Interaction of bovine serum albumin with cationic monomeric and dimeric surfactants: A comparative study

Journal of Molecular Liquids 218 (2016) 421–428

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Interaction of bovine serum albumin with cationic monomeric and dimeric surfactants: A comparative study Srishti Sinha a, Deepti Tikariha b, Jyotsna Lakra a, Toshikee Yadav a, Sunita Kumari c, Subit K. Saha c, Kallol K. Ghosh a,⁎ a b c

School of Studies in Chemistry, Pt. Ravishankar Shukla University, Raipur C.G., India Govt. Naveen College, Nawagarh, Janjgir–Champa C.G., India Department of Chemistry, Birla Institute of Technology and Science, Pilani 333 031, Rajasthan, India

a r t i c l e

i n f o

Article history: Received 24 December 2015 Accepted 18 February 2016 Available online xxxx Keywords: Gemini surfactant Serum albumin Micellization Thermodynamic parameters

a b s t r a c t The interaction of bovine serum albumin (BSA) with a single chain cationic surfactant, dodecyl trimethylammonium bromide (DTAB) and three dimeric surfactants viz., butanediyl-1,4 bis(dimethyldodecylammonium bromide (12–4-12,2Br−), 2- butanol-1,4-bis(dimethyldodecylammonium bromide) (12–4(OH)-12,2Br−), 2,4dibutanol-1,4 bis(dimethyldodecylammonium bromide) (12-4(OH)2-12,2Br−) have been investigated by means of surface tension, conductance, viscosity, fluorescence and UV–Visible spectroscopic measurements. The results obtained depict that the hydroxyl group substitution in linker of gemini surfactants affects the BSA-surfactant interactions. As compared to single chain cationic surfactants, gemini surfactants show more interactions with BSA. The negetive values of thermodynamic parameters viz., Gibbs free energy of micellization (ΔG°m), Gibbs free energy of adsorption (ΔG°ads) and Gibbs free energy of quenching process (ΔG°q) showed spontaneity of micellization process. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Protein–surfactant interactions carry greater importance since they are more relevant in the fields of detergents, cosmetics, biosciences, foods and pharmaceuticals [1,2]. The marginal stability of the native globular conformation of proteins which is a delicate balance of various interactions in the proteins, is influenced by the pH, temperature and additives such as substrates, activators, coenzymes and inhibitors [3, 4]. Studies on the interactions of surfactants with globular proteins can contribute towards an understanding of the action of surfactants as denaturants and as solubilizing agents for membranes of proteins and lipids. Surfactant can be broadly classified according to their binding nature towards proteins [5]. Some of the surfactants only bind to protein and leave their tertiary structure intact while some are initiate protein unfolding known as denaturing surfactants [6]. Commonly used ionic surfactants such as sodium dodecyl sulfate and cetyltrimethylammonium bromide, generally denature proteins whereas non-ionic surfactants do not [7,8]. Binding of surfactants to proteins could either stabilize the structures of these proteins or denature them. Ionic surfactants usually interact strongly with proteins and denature them [9]. The denaturation mechanism of surfactant is different from that of urea and guanidinium chloride. It usually occurs at very low concentration, making them much more efficient than traditional ⁎ Corresponding author. E-mail address: [email protected] (K.K. Ghosh).

http://dx.doi.org/10.1016/j.molliq.2016.02.052 0167-7322/© 2016 Elsevier B.V. All rights reserved.

