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Effect of spacer length of alkanediyl-α,ω-bis(dimethylcetylammonium bromide) gemini homologues on the interfacial and physicochemical properties of BSA
Colloids and Surfaces B: Biointerfaces 77 (2010) 54–59
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Effect of spacer length of alkanediyl-␣,-bis(dimethylcetylammonium bromide) gemini homologues on the interfacial and physicochemical properties of BSA Mohammad Amin Mir a , Javeed Masood Khan b , Rizwan Hasan Khan b , Ghulam Mohammad Rather a , Aijaz Ahmad Dar a,∗ a b
Department of Chemistry, University of Kashmir, Srinagar 190006, J&K, India Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, India
a r t i c l e
i n f o
Article history: Received 26 September 2009 Received in revised form 4 January 2010 Accepted 7 January 2010 Available online 14 January 2010 Keywords: BSA Interfacial tension Circular dichroism Gemini surfactant Denaturation
a b s t r a c t The interactions of bovine serum albumin (BSA) with cationic gemini surfactants alkanediyl␣,-bis(dimethylcetylammonium bromide) (designated as C16 Cs C16 Br2 , s = 4, 5, and 6) and single chain surfactant cetyltrimethylammonium bromide (CTAB) have been investigated with tensiometry, Rayleigh’s scattering, fluorescence spectroscopy, and circular dichroism at physiological pH and 25 ◦ C. The results of the multi-technique approach showed that the gemini surfactants interact more efficiently with the proteins than their conventional single chain counterparts and their efficiency increases with decrease in the length of the spacer. The saturation in interfacial tension occurred at a lower concentration in presence of BSA compared to CMC of the surfactants in absence of BSA and the concentration of gemini surfactants corresponding to interfacial saturation decreases with decrease in the spacer length. Fluorescence and circular dichroism spectroscopy results revealed increase in unfolding of BSA with decrease in spacer length of gemini surfactants. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Protein–surfactant interactions have been a subject of extensive studies because of its relevance to the field of pharmaceutics, paints and coatings, adhesives, oil recovery, etc. [1–8]. Moreover, such studies can provide insight relevant to the solubilizing and denaturing/renaturing action of surfactants on proteins. Protein–surfactant interactions are usually dependent on the surfactant features. Compared to anionic surfactants, cationic surfactants weakly interact with the proteins as a consequence of smaller relevance of electrostatic interactions at the pHs of the interest [9–14]. However, the binding isotherms of both types of surfactants have been found to be similar [14,15]. At low surfactant concentrations ionic surfactants bound to the oppositely charged sites of proteins which cause the protein to unfold resulting in the exposure of more binding sites. As the surfactant concentration is increased the binding becomes cooperative and ultimately the protein is saturated by the surfactants and their aggregates [16]. Cationic gemini surfactants are made up of two hydrophobic chains and two polar head groups covalently linked through a spacer group [17–20]. These kinds of surfactants have number of unique aggregation properties such as lower CMC and kraft
∗ Corresponding author. E-mail address: aijaz [email protected] (A.A. Dar). 0927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2010.01.005
temperature, special aggregation morphology, strong hydrophobic microdomains, etc. [21–24]. Because of these unique properties gemini surfactants strongly interact with the proteins [25–30] compared to conventional ones. The unusual properties of aggregates of gemini surfactants are related to spacer structure because it influences the distances between head groups in the aggregate [31]. In aggregates formed by conventional surfactants, the only distance is the thermodynamic distance between polar head groups, whereas in aggregates formed by gemini, two head group distance control the aggregation properties (bimodal distribution), one corresponding to the single chain surfactant equilibrium distance and one corresponding to the length and nature of the spacer [32]. Various applications of the gemini surfactants relevant to the surfactant–protein interactions viz., antimicrobial, hair conditioning, skin and eye care, etc., are important over the other conventional surfactants [33,34]. The effectiveness of gemini surfactants depends upon the length and type of spacer, and the hydrophobic counterpart. In spite of that, the effect of spacer length of the gemini surfactant on the surfactant–protein interaction has not been given the due attention. Although, Li et al. [25] studied the interaction of BSA with short alkyl chain DTAB and its gemini homologues (C12 Cs C12 Br2 , where s = 3, 6, 12) but due to the large differences and lack of continuity in the spacer lengths, a critical analysis escaped. We hereby report the interaction of BSA with long alkyl chain CTAB and its gemini homologues (C16 Cs C16 Br2 , where s = 4, 5, 6)
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Fig. 1. Tensiometric dilution (squares) and interaction (circles) profiles for CTAB (A), C16 C4 C16 Br2 (B), C16 C5 C16 Br2 (C), and C16 C6 C16 Br2 (D).
