Study on interactions of cationic gemini surfactants

Journal of Molecular Liquids 243 (2017) 369–379

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Journal of Molecular Liquids journal homepage: www.elsevier.com/locate/molliq

Study on interactions of cationic gemini surfactants with folded and unfolded bovine serum albumin: Effect of spacer group of surfactants Sonu a,1, Sayantan Halder a, Sunita Kumari a, Rishika Aggrawal a, Vinod K. Aswal b, Subit K. Saha a,⁎ a b

Department of Chemistry, Birla Institute of Technology & Science (BITS), Pilani, Pilani Campus, Rajasthan 333 031, India Solid State Physics Division, Bhabha Atomic Research Centre (BARC), Mumbai 400085, India

a r t i c l e

i n f o

Article history: Received 26 April 2017 Received in revised form 16 July 2017 Accepted 28 July 2017 Available online 30 July 2017 Keywords: BSA-Gemini surfactant interaction Gemini surfactant with various spacers BSA fluorescence Fluorescence lifetime

a b s t r a c t Interactions of three cationic gemini surfactants, 12-4-12, 2Br− 12-8-12, 2Br− and 12-4(OH)-12, 2Br− with natured and denatured protein, bovine serum albumin (BSA) have been studied by means of UV–Visible absorption, steady-state and time-resolved fluorescence, and circular dichromism (CD) spectroscopy. CD spectroscopic study shows the change in the α-helix and β-strand content of protein with the concentration of gemini surfactants. Gemini surfactant with hydroxyl group in the spacer decreases the α-helix of the BSA more efficiently than that without hydroxyl group in the spacer. Efficiency to decrease the α-helix of the protein increases with decreasing the hydrophobicity of the spacer group of the surfactants at lower concentration range following the order, 12-8-12, 2Br− b 12-4-12, 2Br− b 12-4(OH)-12, 2Br−. However, at higher concentration range of surfactant, the increasing order of providing hydrophobic environment to tryptophan (Trp) and tyrosine (Tyr) residues of the protein is as follows: 12-4(OH)-12 b 12-4-12 b 12-8-12. Gemini surfactant with hydrophobic spacer group provides more hydrophobic environment around Trp and Typ residues of the protein forming micelles like structures along the protein chain. In this concentration range, 12-8-12, 2Br− interacts differently as compared to other two surfactants which are evidenced by the data on excited state lifetime of the protein. It is more efficient to form a particular conformer and/or puckerd ring of Trp as compared to other two surfactants. The microenvironment around Trp residues of BSA is perturbed to a greater extent than that around Tyr residues in presence of gemini surfactants. Fluorescence from Trp and Tyr are quenched by acrylamide to a greater extent in presence of 12-8-12, 2Br−. Interactions of denatured BSA with gemini surfactants also have been studied. 12-8-12, 2Br− even interacts with the BSA unfolded by guanidine hydrochloride (GdHCl) to a greater extent than that by 12-4-12, 2Br− and 12-4(OH)-12, 2Br−. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Proteins have fundamental importance in living organism and take part in many life processes. Serum albumins are found in blood plasma and are considered as transport proteins [1,2]. Principal function of serum albumin is to transport general anasthetic, variety of metabolites and fatty acids [3]. Bovine serum albumin (BSA) is one of the proteins that is commonly used for research purposes due to its water solubility, stability, and versatile binding capacity [1]. In the primary structure of BSA there are nine loops which are held together by 17 disulfide bonds, resulting in three domains (I, II, III) each consists of two sub domains (A and B). BSA and human serum albumin (HSA) shares 76% sequence homology. One of the differences between these two proteins is that BSA has two tryptophan (Trp) residues, but HSA has only one Trp residue [4–6]. ⁎ Corresponding author. E-mail address: [email protected] (S.K. Saha). 1 Present address: Department of School Education, Haryana, (India).

http://dx.doi.org/10.1016/j.molliq.2017.07.122 0167-7322/© 2017 Elsevier B.V. All rights reserved.

Protein can bind with various types of ligands such as fatty acids, metal ions, drugs, surfactants, etc. [7–15]. Studying protein-surfactant interactions is an active field of research. Interaction of proteins with surfactants has many applications in biotechnology processes, drug delivery, cosmetic systems, detergents, and biosciences [16–19]. Surfactants unfold the native proteins structure. Proteins have both hydrophobic and hydrophilic amino acids and due to these properties of amino acids, surfactant molecules bind with protein [20]. At low surfactant concentration, surfactants bind to the protein at high energy specific binding sites. At high concentration of surfactant, the binding between surfactant and protein is highly specific and non-cooperative in nature. Even at a high concentration of surfactants, three dimensional structure of a protein is destroyed and the protein is denatured. Interaction between protein and surfactant molecules depends upon surfactant features. Protein denatured by anionic surfactant like sodium dodecyl sulphate is used to determine the molecular weight of the protein by electrophoresis [21]. To prevent aggregation and unwanted adsorption of protein during filtration and storage, non ionic surfactants are used [22]. Cationic surfactants are used as bactericide in various systems by

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Sonu et al. / Journal of Molecular Liquids 243 (2017) 369–379

of cationic single chain and dimeric surfactant with BSA [51]. Kabirud-din et al. have reported the comparative interaction of cationic single chain and gemini surfactant with HSA [20]. These studies have showed that α-helical content of BSA decreases with increasing the concentration of gemini surfactant. Folding of denatured protein into its native structure is important at fundamental and biotechnology level. Genetically engineered cells produced protein in non native form. The native conformation is required for basic and biotechnological applications [52]. In the present work we have demonstrated the effect of spacer group of gemini surfactants, 12-4-12, 12-8-12 and 12-4(OH)-12 (Scheme 1) on interactions of surfactants with folded and unfolded BSA. Although there are reports on the effect of spacer group on interactions of gemini surfactants with folded or natured proteins, but to the best of author's knowledge there are no such systematic reports on interactions of gemini surfactants with unfolded or denatured proteins. It has been reported that the effect of conventional surfactant on binding with BSA is less as compared to gemini surfactant [49,51]. Rather et al. [52] have reported the refolding of BSA by gemini surfactants assisted by artificial chaperones. Mukherjee et al. [53] have studied the effect of β-cyclodextrin on BSA unfolded by sodium dodecyl sulphate. In the present work we have focused on the effect of spacer group of gemini surfactants on their interactions with natured protein and protein denatured by GdHCl using steady-state and time resolved fluorescence methods in absence of any artificial chaperones.