chemical denaturants. Except for few nonionic surfactants, most of them usually do not denature the proteins [10,11]. The most abundant protein in plasma is serum albumin. Serum albumin is synthesized in liver and exported as non-glycosylated protein [12]. They bind to a variety of hydrophobic ligands and thus used as model proteins for many studies like biophysical, biochemical and physico-chemical [13,14]. Albumin plays an important role in the transportation and deposition of a variety of endogenous and exogenous substances in the blood [15,16] and are also used in peritoneal dialysis in fighting against the harmful effect of antibiotics [17]. Studies have shown that the distribution and metabolism of a large number of biologically active compounds, such as metabolites, drugs, and even some toxins, in blood are dependent to a larger extent on their affinities towards serum albumin [18,19]. Bovine serum albumin (BSA) is an expedient and widely studied model globular protein not only for its important roles in biological processes due to its unique ligand binding properties, but also for its structure being well established [20,21]. In its native state, it contains 583 amino acids and 17 disulfide bonds with one free cystein group [22]. It is highly water soluble because of their distribution of amino acid (hydrophobic inside, hydrophilic outside) and large number of ionized amino acids [23]. From the last decades considerable attention have been paid from researchers across the globe to study the interaction of BSA and surfactant of different kind viz., ionic, nonionic, gemini etc. [24–27]. However, most researches mainly focused on the interactions of protein with traditional surfactants such as sodium dodecyl sulfate, cetyltrimethylammonium

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bromide and the researches on gemini surfactants, a new generation of surfactants, with proteins are relatively limited [28]. In general, the gemini surfactants show comparatively stronger interaction with the BSA molecule than the single-chain surfactants [25]. Gemini surfactants are made up of two polar headgroups and two hydrophobic alkyl tails linked by a spacer at the level of headgroups or close to the headgroups. This unique structure makes gemini surfactants to possess different protein binding behaviors than the conventional surfactants [29–33]. Different types of physicochemical techniques have been employed to investigate the interactions between cationic and anionic surfactants with proteins in vitro [34–36]. The mechanism of such type of interactions has become an essential field of research in colloidal science. Li et al. [27] have reported that the cationic gemini surfactant stabilizes the secondary structure of bovine serum albumin (BSA) at low surfactant concentration, while the corresponding monomeric surfactant (dodecyltrimethylammonium bromide) did not show such effect. Ahluwalia et al. [1] have studied the conformational changes induced by conventional anionic surfactants. Niu et al. [37] have also done some spectroscopic studies on interaction of BSA and gemini surfactant with different spacer length. Recent studies revealed that gemini surfactants interact more efficiently with proteins and denature them at lower concentration as compared with conventional surfactants [30,31]. Owing to superior performance of gemini surfactants, they have been creating special interest in the field of protein–surfactant studies. The present study is aimed to explore the interactions of BSA with structurally different monomeric and gemini surfactants. In the present investigation, we have performed a comparative study to evaluate the binding efficacy of hydroxyl group substituted gemini surfactants with BSA against conventional monomeric cationic surfactants. Structure of the surfactants are presented in Scheme 1.

2. Materials The gemini surfactants, 12-4-12, 2Br−, 12-4(OH)-12, 2Br− and 124(OH)2-12, 2Br− were synthesized following the same method as reported in the literature [38,39]. Bovine serum albumin (BSA) with the molar mass of 66.4 kDa and DTAB were purchased from Sigma Aldrich and used as received. Millipore water was used for preparing the solutions. The BSA concentration was determined by Varian Cary 50 UV–visible spectrophotometer, using a molar extinction coefficient of 4.4 × 104 cm−1 at 280 nm [27]. 3. Method 3.1. Conductivity Conductometric measurements were carried out using a Systronics direct reading conductivity meter (Type 306). The conductivity cell constant was calibrated with KCl solutions in appropriate concentration range. The temperature of the solution was carefully controlled by a thermostat (having a temperature accuracy of ±0.01 °C). A concentrated protein–surfactant solution was added in 10 ml of aqueous medium using a micropipette. After ensuring temperature equilibration, the specific conductance (κ) was measured. At each concentration, the conductivity measurement was repeated three times and the average value was obtained. 3.2. Surface tension The surface tension measurement was measured using surface tensiometer (Jancon, India) employing ring detachment technique at

Scheme 1. Structure of surfactants and protein.

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300 K. For calibration of the tensiometer milipore water was used. Platinum ring was thoroughly clean and washed with milipore water. The γ values of surfactant in the presence and absence of BSA were taken by adding their stock solution. The results were accurate within ±0.1 mN m−1.