using tensiometry, spectrofluorometry and circular dichroism at physiological pH and 25 ◦ C. Gemini surfactants with comparatively rigid and closely related spacer lengths were selected to have meaningful insight into the effect of spacer length/bimodal distribution of gemini surfactants on the surfactant–protein interactions. BSA, a globular protein, has a molar mass of 66.4 kDa and posses about 583 amino acids [35]. It has relatively high water solubility because of its larger number of ionizable amino acids [36]. BSA can bind many different types of biological amphiphilic molecules, which are believed to play an important role in determining the physiological functions [37]. 2. Experimental 2.1. Materials Bovine serum albumin (BSA, Sigma) and cetyltrimethylammonium bromide (CTAB, Sigma) were used as received. The gemini bis(cetyldimethylammonium)butane dibromide, C16 H33 (CH3 )2 N+ –(CH2 )s –N+ (CH3 )2 C16 H33 .2Br− , (where s = 4, 5, 6) were synthesized and characterized as described elsewhere [38]. All the stock solutions of BSA, CTAB, gemini surfactants (C16 Cs C16 Br2 ) were prepared in 60 mM phosphate buffer of pH 7.4 with triple distilled water and utilized to prepare the samples of
desired concentrations. The concentration of the BSA was determined by measuring the absorbance of protein at 280 nm on a Hitachi U-1500 spectrophotometer and kept constant (0.2 mg/mL) throughout the study. 2.2. Methods 2.2.1. Tensiometry Surface tension measurements were made with a Kruss 9 tensiometer by the Whilhemy plate method. 10 mL of 0.2 mg/mL BSA solution in phosphate buffer was taken in sample vessel and the desired concentrations of buffered surfactant solutions was added in small installments using a Hamilton microsyringe, and readings were taken after thorough mixing and temperature equilibration. No more than 1.0 mL of surfactant solution was added to BSA solution. The temperature was maintained at the 25 ◦ C within 0.1 ◦ C by circulating water from a HAAKE GH thermostat. The accuracy of measurements was within (0.1 dyn cm−1 ). 2.2.2. Aggregation studies Rayleigh’s scattering measurements were performed by observing emission at 350 nm after exciting at same wavelength on Hitachi spectrofluorometer (model 2500) equipped with a PC. The
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fluorescence spectra were collected at 25 ◦ C with a 1 cm pathlength cell. 2.2.3. Fluorescence measurements The fluorescence spectra were collected at 25 ◦ C with a 1 cm path-length cell using a Hitachi spectrofluorimeter (model 2500) equipped with a PC. The excitation and emission slits were set at 5 nm. The reference sample consisting of the buffer and the detergent did not give any fluorescence signal. Intrinsic fluorescence was measured by exciting the protein solution at 280 and 295 nm and the emission spectra were recorded in the range of 300–400 nm. 2.2.4. Circular dichroism CD measurements were carried out with a Jasco spectropolarimeter, model J-720, equipped with a microcomputer. The instrument was calibrated with D-10-camphorsulfonic acid. All the CD measurements were carried out at 25 ◦ C with a thermostatically controlled cell holder attached to a Neslab RTE-110 water bath with an accuracy of ±0.1 ◦ C. Far-UV CD spectra were acquired with use of a cell of 1 mm path length over the wavelength range between 200 and 250 nm. A reference sample containing buffer and the detergent was subtracted from the CD signal for all measurements. The high-tension voltage for the spectra obtained was found to be less than 600 V. Spectra were collected with a scan speed of 20 nm/min and response time of 1 s. Each spectrum is the average of four scans 3. Results and discussion 3.1. Tensiometry In tensiometry the threshold surfactant concentration required to saturate the air/solution interface is called the critical micelle concentration (CMC), which corresponds to the break point in the surface tension () versus log [surfactant] plot. Tensiometric profiles for the addition of CTAB and C16 Cs C16 Br2 (s = 4, 5, 6) to buffer solution under identical experimental conditions are illustrated with squares in Fig. 1 and the corresponding CMC values are given in Table 1. The ionic strength of the buffer solution lowered the CMC of the CTAB and C16 Cs C16 Br2 