protein denaturation [23–25]. Various techniques have been used for the study of protein surfactant interactions [26–33]. BSA has two Trp residues at position 134 and 213 of amino acid sequence. Trp 134 located on the surface of BSA [34]. Intrinsic fluorescence of Trp is helpful to study the structure and dynamics of BSA and also to investigate the isotherm of protein and surfactant interactions [35]. Many reports are available for study on interaction of various types of single chain surfactants with protein [36–39]. Another class of surfactant called gemini surfactant has gained now-a-days more importance over conventional monomeric surfactants. Gemini surfactants are attracting special attention in material sciences, biological sciences, nanotechnology, supramolecular chemistry etc. [40]. Gemini surfactants are made up of two hydrophobic tails and two hydrophilic headgroups, covalently joined by a spacer group at their headgroups. Some properties of gemini surfactants are superior to their conventional counterpart [41]. The spacer part plays a significant role for the aggregation properties of a gemini surfactant. The critical micelle concentration (cmc), counter ions binding, thermodynamic properties, microviscosity and micropolarity, rheological behavior, aggregation number etc. of gemini surfactants vary with any change in the spacer part [40,42–47]. The spacer group of a gemini surfactant can be long or short, hydrophilic or hydrophobic, flexible or rigid [40]. Few reports are available for the study on interaction of gemini surfactant with proteins. Xu et al. have studied the interaction between BSA and gelatine with gemini surfactant [48]. Jiange et al. [49] also have studied the interaction of gemini surfactants with BSA. They have reported the effect of spacer chain length of gemini surfactant. The gemini surfactant with longer spacer chain length interact strongly with BSA due to stronger hydrophobic interaction. Sinha et al. [50] have reported the effect of hydroxyl group substituted spacer group of gemini surfactant on protein-surfactant interactions. Moya et al. studied the binding

2. Materials and methods Gemini surfactants, 12-4-12, 12-8-12 and 12-4(OH)-12 were synthesized according to the reported method [44,47]. The synthesized compounds were recrystallized several times with the mixture of

2BrH3C

+ N

+ N

CH3 CH3

H3C

C12H25 C12H25

1,4-bis(dodecyl-N,N-dimethylammonium bromide)-butane (12-4-12) 2Br H3C

CH3

N

N

H3C

CH3

C12H13

C12H13

1,8-bis(dodecyl-N,N-dimethylammonium bromide)-octane (12-8-12) 2BrH3C

+ N

+ N

CH3 CH3

H3C

C12H25 C12H25

OH

Scheme 1. Chemical structure of gemini surfactants.


3. Results and discussions 3.1. UV–Visible absorption study Surfactants bind strongly to proteins and bring a substantial change in the protein conformation. Fig. 1 shows the UV–Visible absorption spectra of BSA in absence and presence of gemini surfactant, 12-4-12 as a representative one. An absorption spectrum of pure BSA shows two absorption peak maxima one at 210 nm and other at 279 nm. The absorption peak maximum at 210 nm corresponds to π → π⁎ transition of characteristic of C_O group in polypeptide backbone structure [54] and the absorption peak maximum at 279 nm corresponds to n → π⁎ transition of aromatic amino acids, i.e., Phenylalanine (Phe), Trytophan (Trp), and Tyrosine (Tyr). In presence of a gemini surfactant, the absorption spectra of BSA show changes in absorption peak maxima and absorbance values i.e. UV–Visible absorption properties are very much sensitive to the concentration of gemini surfactants. The absorption peak maximum for pure BSA located at 210 nm shifts towards longer wavelength with increasing the concentration of gemini surfactant.

4.0 Pure BSA [12-4-12] 0.08 mM 0.60 mM 8.00 mM 20.0 mM

3.5 3.0

Absorbance

methanol and ethylacetate and their structures were confirmed by FTIR and 1H NMR data (Table S1, Supporting information). BSA was purchased from Sigma Chemical Company and was used as received. GdHCl and acrylamide were purchased from Sd-fine Chemical Company, India and were used as received. All solutions were prepared in 25 mM Na-phosphate buffer of pH = 7.25. Concentration of BSA in all measurements was kept at 5.0 μM. The absorption spectra were recorded using Shimadzu (UV-1800) UV–Visible spectrophotometer with the sample in a 1 cm path length quartz cuvette. The steady-state fluorescence spectra were measured using a Horiba Jobin Yvon Fluoromax-4 scanning spectroflourimeter with a 1 cm path length quartz cuvette. The slit width of 3 nm was kept for all the fluorescence measurements. In steady-state fluorescence experiments, all the samples were excited at 295 nm. Synchronous fluorescence spectra were recorded on the same spectrofluorimeter. The differences between excitation and emission wavelength (Δλ) were kept at 15 nm and 60 nm to get the contribution of Tyrosin (Try) and Trp residues, respectively [48]. For time-resolved fluorescence measurements, Horiba Jobin Yvon Fluorocube-01-NL picosecond time-correlated single-photon counting (TCSPC) experimental setup was used. A diode of wavelength 300 nm (NanoLED 300, IBH, UK) was used as a light source. The fluorescence decays were collected at a magic angle (54.7°) with a vertically excitation beam using a TBX photon detection module (TBX-07C). The typical Full Width at Half Maximum (FWHM) after deconvolution using a liquid scatterer of the system response was about 900 ps for 300 nm diode. IBH DAS-6 decay analysis software has been used for the analysis of decays. All the decays were fitted with a biexponential function. The goodness of fits was analyzed by both χ2 criterion and visual inspection of the residuals of the fitted function to the data. The far –UV circular dichromism (CD) spectra of all the studied systems were performed using Chirascan CD spectropolarimeter (Applied Photophysics Ltd.) in the wavelength range of 200–260 nm. To fill the sample a cuvette of path length of 0.1 cm was used. Each CD spectrum was average of three scans with a scan speed of 200 nm min−1 with a spectral bandwidth of 10 nm. Surfactant buffer spectra were subtracted from the recorded spectra of BSA and gemini surfactant for background correction. The UV–Visible absorption, steady-state and time-resolved fluorescence and circular dichroism (CD) spectra of BSA in presence of gemini surfactants at concentrations below and well above their respective cmc values have been recorded. The cmc values of 12-4-12, 12-8-12 and 124(OH)-12 are 1.17 mM, 0.72 mM, and 0.78 mM, respectively [46,47].The changes in the spectroscopic properties of BSA due to the presence of gemini surfactants have been discussed in respective sections. All the measurements were performed at 298 ± 0.1 K.