3.3. Viscosity An Ostwald viscometer, calibrated with redistilled water and used to measure the viscosities. It is a U-shaped glass tube. A liquid was allowed to flow through its capillary tube between two etched marks and the time of flow of the liquid was measured using a stopwatch. An electronic digital stopwatch with an uncertainty of ±0.01 s was used to measure the flow of time. The measurements were carried out at 300 K temperature.

3.4. UV–visible spectroscopy The absorption spectra were recorded on Varian Cary 50 UV–visible spectrophotometer. The measurements were performed by successive additions of 0.01 M stock solutions of surfactants directly into the 1 cm path length quartz cell containing 0.6 ml of (2.0 × 10−6 mol/L) BSA solution. The temperature was maintained at 300 K.

3.5. Fluorescence The fluorescence quenching measurements were performed on a Cary Eclipse Fluorescence Spectrophotometer. The temperature was maintained at 295 K, 300 K and 305 K. To investigate the effect of surfactant addition on intrinsic fluorescence of BSA, the fluorescence spectra of solution containing a fixed concentration (2.0 × 10−6 mol/L) of BSA and various surfactant concentrations were monitored with a 1 cm path length quartz cell. The excitation wavelength for BSA fluorescence was 280 nm and its emission spectra were scanned in the range of 290– 450 nm. Both excitation and emission slit widths were fixed at 5 nm. The fluorescence intensities diminish with increasing surfactant concentration (shown in Fig. 4). Critical micelle concentration (CMC) of some gemini surfactants was determined by fluorescence measurements by using pyrene as a probe. The excitation intensity was fixed at 334 nm and its emission spectra were scanned between 373 and 384 nm, which corresponded to the first and third vibrational peaks, respectively. The slit widths were also fixed at 5 and 2.5 nm. Fluorescence emission of pyrene was studied by monitoring the emission spectra of solution containing a fixed concentration of pyrene (4.1 × 10− 7 mol/L) and increasing the surfactant concentration.

4. Results and discussion

Fig. 1. Dependence of the specific conductance (κ) on concentration of surfactant, [C] in surfactant solutions in the presence of protein at (2.0 × 10−6 mol/L) BSA.

4.2. Surface activity of the system The dependence of surface tension, γ, on log[C] for the buffered surfactant solutions (0.01 mol dm−3 phosphate buffer) in the absence and presence of BSA is displayed in Fig. 2. In the absence of protein, a welldefined break point is observed, corresponding to the CMC. The observed surface tension of BSA contained surfactant solution is lower than the pure surfactant because of surface active nature of BSA. The γ vs log[C] curves in the presence of BSA exhibit one break point which marks the concentration above which the aggregation of surfactants occurs. The CMC values are summarized in Table 1. At low surfactant concentration, the interaction between the protein and the cationic surfactant at the interface is expected to be dominated by electrostatic interaction, and then protein interacts with surfactant via hydrophobic force as the surfactant concentration increases. The attenuation in γ with an increasing surfactant concentration would be caused by the formation of protein−surfactant complexes of enhanced surface activity compared to that of the native protein [40]. The slope of the initial linear decrease in γ with an increasing surfactant concentration is a measure of the interfacial adsorption efficacy of the surfactant, which can be quantified by the Gibbs surface excess concentration (Γmax). To calculate Γmax and minimum area per molecule at the air– water interface (Amin), the following Gibbs equation has been used [41]: Γ max ¼ −

  1 dγ 2:303nRT d log C T;P

ð1Þ

A min ¼ 1=NΓ max

ð2Þ

where R is the gas constant (8.314 Jmol−1 K−1), T is the absolute temperature, C is the surfactant concentration, (dγ/dlogC) is the slope from γ vs log C plot and N is the Avogadro's number. The constant n

4.1. Criticle micelle concentration For the understanding of protein–surfactant interactions the micellization process of the surfactants in aqueous solution is required. The electrical conductivity method was used to determine the critical micellization concentration (CMC) of surfactants and degree of ionization (α) of micelles in absence and presence of BSA. The CMC values have been evaluated from the break points in the specific conductivity (κ) versus surfactant concentration plot (Fig. 1). The values are summarized in Table 1. Data in Table 1 show that the CMC of geminis is lower than that of DTAB. This result could be explained by considering the unique aggregation morphology of the gemini surfactants, when compared to that of monomeric surfactant.