compared to that in aqueous solution [39–41], by way of decreasing the electrostatic repulsion between the charged head groups. The CMC values of gemini surfactants increase with increase in the spacer chain length due to increase in the head group area [22,41,42]. The tensiometric interaction profiles for the CTAB-BSA and C16 Cs C16 Br2 -BSA are included in Fig. 1(circles). The tensiometric profiles of surfactants in presence of BSA show lower surface tension values than the tensiometric profiles of surfactants in absence of BSA, due to the surface activity of BSA ( = 56 mN/m for 0.2 mg/mL BSA solution). The tensiometric profile for CTAB and C16 Cs C16 Br2 in presence of BSA consists of a single break at a lower concentration compared to their CMC values in the buffer solution. This concentration corresponding to the saturation in interfacial tension is denoted by C1 and marks the concentration above which the aggregation of surfactants on the BSA backbone occurs [43]. At physiological pH, BSA has an overall negative charge [44]. The interaction between
proteins and ionic surfactants at the interface is dominated by the electrostatic interactions [45,46]. With increase in concentration of CTAB and C16 C4 C16 Br2 the available charges in the protein molecules are compensated by the oppositely charged surfactant ions, thus forming electroneutral complexes of enhanced surface activity compared to native protein [45,46]. The surface excess concentration ( max ) in mol m−2 at CMC or C1 and minimum surface area per molecule (Amin ) in nm2 at air/solution interface both in absence as well as presence of BSA were calculated from the equations [47] max = −
Amin =
1 lim 2.303nRT C→CMC/C1
1018 Nmax
d d log C
(1)
(2)
where C is the total surfactant concentration, N the Avogadro’s constant and R and T have their usual meanings. n is the number of species whose interfacial concentration changes with the change in bulk phase concentration of surfactant. Because of swamping amount of electrolyte used (60 mM buffer), n can be assumed to be unity for all the surfactant systems [43,46,48]. The variation of 1 ACMC /ACmin with the spacer length of the gemini surfactant is premin 1 sented in Fig. 2. Lower values of ACMC than the ACmin signify less min compact monolayer in presence of BSA. This indicates the presence of BSA–surfactant complexes at the air/solution interface [43]. Fig. 2 indicates that gemini surfactants, both in absence and presence of BSA, form more compact monolayer compared to single chain surfactants but the compactness decreases slightly with increase in the spacer length due to increase in head group area. However, the ACMC values of gemini surfactants were found much lower than the min reported ACMC values for gemini surfactants [49,31]. The discrepmin ancies in ACMC values of gemini surfactants are related to the value min of n in Eq. (1). The exact value of n has been found to be function of state of the surfactant in the solution below CMC, nature of the surfactant and concentration of the surfactant in the submicellar range [50,51]. Therefore, very small ACMC values in our case may min be due to cumulative effect of screening of electrostatic repulsion between the gemini head groups by dense ionic atmosphere furnished by 60 mM phosphate buffer, use of n = 1 instead of 2 or 3 and formation of premicellar aggregates [52,53] which induce abrupt decrease in surface tension near CMC.
Table 1 Critical micelle concentration (CMC) and the concentration corresponding to saturation in interfacial tension in presence of BSA (C1 ) at physiological pH and 25 ◦ C. Surfactant
CMC (mM)
C1 (mM)
CTAB C16 C4 C16 Br2 C16 C5 C16 Br2 C16 C6 C16 Br2
0.2300 0.0052 0.0058 0.0063
0.0890 0.0048 0.0050 0.0056
1 in absence and presence of BSA with the spacer length Fig. 2. Variation of ACMC /ACmin min of the gemini surfactants at physiological pH and 25 ◦ C.
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Fig. 3. Fluorescence spectra (excited at 350 nm) of BSA in native state and in presence of 0.1 mM CTAB, 0.1 mM C16 Cs C16 Br2 (s = 4, 5, 6) at physiological pH and 25 ◦ C.
Fig. 5. Fluorescence spectra (excited at 280 nm) of BSA in native state and in presence of 0.1 mM CTAB, 0.1 mM C16 Cs C16 Br2 (s = 4, 5, 6) at physiological pH and 25 ◦ C.