371

2.5 2.0 1.5 1.0 0.5 0.0 180

210

240

270

300

330

360

Wavelength (nm) Fig. 1. Absorption spectra of BSA at different concentrations of 12-4-12. [BSA] = 5.0 μM.

Absorbance at 210 nm decreases continuously with increasing the concentration of gemini surfactant. The observed changes in the spectroscopic properties of BSA are due to the interaction of gemini surfactant with BSA. In presence of gemini surfactant the microenvironment around amide group of BSA is different as compared to the native protein. When amide groups of BSA are exposed to water environment the energy of π → π⁎ transition is decreased. The chromophore, C_O has higher polarity in the excited state than that in the ground state. Polar water molecules stabilize the energy of excited state more than that of ground state and that is why absorption spectrum of BSA shows bathochromic shift with increasing the concentration of gemini surfactant. Absorption maximum at 279 nm shows very little change in absorbance with increasing the concentration of gemini surfactant. Similar behaviors in the absorption spectra are observed in presence of 12-8-12 and 12-4(OH)-12 gemini surfactants as well. 3.2. Steady-state fluorescence study BSA has three types of aromatic amino acids residues, Phe, Trp, and Tyr. These amino acids give intrinsic fluorescence of BSA. The intrinsic fluorescence of BSA is mainly contributed by Trp and Tyr residues. Phe has very low quantum yield and therefore fluorescence from Phe can be ignored [55]. BSA has two Trp amino acid residues at 134 and 213 (Trp 134 and Trp 213) positions of amino acid sequence in domains I and II, respectively. Trp 134 is located at the protein surface in domain I (subdomain IC), and Trp 213 is located in the hydrophobic binding pocket of protein in domain II (subdomain IIA). The intrinsic fluorescence of BSA is sensitive towards the change in microenvironment. Thus, fluorescence analysis is used as an effective method for the study of interaction of BSA with surfactant molecules [48,49,51,54,56]. Fig. 2 shows the emission spectra of BSA in absence and presence of gemini surfactant, 12-4-12 as a representative one. Spectrum of pure BSA shows fluorescence peak maximum at 347 nm. Fig. 3 shows the variation in fluorescence intensity of BSA with changing the concentration of all three surfactants. With increasing the concentration of gemini surfactant (12-4-12) upto 0.2 mM, the fluorescence intensity of BSA is decreased with a shift in the fluorescence peak maximum towards shorter wavelength at 335 nm (Fig. 4). Further increase in the concentration of 12-4-12 upto 4.0 mM, a rise in fluorescence intensity has been observed with a red shift in fluorescence peak maximum. Beyond 4.0 mM, fluorescence intensity remains almost constant. Similar type of variation in the fluorescence intensity of BSA has been observed for other gemini surfactants, 12-8-12 and 12-4(OH)-12 as well. At very low concentration (0.0 mM–0.005 mM) of all three gemini surfactants,


Fluorescence Intensity (a.u.)

Pure BSA [12-4-12] 0.001 mM 0.01 mM 0.08 mM 0.2 mM 0.8 mM 6.0 mM

350

12-4-12 12-4(OH)-12 12-8-12

348 346 344 fl max

372

342 340 338 336 334 0

2

4

6

8

10

[Gemini Surfactant]/mM 320

340

360

380

400

420

440 Fig. 4. λflmax of BSA in presence of various concentration of 12-4-12, 12-4(OH)-12 and 128-12 at λex = 295 nm. [BSA] = 5.0 μM.

Wavelength (nm) Fig. 2. Steady-state fluorescence spectra of BSA in presence of various concentrations (0.001 mM to 6.0 mM) of 12-4-12. [BSA] = 5.0 μM. λex = 295 nm.

the fluorescence intensity of BSA is increased without much change in the fluorescence peak maximum. The rise in fluorescence intensity is due to the interaction of gemini surfactant at very high energy binding sites of BSA which gives rise to compactness to the BSA native structure [53,57]. It indicates that at a low concentration of surfactant (1:1 molar ratio of BSA and gemini) the secondary structure of BSA remains unaltered and the tertiary structure gets affected [53]. The high energy binding interaction is found to be more significant in case of 12-4(OH)-12 as compared to other two surfactants, 12-4-12 and 12-8-12. These observations indicate that gemini surfactants, 12-4-12, 12-812 and 12-4(OH)-12 interact with BSA and alter the native structure of BSA. Variation in the fluorescence intensity of fluorophores indicates that microenvironment around fluorophores change with the concentration of gemini surfactant. Surfactant molecules unfold the native structure of protein [58]. Ionic surfactants interact strongly with protein by hydrophobic interaction between surfactant tail and nonpolar amino acids, and by electrostatic attraction of headgroups of surfactant and oppositely charged amino acids. The secondary structure of BSA is altered at high concentration of surfactant [48]. It has been observed that the percentage of α-helix decreases with increasing the amount of surfactant [48]. As mentioned above for 12-4-12 surfactant, the fluorescence