Table 1 Critical micelle concentration (CMC) and degree of ionization (α) of some monomeric and gemini surfactants in the absence and presence of BSA (2.0 × 10−6 mol/L) at pH 7 and 300 K. Surfactants

Without BSA

With BSA Α

CMC (mM)

DTAB 12-4-12, 2Br− 12-4(OH)-12, 2Br− 12-4(OH)2-12, 2Br−

Cond.

S.T.

FL

14.0 1.22 0.99 0.78

14.0 1.15 0.99 0.74

– 1.15 1.00 –

0.54 0.28 0.26 0.22

CMC (mM) Cond.

S.T.

10.0 0.69 0.52 0.15

11.0 0.90 0.56 0.29

Cond. — Conductivity, S.T. — Surface Tension, FL — Fluoroscence.

α

0.86 0.73 0.37 0.11

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Fig. 2. Dependence of the surface tension,γ, on log[C] in surfactant solutions in the presence and absence of protein at (2.0 × 10−6 mol/L) BSA (a) 12-4-12, 2Br−, (b) 12-4(OH)-12, 2Br−, (c) 12-4(OH)2-12, 2Br−.

for an ionic surfactant having univalent counter ion and surfactant ion takes the value of 2. For dimeric surfactant having divalent surfactant ion and two counter ion takes the value of 3. The observed Γmax (surfactant) N Γmax (surfactant + protein) relation and the consequent Amin (surfactant) b Amin (surfactant + protein) relation can be explained by the reduced compactness of the interfacial monolayer at the air−solution interface in the presence of BSA, which comes from the presence of the solubilized protein −surfactant complexes at the interfacial monolayer [40] (See Table 2). 4.3. Viscocity of the system Viscometry is effective in probing conformational and rheological changes in interaction of protein with ionic surfactant. The relative

viscosity of surfactant-doped BSA solution (ηr) vs surfactant concentration is displayed in Fig. 3, where the relative viscosity is defined as the ratio of the viscosity of the surfactant-doped protein solution to that of the surfactant solution. Two characteristic concentrations in each plot are identified in Fig. 3. For example, the first break occurs at a concentration of 0.002 mol dm−3 of 12-4(OH)2-12 is called the threshold concentration, marking the commencement of thickening. The second break occurs at 0.004 mol dm−3 concentration and then the relative viscosity curve rises smoothly at a slower rate than the previous one with a higher concentration of surfactant. The initial increase in viscosity is due to the charging up of the protein molecule by binding of surfactants. The subsequent increase of viscosity at high surfactant concentration is possibly due to the cross-linking of the several aggregates by free surfactant micelles in the solution [41]. Similar result has found for 12-4-12 and 12-4(OH)-12.

Table 2 Surface excess parameter (Γmax), surface pressure at CMC (πCMC), minimum surface area per molecule (Amin) of some monomeric and gemini surfactants with BSA (2.0 × 10−6 mol/L) and without BSA at 300 K. Surfactants

Without BSA 6

Γmax × 10 mol.m DTAB 12-4-12, 2Br− 12-4(OH)-12, 2Br− 12-4(OH)2-12, 2Br−

0.64 1.08 0.90 0.61

With BSA −2

Amin × 10 259.0 153.3 184.1 273.1

20

−2

m

.mol

−1

πCMC mNm

Γmax × 106 mol.m−2

Amin × 1020 m−2.mol

πCMC mNm−1

42.4 33.0 36.0 34.0

0.628 0.881 0.698 0.407

264.4 188.4 237.8 407.9

32.0 31.0 29.5 33.0

S. Sinha et al. / Journal of Molecular Liquids 218 (2016) 421–428

Fig. 3. The profile of ηr vs [Surfactant] (relative to surfactant) for BSA-surfactant mixture at pH 7 and 300 K.