3.2. Aggregation studies
tryptophan residues get excited and are used to probe changes in their respective microenvironments and provide a picture of local change in the protein. At 280 nm both tryptophan as well as tyrosine residues get excited and provide a picture of global change in the protein. The change in the wavelength of maximum emission by excinm , is illustrated in Fig. 4 and indicate that tation at 280 nm, 280 max BSA exhibits a blue shift in presence of the surfactants. The blue shift increases with increase in the concentration of the surfactant and then attains a constant value at higher surfactant concentranm with increase in surfactant concentration tions. Decrease in 280 max indicates shift of fluorophores towards more hydrophobic environnm thereafter means no change in ment [55]. The constancy in 280 max the hydrophobicity and suggests saturation of BSA backbone by the micelles, in corroboration with the binding isotherms suggested for protein–surfactant interactions [14]. Although the decreasing nm is similar in both CTAB and C C C Br , but the trend of 280 16 s 16 2 max nm is more when the BSA interacts with rate of decrease of 280 max nm (Fig. 5) C16 Cs C16 Br2 than the CTAB. The rate of decrease of 280 max of BSA slows down as the of the spacer length of the gemini surfactant increases. These changes in the microenvironment of BSA are harmonious with the variation of C1 as a function of spacer length of gemini surfactants.
The change in the scattering intensities at 350 nm of BSA mainly arises from the change in number and/or size of BSA–surfactant aggregates [54]. Fig. 3 illustrates the effect of surfactant on the BSA–surfactant aggregation at a particular surfactant concentration (0.1 mM). In conformity with the earlier reports [25,29,30], gemini surfactants, because of their unique aggregation properties, interact efficiently with BSA than their single chain counterparts. It is clear from the Fig. 3 that the interaction of gemini surfactants with the BSA decreases with increase in the spacer length. These results are in agreement with the tensiometric results that the C1 increases with increase in spacer length of gemini surfactants. Lower C1 implies earlier aggregation of surfactant with BSA and hence large number or increased size of BSA–surfactant aggregates at a given surfactant concentration. 3.3. Intrinsic fluorescence The change in the wavelength of emission maximum (max ), a parameter sensitive to the protein conformations and fluorescence intensities at 340 nm by excitation at 280 and 295 nm can be used to examine the protein–surfactant interactions [55]. At 295 nm only
nm nm Fig. 4. Variation of 280 (left) and 295 (right) of BSA with the concentration of CTAB and C16 Cs C16 Br2 (s = 4, 5, 6) gemini surfactants at physiological pH and 25 ◦ C. max max
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The less significant blue shift at the excitation wavelength of 295 nm than at 280 nm is attributed to the fact that the tryptophan residues are buried in the inner core of BSA while the tyrosine residues are distributed throughout. Binding of surfactant to BSA increases the hydrophobic environment around the fluorophores resulting in decreased fluorescence intensity (FI). The variations nm , is found to folin the wavelength of emission maximum, 280 max low the fluorescence intensity (FI) changes faithfully. A decrease nm is observed as the FI decreases as shown in Fig. 5 for in 280 max 0.1 mM surfactant concentration. Gemini surfactants because of their strong hydrophobic microdomains are more effective as comnm of BSA. Like 280 nm , pared to CTAB in decreasing the FI and 280 max max FI intensity of BSA decreases as the spacer length of the gemini surnm and FI suggest decrease factant decreases. These changes in 280 max in hydrophobic environment around the fluorophores of BSA with increase in spacer length of the gemini surfactants. Decrease in hydrophobic environment is attributed to decrease in number of micelle like clusters around the fluorescent residues of BSA due to increase in C1 with the spacer length. 3.4. Far-UV circular dichroism Circular dichroism can be used to probe transitions in the secondary structure of the proteins. BSA exhibits two negative bands in the far-UV CD spectrum at 208 and 222 nm, characteristic of ˛-helical structure [56]. Alterations in ellipticity at 222 nm ( 222 ) are used to monitor the change in ˛-helical content of BSA [57] by CTAB and C16 C4 C16 Br2 . Fig. 6 shows typical far-UV CD spectra of native BSA and BSA in presence of 0.1 mM CTAB and 0.1 mM C16 Cs C16 Br2 (where s = 4, 5, 6) at physiological pH. It is clear from the figure that the CTAB as well as C16 Cs C16 Br2 denature BSA but the gemini surfactants are more effective than CTAB. These results are in conformity with previous results [25,29,30] and have been attributed to the unique characteristics of gemini surfactants. The denaturation of BSA by the ionic surfactants arises from the replacement of hydrophobic interaction in the protein by the electrostatic repulsion between the micelle like aggregates formed on the BSA [30]. Among the gemini surfactants, the denaturation of BSA increases in the direction of decreasing spacer length. As the spacer length of gemini surfactant increases, the charge density decreases due to decrease in the compactness of micelle and hence reduces the electrostatic repulsion between the micelle like aggregates responsible for the denaturation of BSA. Increase in C1 with increase in spacer length of gemini surfactants also decreases the
Fig. 7. Variation of ellipticity of BSA with the concentration CTAB and C16 Cs C16 Br2 (s = 4, 5, 6) at physiological pH and 25 ◦ C.