1.2 1.1 1.0

F/F0

0.9 0.8 0.7 0.6

12-4-12 12-4(OH)-12 12-8-12

0.5 0.4 0

2

4

6

8

10

[Gemini Surfactant]/mM Fig. 3. Plot of fluorescence intensity of BSA with different concentration of gemini surfactants. [BSA] = 5.0 μM. λex = 295 nm.

intensity is decreased upto 0.2 mM which is due to the unfolding of BSA. In the unfolded state of BSA, Trp and Tyr residues are exposed to the polar environment and that is why fluorescence intensities of Trp and Tyr residues are reduced [7]. Enhancement in the fluorescence intensities at comparatively higher concentration of gemini surfactant indicates that Trp and Tyr residues get located in the nonpolar environment [51]. Fig. 4 shows variations of fluorescence peak maxima of BSA at various concentrations of gemini surfactants. These variations are in accordance with the change in fluorescence intensities. It is very clear from the Fig. 3 that up to 0.2 mM concentration of surfactant, the change in fluorescence intensity is lowest with 12-8-12 and the same is highest with 12-4(OH)-12 i.e. the increasing order of interactions of gemini surfactants with protein is 12-8-12 b 12-4-12 b 124(OH)-12. It indicates that the chemical nature of spacer group of gemini surfactant has effect on the denaturation of protein i.e. to decrease the α-helix. Sinha et al. [50] have also indicated the similar effects. The present study demonstrates that the extent of denaturation of protein increases with decreasing hydrophobicity of the spacer group of gemini surfactants at lower concentration range keeping the hydrocarbon tail lengths of surfactants fixed. We have also observed that 12-4(OH)-12 even shows comparatively stronger high energy binding site interaction. This is due to the presence of one hydroxyl group in the spacer part of 12-4(OH)-12. Gemini surfactant, 12-4(OH)-12 may interact with BSA through hydrogen bonding and through lone pair of oxygen atom [59]. It is noteworthy that the concentrations of 12-8-12, 12-412 and 12-4(OH)-12 at which the minimum of F/Fo occurs in Fig. 3 are 0.04 mM, 0.20 mM and 0.30 mM, respectively. It infers that the spacer chain length has control over the efficiency of a surfactant molecule to decrease the α-helix. 12-8-12 with maximum hydrophobicity in the spacer group is least efficient and 12-4(OH)-12 with minimum hydrophobicity in the spacer group is most efficient to decrease the α-helix. However, 12-8-12 surfactant at a concentration above 0.5 mM (Fig. 3) provides an environment around fluorophore which is more hydrophobic than that in folded protein. Thus the micelles of 12-8-12 formed along the protein chain provides comparatively more hydrophobic environment to the fluorophore because the spacer group of 12-8-12 is more hydrophobic than that of 12-4-12 and 12-4(OH)-12. 3.3. Synchronous fluorescence spectroscopy study Synchronous fluorescence spectroscopy is an effective technique to explore the microenvironment of amino acid residues [48,60]. Synchronous fluorescence technique involves simultaneous scanning of excitation and emission monochromators while maintaining a constant wavelength interval between them. When the wavelength interval


then the fraction (α) of BSA occupied by surfactant is given by α = n/ n0 [56,61]. At saturation binding condition, when all the possible sites are occupied by surfactant then α = 1, and in the absence of the surfactant, α = 0. The fractional change in the fluorescence of BSA (α) due to the binding of gemini surfactant has been calculated by Eq. (1) [56,61, 62]

(Δλ, difference between excitation and emission wavelength) is kept at 15 nm and 60 nm, synchronous fluorescence spectra give information about Tyr and Trp residues, respectively [48,60]. Fig. 5 shows the synchronous fluorescence spectra of BSA for Δλ = 60 nm in presence of 12-4-12, 12-8-12 and 12-4(OH)-12. With increasing the concentration of each of 12-4-12, 12-8-12 and 12-4(OH)-12, there is a tendency for decrease in fluorescence intensity with blue shift in fluorescence peak maxima. It indicates that the conformation of BSA is changed in the presence of gemini surfactant. Fig. 6 shows the synchronous fluorescence spectra of BSA for Δλ = 15 nm in presence of each of 12-4-12, 12-8-12 and 12-4(OH)-12. At Δλ = 15 nm, fluorescence intensity increases with increasing the concentration of gemini surfactants [48]. In addition, the fluorescence intensity for Trp is higher than that for Tyr with greater extent of variation in the fluorescence intensity of the former as compared to the latter. These results can be depicted based on discussion made by Ruiz et al. [61] in their work on protein-surfactant interactions as follows: (i) Major intrinsic fluorescence is contributed by the Trp residue of BSA, (ii) There is resonance energy transfer from Phe to Tyr to Trp, (iii) With increasing concentration of surfactant the conformation of BSA is changed. As a result of that the distance between donor and acceptor increases. Thus the energy transfer from Tyr to Trp is reduced leading to enhancement in Tyr fluorescence and quenching of Trp fluorescence. It is noteworthy that when Δλ = 60 nm, the synchronous fluorescence peak maximum (337 nm) is close to that as observed in Fig. 2 (347 nm). In fact in case of 12–8-12 the peak maximum is exactly 347 nm. From these observations it is clear that the microenvironment around Trp residues of BSA is perturbed to a greater extent than that around Tyr residues in presence of gemini surfactants and 12-8-12 has strongest effect on it.