4.4. Fluorescence quenching of BSA BSA has three intrinsic fluorophores, viz. tryptophan (Trp), tyrosine (Tyr) and Phenylalanine (Phe). Phe gives quite low quantum yield and in most of the cases it is not excited. So emission from this residue can be overlooked [42]. Fig. 4 shows the fluorescence quenching response of BSA to surfactants, where the addition of surfactant quenched the fluorescence emission of BSA with a small blue shift, which resulted from the conformational change of BSA. This phenomenon is attributed to the exposure of the Trp and Tyr residues of BSA to hydrophobic environment of surfactant, which leads to the interaction and energy transfer between BSA and surfactant. Fig. 4 indicating the blue shifts of the fluorescence bands at high surfactant concentrations for DTAB/BSA

Fig. 5. A Stern–Volmer constant plots of F0/F versus [Q] for BSA/12-4(OH)2-12 complex.

and 12–4(OH)2–12/BSA systems. It suggests that the fluorophore residues are enclosed by a more hydrophobic surroundings resulted from the absorption of the surfactant molecules near the amino acid residues. The fluorescence intensity decreases gradually with the increase in surfactant concentration above CMC as illustrated by Fig. 4 where the gemini surfactants exhibit the stronger ability to decrease the intensity of intrinsic fluorescence of BSA. This may be due to partially denatured BSA. This decrease in the intensity of BSA fluorescence accompanied by a blue shift of the maximum emission peak, suggesting that fluorophore residues are exposed to a more hydrophobic environment. This phenomenon is ascribed to the formation of BSA/gemini complex. The fluorescence quenching is usually divided into dynamic quenching and static quenching. Dynamic quenching results from collisional encounters between the fluorophore and quencher, while static quenching results from the formation of a ground-state complex between the fluorophore and quencher [43]. Dynamic and static quenching can be distinguished from the dependence of binding constants on temperature and viscosity. Generally the quenching mechanism of BSA with cationic surfactants is dynamic in nature [43]. For dynamic quenching, a decrease in intensity is described by the Stern– Volmer equation [44]: F0 =F ¼ 1 þ K sv ½Q 

Fig. 4. Emission spectra of BSA (2.0 × 10−6 mol/L) in the presence of various surfactant concentrations of (A) 12-4(OH)2-12 (B) DTAB (λexc = 280 nm) at pH 7 and 300 K.

425

ð3Þ

Fig. 6. Stern–Volmer plots of fluorescence quenching of BSA by 12-4(OH)2-12; λex = 280 nm.

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[Surfactant] NN [B0], by introducing the approximate concentration [(Surfactant)nB] = [B0] − [B], so that Eq. (4) becomes: Ka ¼

½B0 −½B : n ½Surfactant ½B

Using the spectral ratio between fluorescence intensity and the unbound biomolecule [B]/[B0] = F/F0 and transforming the equation we obtain [43]: log½ð F0 −FÞ=F ¼ log K a þ n log ½Surfactant

ð5Þ

where Ka is the apparent binding constant of surfactant with BSA and n is the number of binding sites (binding capacity). Ka and n can be obtained from the plot of log[(F0 − F)/F] vs log[Surfactant] by using a least-square algorithm for data-fitting (See Fig. 7). The data obtained are given in Table 3. Fig. 7. A plot of log[(F0 − F)/F] vs log[Surfactant] for binding constant of BSA and gemini surfactants i.e., 12-4-12, 2Br−, 12-4(OH)-12, 2Br− and 12-4(OH)2-12, 2Br− and monomeric surfactant DTAB.

where Ksv and [Q] are dynamic Stern–Volmer quenching constant and concentration of quencher, respectively. F0 is the fluorescence intensities in the absence of quencher and F is in the presence of quencher. Stern–Volmer quenching constant (Ksv) can be obtained from linear Stern–Volmer plot of F0/F against [Q] (Eq. (3)) at 300 K temperature (Fig. 5). The linear relationship between F0/F and the concentration of surfactant at different temperatures (Fig. 6) show that the fluorescence quenching follows the Stern–Volmer equation, i.e. dynamic quenching. 4.5. Binding constant and binding capacity If ligand molecules (surfactants) interact independently with a set of equivalent sites on a biomolecule (B) and forms a compact structure [(surfactant)nB], the following equilibrium should be reached:

4.6. UV–visible studies UV–visible absorption is a useful tool to probe protein–ligand complex formation [28,45]. This absorption method is another method of calculating binding constant. In the absorption spectra of BSA absorbance at 280 nm decreases upon addition of surfactant (i.e., 12-4-12, 12-4(OH)-12 and 12-4(OH)2-12) (See Fig. 8). The absorption around 280 nm is due to the absorption of aromatic amino acids (Trp, Tyr, and Phe) [45]. The peak positioned around 280 nm is red shifted. Based on the peak shift and decrease in absorbanse, it can be concluded that all gemini surfactants can form complex with BSA. This observable fact offers another evidence for quenching of BSA fluorescence by surfactants. 4.7. Determination of binding constant of BSA/Surfactant complex The binding constant BSA–Surfactant complexes were estimated from Benesi–Hildebrand (B–H) equation [46]. The change in absorbance is depend on the concentration of surfactants, according to the following equation, 1 1 1 þ ¼ A−A0 KðA max −A0 Þ½Surfactant A max −A0

ð6Þ

Ka

B þ nðsurfactantÞ ⇄ ðsurfactantÞn B when the system achieves equilibrium, the binding constant can be written as:   ðSurfactantÞn B Ka ¼  n : ðSurfactantÞ B

ð4Þ

Here, [B] is the concentration of free (unbound) biomolecule. If the overall amount of biomolecules (bound and unbound with the surfactant) is B 0, thereupon [B0 ] = [(Surfactant)n B] + [B], when

where A0, A, Amax are the absorbance in the absence of and at intermediate concentration of surfactant, and at saturation point, respectively and K is the binding constant. The plot of 1/[A − A0] vs.1/[Surfactant] gives straight lines (Fig. 9), which further indicates the formation of 1:1 complex between protein (BSA) and gemini surfactants. The values of the binding constant obtained from the intercept-to-slope ratio of the Bensei–Hildebrand plot (Fig. 9) for BSA/Surfactant complexes show that the 12-4(OH)2-12 gemini shows more binding affinity towards BSA than other gemini surfactants as they readily interact and form hydrogen bond with water. Owing to substituted hydroxyl group, it reduces the unfavorable

Table 3 Binding constant (Ka), Stern–Volmer quenching constant (Ksv), binding capacity (n), correlation coefficient (R) and corresponding thermodynamic parameter (ΔGq) of surfactant + BSA systems at different temperatures 295 K, 300 K and 305 K. Surfactants

DTAB 12-4-12, 2Br− 12-4(OH)-12, 2Br− 12-4(OH)2-12, 2Br−

295 K

300 K

305 K

Ka × 10−4 M

Ksv × 10−4 M

R

n

ΔGq kJ/mol

Ka × 10−4 M

Ksv × 10−4 M

R

n

ΔGq kJ/mol

Ka × 10−4 M

Ksv × 10−4 M

R

n

ΔGq kJ/mol

0.31 0.69 0.53 0.92

0.45 0.61 1.29 1.12

0.978 0.989 0.969 0.989

0.81 1.03 0.70 0.86

−20.63 −21.38 −21.03 −22.76

0.39 0.97 1.30 2.15

0.36 0.81 1.12 1.47

0.977 0.976 0.997 0.982

1.00 1.03 1.11 1.01

−20.42 −22.89 −23.62 −24.88

0.82 1.08 1.51 1.59

1.15 1.44 2.10 2.55

0.984 0.968 0.950 0.986

0.74 0.46 0.89 0.96

−23.71 −24.28 −25.24 −25.73

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Table 4 Binding constant (K) and correlation coefficient (R) of surfactant + BSA systems at 300 K and pH 7.