number of micelle like aggregates at a given surfactant concentration available for denaturation of BSA. Fig. 7 depicts the variation of denaturation of BSA as a function of concentration of CTAB and C16 Cs C16 Br2 . BSA denatures more and more with increase in surfactant concentration and then attains a constant value. From the inset of the figure, it is clearly shown that lower spacer gemini are more effective in denaturation of BSA, due to the above mentioned reasons. 4. Conclusions The study reveals that, compared to single chain surfactants, gemini surfactants interact strongly with BSA. Like other properties of gemini surfactants, their interaction with BSA is also dependent on the spacer length. Our multi-technique approach showed that the interaction of gemini surfactants with BSA decreases with increase in spacer length. The decrease in interaction has been attributed to increase in C1 with increase in spacer length/head group hydrophobicity of the gemini surfactants. Keeping in view the results presented, the present study may prove fruitful in selection of gemini surfactants for solubilizing agents to recover proteins from the inclusion bodies, efficient and inexpensive folding catalysts, skin care formulations as they act at lower concentrations compared to conventional surfactants. Acknowledgements We are thankful to Head, Department of Chemistry, University of Kashmir, for providing the laboratory facilities and his constant encouragement and inspiration. MAM (JRF) acknowledges the financial support [File No. 09/251(0021)/2008-EMR-I], from the Council of Scientific and Industrial Research, India. References
Fig. 6. Far-UV CD spectra of native BSA and in presence of 0.1 mM CTAB, 0.1 mM C16 C4 C16 Br2 at physiological pH and 25 ◦ C.
[1] K.P. Ananthapadmanabhan, in: E.D. Goddard, K.P. Ananthapadmanabhan (Eds.), Interactions of Surfactants with Polymers and Proteins, CRC Press, Inc., London, UK, 1993, Chapter 8. [2] J.A. Reynolds, C. Tanford, J. Biol. Chem. 245 (1970) 5161. [3] C. Tanford, J. Mol. Biol. 67 (1972) 59. [4] K. Shirahama, K. Tsujii, T. Takagi, J. Biochem. (Tokyo) 75 (1974) 309. [5] M.N. Jones, P. Manley, J. Chem. Soc. Faraday Trans. 1 75 (1979) 1736. [6] M.N. Jones, Biochem. J. 151 (1975) 109. [7] P.C. Griffiths, J.A. Roe, B.L. Bales, A.R. Pitt, A.M. Howe, Langmuir 16 (2000) 8248. [8] N.J. Turro, X.G. Lie, K.P. Ananthapadmanabhan, M. Aronson, Langmuir 11 (1995) 2525.
M.A. Mir et al. / Colloids and Surfaces B: Biointerfaces 77 (2010) 54–59 [9] D. Kelley, D.J. McClements, Food Hydrocolloids 17 (2003) 73. [10] A. Valstar, M. Almgren, W. Brown, Langmuir 16 (2000) 922. [11] C. Honda, H. Kamizono, K. Matsumoto, K. Endo, J. Colloid Interf. Sci. 278 (2004) 310. [12] S.F. Santos, D. Zanette, H. Fischer, R. Itri, J. Colloid Interf. Sci. 262 (2003) 400. [13] A. Wangsakan, P. Chinachoti, D.J. McClements, Langmuir 20 (2004) 3913. [14] A.V. Few, R.H. Ottewill, H.C. Parreira, Biochim. Biophys. Acta 18 (1955) 136. [15] Y. Nozaki, J.A. Reynolds, C. Tanford, J. Biol. Chem. 249 (1974) 4452. [16] M.N. Jones, Chem. Soc. Rev. 21 (1992) 