α¼

Pure BSA [12-4-12] 0.01 mM 0.05 mM 0.08 mM 0.10 mM

50000000 40000000 30000000 20000000 10000000

320

330

340

350

360


ð1Þ

2000000

Pure BSA [12-8-12] 0.01 mM 0.04 mM 0.3 mM 0.5 mM

b 1500000

1000000

500000

320

370

Wavelength (nm)

60000000

0

0

310



The nature of protein-surfactant interaction can be understood by using the binding isotherm of protein and surfactant. If a protein (BSA in the present study) has n0 available sites for binding of a given surfactant, and at a certain stage surfactant molecules bind to n sites at BSA,

a

I−Io Imin −Io

where I is the fluorescence intensity at any surfactant concentration, Io is the fluorescence intensity in the absence of surfactants and Imin is the fluorescence intensity at saturation level. Fig. 7 displays the variation in α with the concentration of studied gemini surfactants, 12-4-12, 12-8-12 and 12-4(OH)-12. With increasing the concentration of surfactant, generally the protein-surfactant isotherm shows three regions prior to the saturation region [50,56] viz. (1) specific binding, (2) noncooperative binding, and (3) co-operative binding. In Fig. 7, region 1 (not shown) includes the total surfactant concentration from 0 mM to 0.03 mM. In this concentration region the binding isotherm rises very slowly due to ionic binding of surfactant molecules with protein at specifically high energy binding sites [56] (Scheme S1). Region 2 shows the non-cooperative binding region. In this region, the binding isotherm rises sharply with surfactant concentration from 0.03 mM to 0.4 mM. As mentioned above in this concentration range the fluorescence intensity is dropped drastically and BSA conformation is altered. Above 0.4 mM the binding isotherm rises gradually. This represents the massive cooperative binding between BSA and surfactant molecules [51]. In this region, the surfactant molecules form micelles like structure along the protein chain (Scheme S1). Beyond region 3, a plateau has been observed which indicates the saturation of binding of gemini surfactant. Hence, binding of surfactants with BSA passes through four different stages. Das et al. [56] also observed four regions of binding of surfactant with protein.

3.4. Binding of gemini surfactant with BSA: Study of binding isotherm

60000000

373

330

340 350 360 Wavelength(nm)

370

380

pure BSA [12-4(OH)-12] 0.01 mM 0.04 mM 0.20 mM 0.50 mM

C

50000000 40000000 30000000 20000000 10000000 0

310

320

330

340

350

360

370

380

Wavelength (nm) Fig. 5. Synchronous fluorescence spectra of BSA at Δλ = 60 nm in presence of (a) 12-4-12, (b) 12-8-12 and (c) 12-4(OH)-12. [BSA] = 5.0 μM.


16000000 14000000

Pure BSA [12-4-12] 0.01 mM 0.05 mM 0.08 mM 0.60 mM 3.00 mM 8.00 mM 15.0 mM

a

12000000 10000000 8000000 6000000 4000000 2000000

600000



374

Pure BSA [12-8-12] 0.01mM 0.04 mM 0.3 mM 2 mM 6 mM 10 mM

b

500000 400000 300000 200000 100000 0

0

280

290

300

310

320

330

280

290

300

310

320

330

wavelength(nm)

Wavelength (nm) Fluorescence Intensity (a.u.)

18000000

Pure BSA [12-4(OH)-12] 0.01 mM 0.04 mM 0.20 mM 2.00 mM 6.00 mM 12.0 mM

16000000 14000000

c

12000000 10000000 8000000 6000000 4000000 2000000 0

280

290

300

310

320

330

Wavelength (nm) Fig. 6. Synchronous fluorescence spectra of BSA at Δλ = 15 nm in presence of (a) 12-4-12, (b) 12-8-12 and (c) 12-4(OH)-12. [BSA] = 5.0 μM.

3.5. Circular dichroism study Circular dichroism (CD) study reveals the informations about the change in the conformation of the protein. The far-UV CD spectra in the range of 200 nm to 260 nm have been recorded for pure BSA and BSA in presence of gemini surfactants. The representative CD spectra are given in Fig. S1. CD spectra show two characteristic negative bands for α-helix of BSA at 210 nm and 223 nm [63]. There is a decreasing tendency of negative molar ellipticity with increasing the concentration of gemini surfactant which shows that the secondary structure of BSA is altered in the presence of a gemini surfactant. The changes in the α-helical and β-sheet content upon addition of gemini surfactant have been calculated using K2D3 secondary structure analysis software and the data

1.0

4

3

obtained are given in Table 1. In case of pure BSA the α-helix content is 67.74% which is comparable to the reported value [63,64]. The decrease in the values of % of α-helix of BSA with gemini surfactant concentration indicates that gemini surfactant interacts with BSA and unfolds it. In case of 12-8-12 we could not calculate the values of % of α-helix and % of β content beyond 1.00 mM of its concentration because of appearance of excessive noise in the CD spectra at those concentrations. However, while comparing these values at 1.00 mM concentrations of all three surfactants and at 5.00 mM concentrations of 12-412 and 12-4(OH)-12, it is revealed that the increasing order of alteration of secondary structure of BSA is as follows: 12-8-12 b 12-4-12 b 124(OH)-12. The presence of hydroxyl group in the spacer group of 124(OH)-12 enhances the unfolding of BSA to a greater extent than that by the spacer groups present in other two surfactants without any hydroxyl group in them. 3.6. Time-resolved fluorescence spectroscopic study Time resolved fluorescence measurement is an effective method to study protein conformational dynamics. Trp exists in various rotational conformers and due to this Trp exhibits multiple exponential decays

0.8

0.6

BSA + 12-4-12 BSA + 12-4(OH)-12 BSA + 12-8-12

0.4

Table 1 α-Helix and β strand content of BSA in the presence and absence of gemini surfactant. BSA/gemini surfactant system

[Gemini surfactant] (mM)

% of α-helix

% of β strand

BSA/12-4(OH)-12

0.00 0.10 1.00 5.00 0.10 1.00 5.00 0.01 0.05 0.20 1.00

67.74 67.79 66.58 3.28 67.60 66.62 18.95 67.73 67.45 67.54 66.74

8.74 8.47 9.07 32.3 8.69 9.08 28.74 8.72 8.61 8.37 8.86

2 0.2 BSA/12-4-12

0.0 0.000

0.005

0.010

0.015

0.020

0.025

[Gemini surfactant] /M Fig. 7. Binding isotherms of 12-4-12, 12-8-12 and 12-4(OH)-12 with BSA.