Fig. 8. Absorption spectra of BSA (2.0 × 10−6 mol/L) in the presence of various surfactant concentrations of 12-4(OH)2-12 (λexc = 280 nm) at pH 7.0 and 300 K.

hydrocarbon-water contact which leads to quick formation of micelle [43] (See Table 4). 4.8. Energetics of micellization The Gibbs free energy of micellization (ΔG°m) of ionic micelles depends on both the CMC and degree of micellar ionization (α). In the case of ionic gemini surfactants, it can be calculated by following equation [44,47]: ΔG m ¼ 2ð1:5–αÞRT lnXCMC

ð7Þ

and for monomeric ΔG m ¼ ð2–αÞRT lnXCMC :

ð8Þ

XCMC is the CMC of the surfactant in mole fraction unit and α is the micellar ionization. The standard free energy of micellization (ΔG°m) is translated into the standard free energy of adsorption (ΔG°ads) at the air/water interface using Eq. (8) [44]: ΔG ads ¼ ΔG m −πCMC =Γ max

ð9Þ

Both ΔG°ads and ΔG°m values are negative and listed in Table 5. All the negative ΔG°ads values imply that the adsorption of the surfactant molecules at the air/water interface takes place spontaneously. Table 5

Surfactants

K × 10−4 M

R

DTAB 12-4-12, 2Br− 12-4(OH)-12, 2Br− 12-4(OH)2-12, 2Br−

0.286 0.488 0.567 0.615

0.984 0.991 0.996 0.995

reveals that the values of ΔG°ads are greater than ΔG°m (in magnitude) confirming the micellization is secondary in nature with respect to surface adsorption, thus work has to be done in transferring molecules from the monomeric form to the micelle form. The interaction forces between amphiphiles and biological macromolecules include hydrophobic force, electrostatic interactions hydrogen bond and Vander Waals' force [43]. For this reason, the temperature-dependent thermodynamic parameters were studied by fluorescence measurement. The temperatures chosen were 295,300 and 305 K. The free energy change for the quenching process (ΔGq) at different temperatures can be calculated from the following relationship [46]: ΔGq ¼ −RT ln Ksv

ð10Þ

The negative values for ΔGq reveals that the binding process of BSAsurfactant systems were spontaneous and favorable for the mixtures studied. 5. Conclusion An estimation of the interactions between the surfactants (DTAB, 12–4-12, Br−, 12–4(OH)-12, Br− and 12-4(OH)2-12, Br−) and BSA is important to understand the role of these amphiphiles in biological processes. In summary, we have attempted to provide a simple and rapid approach for investigating the binding of surfactant to BSA. The CMC values for pure amphiphiles are in a good agreement with literature. The negative values of ΔG°m and Δ G°ads show energetically favorable micelle formation and adsorption at air/solution interface. Synchronous fluorescence spectra and UV–Visible spectra of BSA revealed that the conformation and microenvironment of BSA were changed by the binding of surfactant. At low concentration, the interactions are estimated to be mainly electrostatic but when the concentration increased hydrophobic interaction are also cooperative. At high surfactant concentration BSA-surfactant interaction becomes saturated and successive increase results in formation of micelles. For gemini surfactants electrostatic as well as hydrophobic interactions are estimated to be more stronger than the single chain surfactant, so the geminis are more efficient as protein denaturants than the monomeric surfactant. This investigation indicates that the gemini surfactants act as a better denaturant than monomeric surfactants and will find enormous applications in chemical, biological and medical field.

Table 5 Thermodynamic parameters Gibbs free energy of micellization (ΔG°m) and Gibbs free energy of adsorption (ΔG°ads) of surfactant/BSA systems at 300 K. Surfactant

Fig. 9. Benesi–Hildebrand plot using changes in absorption spectra of BSA (measured at 280 nm for 12-4-12, 12-4(OH)-12 and 12-4(OH)2-12).

DTAB 12-4-12, 2Br− 12-4(OH)-12, 2Br− 12-4(OH)2-12, 2Br−

Without BSA

With BSA

ΔG°m kJ/mol

ΔG°ads kJ/mol

ΔG°m kJ/mol

ΔG°m kJ/mol

−30.18 −65.28 −67.65 −71.35

−96.33 −95.75 −107.56 −127.27

−24.52 −43.39 −65.27 −88.91

−24.52 −43.39 −65.27 −88.91

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