, Ka , and the number of binding sites, n, can be calculated from a modified version of the Stern–Volmer equation [26,27,50,66]: log

F0 − F = log Ka + n log[Q ] F

(5)

The values of n and Ka were thus obtained from the slope and the y-axis intercept of the linear plot of log(F0 − F)/F0 versus log[Q], respectively. These values are shown in Table 2, along with the value of the Gibbs energy for the binding of the surfactant to the protein, Gb , calculated from equation [65,70]: Gb = −2.303RT log Ka

(6)

The Ka values obtained for BSA–(C12 Cys)2 complexes suggest lower binding affinity compared to other strong protein–ligand complexes with binding constants ranging from 105 to 108 L mol−1 [71,72] However, lower binding constants (102 –104 L mol−1 ) have been reported for several other protein–ligand complexes [26,27,50,65–67,70,73]. In fact, the binding constants for imidazolium ionic liquids (ILs) and BSA are very low (in the order of

102 L mol−1 ) which indicate a very weak interaction between the ILs and the protein [58]. This situation clearly contrasts with the pyrrolidinium-based IL synthesized by Kumari et al. [72] which showed KSV and Ka values of 3.45 × 105 and 3.33 × 105 L mol−1 , respectively, in phosphate buffer (pH 7.4) at 298 K. Khan et al. [65,70] studied the binding of amphiphilic drugs amitriptyline hydrochloride (AMT), promethazine hydrochloride (PMT), nortriptyline hydrochloride (NOT) and promazine hydrochloride (PMZ) with serum albumins HSA and BSA. Fluorescence quenching data for the drug–protein complexes yielded values of KSV = (2.51–5.28) × 104 L mol−1 , Ka = (0.46–32.5) × 104 L mol−1 and Gb values between −21.00 and −31.60 kJ mol−1 in Tris–HCl buffer solution (pH 7.4) at 310 K [65,70]. The value of Ka is particularly relevant for the understanding of the distribution of the drug in plasma since weak binding can lead to a short lifetime or poor distribution while strong binding is responsible for the reduction in the plasmatic concentration of free drug. The obtained quenching rate constant values (kq ) suggested a static quenching process for all the drug-serum albumin interactions. For most of the drug–serum albumin complexes n was almost unity indicating the existence of only one class of binding sites in BSA/HSA for the amphiphilic drugs. The change in protein conformation for both proteins was more prominent in the presence of PMT and PMZ than in the presence of NOT or AMT because of the presence of a sulphur atom in the former that was found to form disulfide bond with the serum albumins [65,70]. The KSV and Ka values obtained for the BSA–(C12 Cys)2 system are similar to the ones that have been reported in the literature for cationic gemini surfactants of the same alkyl chain length, which are in the order of 104 L mol−1 [26,27]. The KSV value obtained is higher than the one for the single chain surfactant dodecyldimethylethylammonium bromide (DDAB), which is 1.09 × 104 L mol−1 for a BSA concentration of 0.50 g L−1 [50]. The cationic surfactant 1-dodecyl-N-[2-(acetyloxy)ethyl]N,N-dimethyl ammonium bromide (DTAAB) and the correspondent gemini have even lower KSV values of 1.06 × 103 and 7.75 × 103 L mol−1 , respectively [67]. The number of binding sites n equals 1, indicating that there is one binding site in BSA for (C12 Cys)2 , a result that has also been observed for quaternary ammonium gemini surfactants with dodecyl chains [27,73] and for amphiphilic drug–serum albumin complexes [65,70].

4. Conclusions A novel cationic gemini surfactant derived from the amino acid cysteine, (C12 Cys)2 , was synthesized and its supramolecular behaviour under physiological mimetic conditions (PBS, pH 7.4) was characterized both in the absence and in the presence of BSA. The gemini surfactant was found to associate with the model protein according to data obtained from surface tension measurements, fluorescence quenching, UV–vis spectroscopy. The strength of the interaction was relatively weak as revealed by the association constant obtained from fluorescence quenching data, which is in the order of 104 L mol−1 . However, this value is in agreement with similar ones obtained for cationic gemini surfactants [26,27] of the same alkyl chain length of C12 (Cys)2 and is higher than the association constant found for several amphiphilic drugs with serum albumins [65,70].