127. [17] F.M. Menger, C.A. Littau, J. Am. Chem. Soc. 113 (1991) 1451. [18] M.J. Rosen, Chemtech 23 (1993) 30. [19] F.M. Menger, C.A. Littau, J. Am. Chem. Soc. 115 (1993) 10083. [20] R. Zana, J. Xia, Gemini Surfactants, Marcel Dekker, New York, 2004. [21] R. Zana, J. Xia, Gemini Surfactants, Marcel Dekker, New York, 2003. [22] R. Zana, Adv. Colloid Interf. Sci. 97 (2002) 205. [23] U.S. Siddiqui, G. Ghosh, Kabir-ud-Din, Langmuir 22 (2006) 9874. [24] S.D. Wettig, R.E. Verrall, M. Foldvari, Curr. Gene Ther. 8 (2008) 9. [25] Y. Li, X. Wang, Y. Wang, J. Phys. Chem. B 110 (2006) 8499. [26] Y. Pi, Y. Shang, C. Peng, H. Liu, Y. Hu, J. Jiang, Biopolymers 83 (2006) 243. [27] D. Wu, G. Xu, Y. Feng, Y. Li, Int. J. Biol. Macromol. 40 (2007) 345. [28] D. Wu, G. Xu, Y. Sun, H. Zhang, H. Mao, Y. Feng, Biomacromolecules 8 (2007) 708. [29] N. Gull, P. Sen, R.H. Khan, Kabir-ud-Din, J. Biochem. 145 (2009) 67. [30] N. Gull, P. Sen, R.H. Khan, Kabir-ud-Din, Langmuir 25 (2009) 11686. [31] R. Zana, J. Colloid Interf. Sci. 248 (2002) 203. [32] D. Danino, Y. Talmon, R. Zana, Langmuir 11 (1995) 1448. [33] S.K. Hait, S.P. Moulik, Curr. Sci. 82 (May (9)) (2002). [34] M.J. Rosen, D.J. Tracy, J. Surfactants Deterg. 1 (October (4)) (1998). [35] T.J. Peters, in: T.J. Peter (Ed.), All about Albumin Biochemistry, Genetics, and Medical Applications, Academic Press, San Diego, CA, 1996.
59
[36] C. Giancola, C. De Sena, D. Fessas, G. Graziano, G. Barone, Int. J. Biol. Macromol. 20 (1997) 193. [37] S. Magdassi, Y. Vinetsky, P. Relkin, Colloids Surf. B 6 (1996) 353. [38] Kabir-ud-Din, W. Fatma, Z.A. Khan, A.A. Dar, J. Phys. Chem. B 111 (2007) 8860. [39] K. Kabir-ud-Din, M.S. Sheikh, A.A. Dar, J. Colloid Interf. Sci. 333 (2009) 605. [40] A.A. Dar, G.M. Rather, A.R. Das, J. Phys. Chem. B 111 (2007) 3122. [41] De. Soma, V.K. Aswal, S. Prem, G.S. Bhattacharya, J. Phys. Chem. 100 (1996) 11664. [42] Kabir-ud-Din, U.S. Siddiqui, S. Kumar, A.A. Dar, Colloid Polym. Sci. 284 (2006) 807. [43] T. Chakraborty, I. Chakraborty, S.P. Moulik, S. Ghosh, Langmuir 25 (2009) 3062. [44] C. Ted Lee, K.A. Smith, T.A. Hatton, Biochemistry 44 (2005) 524. [45] R. Miller, V.B. Fainerman, A.V. Makievski, J. Kragel, D.O. Grigoriev, N. Kazakov, O.V. Sinyachenko, Adv. Colloid Interf. Sci. 86 (2000) 39. [46] V.B. Fainerman, S.A. Zholob, M.E. Leser, M. Michel, R. Miller, J. Phys. Chem. 108 (2004) 16780. [47] Q. Zhou, M.J. Rosen, Langmuir 19 (2003) 4555. [48] D.K. Chattoraj, R.P. Pal, Indian J. Chem. 10 (1972) 410. [49] E. Alami, G. Beinert, P. Marie, R. Zana, Langmuir 9 (1993) 1465. [50] Z.X. Li, C.C. Dong, R.K. Thomas, Langmuir 15 (1999) 4392. [51] Z.X. Li, C.C. Dong, J.B. Wang, R.K. Thomas, Langmuir 18 (2002) 6614. [52] L.D. Song, M.J. Rosen, Langmuir 12 (1996) 1149. [53] J.H. Mathias, M.J. Rosen, L. Davenport, Langmuir 17 (2001) 6148. [54] J. Xia, H. Zhang, D.R. Rigsbee, P.L. Dubin, T. Shaikh, Macromolecules 26 (1993) 2759. [55] S. Deep, J.C. Ahluwalia, Phys. Chem. Chem. Phys. 3 (2001) 4583. [56] J. Carbin, N. Methlot, H.H. Wang, J.E. Baenziger, M.P. Blanton, J. Biol. Chem. 273 (1998) 771. [57] R.F. Chen, Arch. Biochem. Biophys. 160 (1974) 106.