BSA/12-8-12


[55,65]. The three rotational conformers (1, 2 and 3) of Trp are shown by Scheme S2. It is believed that the conformer 3 is quite stable and conversion from 3 to either 1 or 2 and vice versa is difficult in nanosecond time scale. The shorter excited state lifetime of Trp is due to the existence of conformer 3 and the longer excited state lifetime arises due to the rapid inter-converting conformers, 1 and 2. It is also believed that in the ground state the indole ring is slightly puckered, but it becomes planer in the excited state, which leads to the delocalization of lone pair of electron from nitrogen atom to the aromatic ring. Upon unfolding process of protein, the interacting ligand approaches to the Trp residues of protein and distorts the planarity of indole ring. Trp residue is also exposed to the bulk solvent and that is why the excited state lifetime of Trp is decreased [54,57]. We have measured the excited state lifetime of Trp moiety of BSA by exciting the sample using a 300 nm light emitting diode (LED). Bi-exponential decays of BSA were observed in presence and absence of studied gemini surfactants. Fig. 8 shows fluorescence decays of native BSA and BSA in presence of various concentration of 12-412 as representative. Tables 2 and 3 represent the excited state lifetime values of BSA in presence of various concentrations of 12-4-12 and 128-12, respectively and Table S2 represents the same for 12-4(OH)-12. Average excited state lifetime, 〈τ〉 has been calculated by using the following Eq. (2) [55,61]:

Table 2 Excited state lifetimesa,b of BSA in presence of 12-4-12. [12-4-12] (mM)

a1

τ1 (ns)

a2

τ2 (ns)

〈τ〉 (ns)

χ2

0.0 0.0005 0.001 0.005 0.01 0.05 0.08 0.1 0.2 0.4 0.5 0.6 0.8 0.9 1.0 2.0 4.0 5.0 6.0 8.0 10.0

0.29 0.32 0.29 0.32 0.41 0.51 0.60 0.58 0.62 0.63 0.51 0.52 0.45 0.52 0.56 0.49 0.54 0.50 0.53 0.47 0.52

3.05 2.34 2.66 2.42 2.57 2.35 2.34 2.23 2.62 2.73 2.61 2.63 2.22 2.61 2.78 2.45 2.62 2.28 2.68 2.37 2.39

0.71 0.68 0.71 0.68 0.59 0.49 0.40 0.42 0.38 0.37 0.49 0.48 0.55 0.48 0.44 0.51 0.46 0.50 0.47 0.53 0.48

6.80 6.29 6.38 6.24 6.35 5.84 5.98 5.73 6.09 6.51 6.17 6.23 5.90 6.20 6.31 5.92 5.99 5.72 5.86 5.68 5.52

6.22 5.70 5.84 5.65 5.52 4.81 4.63 4.51 4.66 4.93 5.08 5.10 5.03 5.08 5.04 4.93 4.85 4.74 4.78 4.79 4.52

1.01 1.00 1.05 1.05 1.02 1.01 1.04 1.01 1.00 1.05 1.04 1.04 0.96 1.02 0.98 1.05 1.08 0.99 1.01 1.00 1.05

a

X i τ¼X

b

ai τ 2i ð2Þ

ai τi

i

where ai is the pre-exponential factor of i-th component and τi is the lifetime of the i-th component. Fig. 9 represents the variation in average excited state lifetime of BSA in presence of the gemini surfactants. Excited state lifetime of pure BSA is close to the reported lifetime value [53, 57]. Excited state lifetime decreases upto 0.1 mM concentration of all three surfactants. It can be noted here that the steady-state fluorescence intensity of Trp is decreased in this concentration range due to the conformational change in the native structure of BSA. Lowering of excited state lifetime of BSA also supports the conformational change of BSA in presence of gemini surfactants. As mentioned above the shorter lifetime is for the conformer 3 and the longer lifetime is due to the rapid interconverting conformers, 1 and 2. The contributions of decay components represent the relative populations of various conformers. The contribution of fast component is increased from concentration 0.01 mM to 0.1 mM for 12-4-12 gemini surfactant and simultaneously the contribution of slow component is

375

λex = 300 nm. λem is the fluorescence peak maximum in respective system.

decreased. It means that in this concentration range due to unfolding of BSA the relative population of conformer 3 is increased and that of interconverting conformers, 1 and 2 is decreased. Similar behavior also has been observed in presence of 12-4(OH)-12 as well (Table S2). However, it is different with 12-8-12 surfactants. Enhancement in the excited state lifetime has been observed with further increasing the concentration of gemini surfactants from 0.2 mM to 1.0 mM for 12-412. Above 0.4 mM of 12-4(OH)-12 the excited state lifetime values remain almost steady. It can be seen in Fig. 9 that at higher concentration of 12-4(OH)-12, the average excited state lifetime values of BSA are lower than that in presence of 12-4-12 gemini surfactant. The effect of hydroxyl group of gemini surfactant, 12-4(OH)-12 on interaction with BSA is also evidenced by the excited state lifetime data. It is noteworthy that the effect of concentration of gemini surfactants on steady-state fluorescence intensities of BSA (Fig. 3) is consistent with that on the excited state lifetime of BSA (Fig. 9) in cases of 12-4-12 and 12-4(OH)-12 i.e. the former one provides more hydrophobic environment around Trp

Table 3 Excited state lifetimesa,b of BSA in presence of 12-8-12.

Counts (a.u.)

10000

Prompt Native BSA [12-4-12] 0.2 mM 2 mM 8 mM 10 mM

8000 6000 4000 2000 0 12

14

16

18

20

Time (ns) Fig. 8. Fluorescence decays of native BSA and BSA in presence of various concentration of 12-4-12. λex = 300 nm, λem is fluorescence peak maximum in respective system, [BSA] = 5 μM.