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The low surfactant concentration region is related to the binding that occurs at specific sites through electrostatic interactions while the higher surfactant concentration region is associated with the cooperative surfactant interaction resulting in the unfolding of the protein. The conformational changes observed in the protein by fluorescence spectroscopy suggest unfolding with increasing surfactant concentrations up to saturation of the protein backbone at a surfactant concentration of 0.70 g L−1 , after which micelle formation occurs. These results are found to be in tune with the ones obtained from surface tension measurements. The fluorescence quenching of BSA is likely to occur through a static quenching mechanism initiated by ground-state complex formation as corroborated by the UV–vis absorption spectra and depicted from the calculated kq value. Further studies in the presence of the ␤-amyloid peptide, both in vitro and in vivo, are necessary to evaluate the efficiency of (C12 Cys)2 as a ␤-amyloid disassembly agent with clinical potential in AD therapy. Acknowledgments The authors acknowledge Isabel Ribeiro at the Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy at the University of Lisbon for the fluorescence measurements. The authors are grateful to Fundac¸ão para a Ciência e Tecnologia (PEst-OE/SAU/UI4013) for financial support. Mafalda A. Branco is thankful to Universidade de Lisboa/Fundac¸ão Amadeu Dias for a research grant. References [1] C. Ballard, S. Gauthier, A. Corbett, C. Brayne, D. Aarsland, E. Jones, Alzheimer’s disease, Lancet 377 (2011) 1019–1031. [2] I.W. Hamley, The amyloid beta peptide: a chemist’s perspective. Role in Alzheimer’s and fibrillization, Chem. Rev. 112 (2012) 5147–5192. [3] A. Aguzzi, T. O’Connor, Protein aggregation diseases: pathogenicity and therapeutic perspectives, Nat. Rev. 9 (2010) 237–248. [4] Y. Han, C. He, M. Cao, X. Huang, Y. Wang, Z. Li, Facile disassembly of amyloid fibrils using gemini surfactant micelles, Langmuir 26 (2010) 1583–1587. [5] C. He, Y. Hou, Y. Han, Y. Wang, Disassembly of amyloid fibrils by premicellar and micellar aggregates of a tetrameric cationic surfactant in aqueous solution, Langmuir 27 (2011) 4551–4556. [6] F.M. Menger, J.S. Keiper, Gemini surfactants, Angew. Chem. Int. Ed. 39 (2000) 1906–1920. [7] R. Zana, Dimeric and oligomeric surfactants. Behaviour at interfaces and in aqueous solution: a review, Adv. Colloid Interface Sci. 97 (2002) 205–253. [8] Y. Han, Y. Wang, Aggregation behaviour of gemini surfactants and their interaction with macromolecules in aqueous solution, Phys. Chem. Chem. Phys. 13 (2011) 1939–1956. [9] A. Pinazo, R. Pons, L. Pérez, M.R. Infante, Amino acids as raw material for biocompatible surfactants, Ind. Eng. Chem. Res. 50 (2011) 4805–4817. [10] M.C. Morán, A. Pinazo, L. Pérez, P. Clapés, M. Angelet, M.T. Garcia, M.P. Vinardell, M.R. Infante, Green amino acid-based surfactants, Green Chem. 6 (2004) 233–240. [11] L. Pérez, A. Pinazo, R. Pons, M.R. Infante, Gemini surfactants from natural amino acids, Adv. Colloid Interface Sci. 205 (2014) 134–155. [12] R. Valivety, I.S. Gill, E. Vulfson, Application of enzymes to the synthesis of amino acid-based bola and gemini surfactants, J. Surfactants Deterg. 1 (1998) 177–185. [13] N. Ménard, N. Tsapis, C. Poirier, T. Arnauld, L. Moine, F. Lefoulon, J.-M. Péan, E. Fattal, Drug solubilisation and in vitro toxicity evaluation of lipoamino acid surfactants, Int. J. Pharm. 423 (2012) 312–320. [14] J. Singh, P. Yang, D. Michel, R.E. Verrall, M. Foldvari, I. Baeda, Amino acidsubstituted gemini surfactant-based nanoparticles as safety and versatile gene delivery agents, Curr. Drug Deliv. 8 (2011) 299–306. [15] P. Yang, J. Singh, S. Wettig, M. Foldvari, R.E. Verrall, I. Baeda, Enhanced gene expression in epithelial cells transfected with amino acid-substituted gemini nanoparticles, Eur. J. Pharm. Biopharm. 75 (2010) 311–320. [16] D. Otzen, Protein–surfactant interactions: a tale of many states, Biochim. Biophys. Acta 1814 (2011) 562–591. [17] C. La Mesa, Polymer–surfactant and protein–surfactant interactions, J. Colloid Interface Sci. 286 (2005) 148–157. [18] A. Stenstam, D. Topgaard, H. Wennerström, Aggregation in a protein–surfactant system. The interplay between hydrophobic and electrostatic interactions, J. Phys. Chem. B 107 (2003) 7987–7992. [19] S. Chodankar, V.K. Aswal, P.A. Hassan, A.G. Wagh, Structure of protein– surfactant complexes as studied by small-angle neutron scattering and dynamic light scattering, Physica B 398 (2007) 112–117. [20] S. De, A. Girigoswami, S. Das, Fluorescence probing of albumin–surfactant interaction, J. Colloid Interface Sci. 285 (2005) 562–573.

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