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Effect of spacer length of alkanediyl-␣,-bis(dimethylcetylammonium bromide) gemini homologues on the interfacial and physicochemical properties of BSA Mohammad Amin Mir a , Javeed Masood Khan b , Rizwan Hasan Khan b , Ghulam Mohammad Rather a , Aijaz Ahmad Dar a,∗ a b
Department of Chemistry, University of Kashmir, Srinagar 190006, J&K, India Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, India
a r t i c l e
i n f o
Article history: Received 26 September 2009 Received in revised form 4 January 2010 Accepted 7 January 2010 Available online 14 January 2010 Keywords: BSA Interfacial tension Circular dichroism Gemini surfactant Denaturation
a b s t r a c t The interactions of bovine serum albumin (BSA) with cationic gemini surfactants alkanediyl␣,-bis(dimethylcetylammonium bromide) (designated as C16 Cs C16 Br2 , s = 4, 5, and 6) and single chain surfactant cetyltrimethylammonium bromide (CTAB) have been investigated with tensiometry, Rayleigh’s scattering, fluorescence spectroscopy, and circular dichroism at physiological pH and 25 ◦ C. The results of the multi-technique approach showed that the gemini surfactants interact more efficiently with the proteins than their conventional single chain counterparts and their efficiency increases with decrease in the length of the spacer. The saturation in interfacial tension occurred at a lower concentration in presence of BSA compared to CMC of the surfactants in absence of BSA and the concentration of gemini surfactants corresponding to interfacial saturation decreases with decrease in the spacer length. Fluorescence and circular dichroism spectroscopy results revealed increase in unfolding of BSA with decrease in spacer length of gemini surfactants. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Protein–surfactant interactions have been a subject of extensive studies because of its relevance to the field of pharmaceutics, paints and coatings, adhesives, oil recovery, etc. [1–8]. Moreover, such studies can provide insight relevant to the solubilizing and denaturing/renaturing action of surfactants on proteins. Protein–surfactant interactions are usually dependent on the surfactant features. Compared to anionic surfactants, cationic surfactants weakly interact with the proteins as a consequence of smaller relevance of electrostatic interactions at the pHs of the interest [9–14]. However, the binding isotherms of both types of surfactants have been found to be similar [14,15]. At low surfactant concentrations ionic surfactants bound to the oppositely charged sites of proteins which cause the protein to unfold resulting in the exposure of more binding sites. As the surfactant concentration is increased the binding becomes cooperative and ultimately the protein is saturated by the surfactants and their aggregates [16]. Cationic gemini surfactants are made up of two hydrophobic chains and two polar head groups covalently linked through a spacer group [17–20]. These kinds of surfactants have number of unique aggregation properties such as lower CMC and kraft
∗ Corresponding author. E-mail address: aijaz [email protected] (A.A. Dar). 0927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2010.01.005
temperature, special aggregation morphology, strong hydrophobic microdomains, etc. [21–24]. Because of these unique properties gemini surfactants strongly interact with the proteins [25–30] compared to conventional ones. The unusual properties of aggregates of gemini surfactants are related to spacer structure because it influences the distances between head groups in the aggregate [31]. In aggregates formed by conventional surfactants, the only distance is the thermodynamic distance between polar head groups, whereas in aggregates formed by gemini, two head group distance control the aggregation properties (bimodal distribution), one corresponding to the single chain surfactant equilibrium distance and one corresponding to the length and nature of the spacer [32]. Various applications of the gemini surfactants relevant to the surfactant–protein interactions viz., antimicrobial, hair conditioning, skin and eye care, etc., are important over the other conventional surfactants [33,34]. The effectiveness of gemini surfactants depends upon the length and type of spacer, and the hydrophobic counterpart. In spite of that, the effect of spacer length of the gemini surfactant on the surfactant–protein interaction has not been given the due attention. Although, Li et al. [25] studied the interaction of BSA with short alkyl chain DTAB and its gemini homologues (C12 Cs C12 Br2 , where s = 3, 6, 12) but due to the large differences and lack of continuity in the spacer lengths, a critical analysis escaped. We hereby report the interaction of BSA with long alkyl chain CTAB and its gemini homologues (C16 Cs C16 Br2 , where s = 4, 5, 6)
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Fig. 1. Tensiometric dilution (squares) and interaction (circles) profiles for CTAB (A), C16 C4 C16 Br2 (B), C16 C5 C16 Br2 (C), and C16 C6 C16 Br2 (D).