[12-8-12] (mM)

a1

τ1 (ns)

a2

τ2 (ns)

〈τ〉 (ns)

χ2

0.0 0.001 0.005 0.01 0.04 0.1 0.3 0.4 0.5 0.6 0.8 0.9 1.0 2.0 4.0 5.0 6.0 8.0 10.0

0.29 0.66 0.61 0.56 0.56 0.62 0.56 0.37 0.58 0.58 0.56 0.54 0.60 0.56 0.61 0.62 0.64 0.66 0.60

3.05 1.44 1.57 1.34 1.34 0.54 1.44 1.38 1.62 1.90 1.48 1.75 1.33 1.29 0.80 0.78 0.65 0.64 0.64

0.71 0.34 0.39 0.44 0.44 0.38 0.44 0.63 0.42 0.42 0.44 0.46 0.40 0.44 0.39 0.38 0.36 0.34 0.40

6.80 6.05 6.09 5.82 5.82 3.15 5.51 4.25 5.77 6.00 5.61 5.81 5.54 5.22 4.54 4.52 4.08 4.17 3.90

6.22 4.59 4.79 4.80 4.80 2.58 4.49 3.79 4.61 4.75 4.57 4.75 4.43 4.28 3.73 3.70 3.32 3.36 3.26

1.01 1.12 1.17 1.19 1.19 1.13 1.18 1.10 1.08 1.14 1.17 1.19 1.19 1.18 1.10 1.15 1.18 1.19 1.19

a b

λex = 300 nm. λem is the fluorescence peak maximum in respective system.

376


6

12-4-12 12-4(OH)-12 12-8-12

6

BSA + 12-4(OH)-12 BSA + 12-4-12 BSA + 12-8-12 BSA

5

5

4

F0/F

< > / ns

4 3 2

3

1

2 0

2

4 6 8 [Gemini Surfactant]/mM

10

0 0.0

Fig. 9. Variation of excited state average lifetime of BSA with the concentration of gemini surfactants. λex = 300 nm, λem is the fluorescence peak maximum in respective system, [BSA] = 5.0 μM.

as compared to the latter. However, lifetime values are lower in case of 12-8-12 as compared to that of other two surfactants. This is in contrary to that is observed in the variation of fluorescence intensities with the concentration of gemini surfactants. In addition to that the data in Tables 2, 3 and S2 show that the contributions of the faster components are mostly lower than that of the slower components in case of 12-4-12 and 12-4(OH)-12. However, the contribution of the faster component is greater than that of the slower components in case of 12-8-12. Also lifetime values of fast components for 12-8-12 are shorter than that for 124-12 and 12-4(OH)-12 with similar lifetime values for longer components for all three surfactants. The synchronous fluorescence spectra at Δλ = 60 nm in case of 12-8-12 is also quite different from that of 12-4-12 and 12-4(OH)-12. It infers that the hydrophobic spacer group interacts strongly with the Trp residues. Although 12-8-12 is not sufficiently effective to decrease the α-helix of protein, but has profound effect to make the indole ring puckered. Thus these results indicate that 12-8-12 surfactant is not efficient to expose the Trp ring to the bulk solvent, but has potential effect to make the indole ring non-planer. Moreover, the comparatively greater contribution of the faster component and shorter lifetime of fast component in case of 12-8-12 might indicate that possibility of having conformer 3 and/or puckered Trp ring is more with 12-8-12 than that with 12-4-12 and 12-4(OH)-12. Zana et al. [44] have reported that the gemini surfactants with spacer group containing N5–6\\CH2 unit form a loop extended towards the hydrophobic core of the micelle. Probably 12-8-12 with a loop in its spacer might be effective to form a particular conformer and/or puckerd ring of an amino acid residue of protein i.e. Trp in the present case. 3.7. Fluorescence quenching of BSA by acrylamide Fluorescence spectra of BSA have been recorded at various concentrations of acrylamide as a quencher in presence and absence of gemini surfactants (5.0 mM). The Stern-Volmer plot in case of pure BSA is deviated from the linearity at high concentration of acrylamide (Fig. 10). The upward curvature might indicate that the quenching of pure BSA is dynamic as well as static in nature [55]. However, both fluorescence intensity and fluorescence lifetime of BSA in presence of gemini surfactants (5.0 mM) continuously decreases with increasing the concentration of acrylamide following the Stern-Volmer [55] Eq. (3): F o = F ¼ τo =τ ¼ 1 þ K SV ½Q ¼ 1 þ kq τo ½Q

ð3Þ

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

[Acrylamide] /M Fig. 10. Stern-Volmer plot of BSA and BSA -12-4-12/12-8-12/12-4(OH)-12 systems. [BSA] = 5.0 μM [12-4-12] = [12-4(OH)-12] = [12-8-12] = 5.0 mM.

where Fo and F are the fluorescence intensities of BSA in absence and presence of acrylamide. KSV and kq are the Stern-Volmer and bimolecular quenching rate constants, respectively. [Q] is the concentration of quencher. τo is the excited state lifetime in absence of quencher. These results infer that the quenching mechanism is only dynamic in nature in presence of surfactants (Fig. 10). The Stern-Volmer quenching constant (KSV) values in all the studied systems have been calculated using Eq. (3). For the calculation of KSV, the linear part of the quenching data has been used. While the KSV value for pure BSA was found to be 4.70 M−1, the KSV values for BSA in presence of each of 12-4-12, 12-812 and 12-4(OH)-12 were found to be 4.78 ± 0.40 M− 1, 4.28 ± 0.33 M−1 and 4.02 ± 0.35 M−1, respectively. The values of bimolecular quenching rate constant (kq) for pure BSA and in presence of each of 124-12, 12-8-12 and 12-4(OH)-12 have been calculated using equation, kq = KSV / τo and found to be 7.56 × 108 M−1 s−1, 1.01 × 109 M−1 s−1, 1.16 × 109 M−1 s−1 and 9.33 ± 0.14 × 108 M−1 s−1, respectively. kq for BSA in presence of each of gemini surfactants is higher than that in pure BSA. In absence of gemini surfactant, Trp and Tyr residues of pure BSA those are accessible to the acrylamide get quenched. In presence of gemini surfactants, there is a change in the conformation of BSA. The buried Trp and Tyr residues are more exposed to the quencher in presence of a gemini surfactant and that is why quenching rate constant increases. The increasing order of quenching rate constant is 12-4(OH)-12 b 124-12 b 12-8-12. This is also the order of hydrophobicity provided to Trp and Tyr residues at higher concentration range of surfactant. The surfactant molecules at 5.0 mM form micelles along the BSA chain. Even if the hydrophobicity of environment around Trp and Tyr residues is high with 12-8-12, the quenching occurred by acrylamide is also high. This might be indicating that the acrylamide molecules first get solubilized in micelles and then quench the fluorescence. It might be that the number of acrylamide molecules solubilized in micelles is maximum for 12-8-12 and minimum for 12-4(OH)-12. 3.8. Interaction of gemini surfactant with unfolded BSA Interaction of gemini surfactant with unfolded BSA has been studied by steady-state and time resolved fluorescence methods. BSA has been denatured by guanidine hydrochloride (GdHCl). Fluorescence spectra of BSA have been recorded in presence of various concentration of GdHCl. Recorded fluorescence spectra of BSA in presence of GdHCl are similar to that reported in the literature [57]. Intensity of fluorescence decreases and fluorescence maximum shifts towards longer wavelength


1.5 1.4 1.3

F/F0

with increasing the amount of GdHCl. At 4.0 M GdHCl, the fluorescence peak maximum of BSA appears at 358 nm. Fig. 11 shows the variation in average excited state lifetime of BSA with increasing concentration of GdHCl. Excited state lifetime initially increases and then decreases with increasing the concentration of GdHCl. These results clearly show the unfolding of BSA in presence of GdHCl. Mukherjee et al. [53] have studied the interaction of BSA by β-cyclodextrin. They have reported that β-cyclodextrin remains inactive for refolding of BSA denatured by GdHCl. Present study have demonstrated the interaction of gemini surfactants, 12-4-12, 12-8-12 and 12-4(OH)12 with unfolded BSA. BSA is first denatured by using 4.0 M GdHCl and then a gemini surfactant of varying concentrations is added. Circular dichroism (CD) spectra do not show any change in peak position with changing concentration of gemini surfactant (spectra not shown). So, refolding does not takes place in presence of gemini surfactants. Fig. S2 shows the steady-state fluorescence spectra to demonstrate the interaction of unfolded BSA with various concentrations of 12-4-12, 12-8-12 and 12-4(OH)-12. Although for 12-4-12 we could go maximum concentration up to 20.0 mM, but for 12-8-12 and 124(OH)-12 the sample with maximum concentration prepared were 8.0 mM and 10.0 mM, respectively beyond which the precipitation in the solutions was observed. Fluorescence intensity of BSA progressively increases with increasing the concentration of gemini surfactants. Therefore, gemini surfactant molecules create hydrophobic environment around Trp and Try residues along the protein chain, may be by forming micelle-like structures. The increasing order of enhancement in fluorescence intensity is as follows: 12-4(OH)-12 b 12-4-12 b 12-812 (Fig. 12) which is similar to that in absence of GdHCl (Fig. 3). Fluorescence peak maximum is shifted from 358 nm to shorter wavelength with increasing the concentration of 12-4-12, 12-8-12 and 12-4(OH)12. In presence of 8.0 mM of each of 12-4-12, 12-8-12, and 12-4(OH)12, the fluorescence peak maximum appears at 348 nm, 343 nm and 353 nm, respectively. At 10.0 mM of each of 12-4-12 and 12-4(OH)12, the fluorescence peak maximum appears at 347 nm and 353 nm, respectively. The fluorescence peak maximum of pure BSA is located at 347 nm. This result shows that 12-8-12 provides an environment around Trp and Tyr residues in the unfolded BSA chain which is more hydrophobic than that with other two surfactants. For 12-8-12, it is even more hydrophobic than that in pure BSA. Thus hydrophobic environment created by gemini surfactants in presence of GdHCl is similar to that in absence of GdHCl.

377

1.2 1.1

12-4-12 12-4(OH)-12 12-8-12

1.0 0.9 0.8 0

2

4

6

8

[Gemini Surfactant]/mM Fig. 12. Plot of variation of fluorescence intensity of BSA unfolded by 4.0 M GdHCl with increasing concentration of gemini surfactants.

In presence of 20.0 mM and 10.0 mM concentration of 12-4-12, the intensity gains are 68% and 63%, respectively. For 12-4(OH)-12 at 10.0 mM concentration, the gain in intensity is about 51%. With 8.0 mM of 12-8-12, the intensity was greater than that for pure BSA. In fact in absence of GdHCl also one can see that at higher concentration of 12-8-12 the fluorescence intensity is higher than that for pure BSA (Fig. 3) which is in contrast to that in presence of other two surfactants. These observations indicate that in presence of gemini surfactants, the denatured BSA regain substantial amount of hydrophobic environment and gain in that environment is more in presence of 12-8-12 than that of 12-4-12 and 12-4(OH)-12. In fact it is substantially different in case of 12-8-12. Excited state lifetimes also have been measured to support the above mentioned process. Fig. S3 shows the fluorescence decays of unfolded BSA in absence and presence of 12-4-12 as representative. Similar decays have been recorded for other three surfactants as well. Lifetime values are tabulated in Tables S3–S5. Fig. 13 shows the variation of excited state lifetime for interaction with various concentrations of gemini surfactants. This variation is similar to that in fluorescence intensities (Fig. 12). As far as 12-8-12 is concerned, it is different from that in absence of GdHCl at higher concentration of surfactant. Different trends in lifetime data for 12-8-12 in presence of GdHCl might be indicating that to what extent the conformation of Trp can be changed by

5.0

6

4.5

4

3.5

< > / ns

/ ns

5