using tensiometry, spectrofluorometry and circular dichroism at physiological pH and 25 ◦ C. Gemini surfactants with comparatively rigid and closely related spacer lengths were selected to have meaningful insight into the effect of spacer length/bimodal distribution of gemini surfactants on the surfactant–protein interactions. BSA, a globular protein, has a molar mass of 66.4 kDa and posses about 583 amino acids [35]. It has relatively high water solubility because of its larger number of ionizable amino acids [36]. BSA can bind many different types of biological amphiphilic molecules, which are believed to play an important role in determining the physiological functions [37]. 2. Experimental 2.1. Materials Bovine serum albumin (BSA, Sigma) and cetyltrimethylammonium bromide (CTAB, Sigma) were used as received. The gemini bis(cetyldimethylammonium)butane dibromide, C16 H33 (CH3 )2 N+ –(CH2 )s –N+ (CH3 )2 C16 H33 .2Br− , (where s = 4, 5, 6) were synthesized and characterized as described elsewhere [38]. All the stock solutions of BSA, CTAB, gemini surfactants (C16 Cs C16 Br2 ) were prepared in 60 mM phosphate buffer of pH 7.4 with triple distilled water and utilized to prepare the samples of
desired concentrations. The concentration of the BSA was determined by measuring the absorbance of protein at 280 nm on a Hitachi U-1500 spectrophotometer and kept constant (0.2 mg/mL) throughout the study. 2.2. Methods 2.2.1. Tensiometry Surface tension measurements were made with a Kruss 9 tensiometer by the Whilhemy plate method. 10 mL of 0.2 mg/mL BSA solution in phosphate buffer was taken in sample vessel and the desired concentrations of buffered surfactant solutions was added in small installments using a Hamilton microsyringe, and readings were taken after thorough mixing and temperature equilibration. No more than 1.0 mL of surfactant solution was added to BSA solution. The temperature was maintained at the 25 ◦ C within 0.1 ◦ C by circulating water from a HAAKE GH thermostat. The accuracy of measurements was within (0.1 dyn cm−1 ). 2.2.2. Aggregation studies Rayleigh’s scattering measurements were performed by observing emission at 350 nm after exciting at same wavelength on Hitachi spectrofluorometer (model 2500) equipped with a PC. The
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fluorescence spectra were collected at 25 ◦ C with a 1 cm pathlength cell. 2.2.3. Fluorescence measurements The fluorescence spectra were collected at 25 ◦ C with a 1 cm path-length cell using a Hitachi spectrofluorimeter (model 2500) equipped with a PC. The excitation and emission slits were set at 5 nm. The reference sample consisting of the buffer and the detergent did not give any fluorescence signal. Intrinsic fluorescence was measured by exciting the protein solution at 280 and 295 nm and the emission spectra were recorded in the range of 300–400 nm. 2.2.4. Circular dichroism CD measurements were carried out with a Jasco spectropolarimeter, model J-720, equipped with a microcomputer. The instrument was calibrated with D-10-camphorsulfonic acid. All the CD measurements were carried out at 25 ◦ C with a thermostatically controlled cell holder attached to a Neslab RTE-110 water bath with an accuracy of ±0.1 ◦ C. Far-UV CD spectra were acquired with use of a cell of 1 mm path length over the wavelength range between 200 and 250 nm. A reference sample containing buffer and the detergent was subtracted from the CD signal for all measurements. The high-tension voltage for the spectra obtained was found to be less than 600 V. Spectra were collected with a scan speed of 20 nm/min and response time of 1 s. Each spectrum is the average of four scans 3. Results and discussion 3.1. Tensiometry In tensiometry the threshold surfactant concentration required to saturate the air/solution interface is called the critical micelle concentration (CMC), which corresponds to the break point in the surface tension () versus log [surfactant] plot. Tensiometric profiles for the addition of CTAB and C16 Cs C16 Br2 (s = 4, 5, 6) to buffer solution under identical experimental conditions are illustrated with squares in Fig. 1 and the corresponding CMC values are given in Table 1. The ionic strength of the buffer solution lowered the CMC of the CTAB and C16 Cs C16 Br2 compared to that in aqueous solution [39–41], by way of decreasing the electrostatic repulsion between the charged head groups. The CMC values of gemini surfactants increase with increase in the spacer chain length due to increase in the head group area [22,41,42]. The tensiometric interaction profiles for the CTAB-BSA and C16 Cs C16 Br2 -BSA are included in Fig. 1(circles). The tensiometric profiles of surfactants in presence of BSA show lower surface tension values than the tensiometric profiles of surfactants in absence of BSA, due to the surface activity of BSA ( = 56 mN/m for 0.2 mg/mL BSA solution). The tensiometric profile for CTAB and C16 Cs C16 Br2 in presence of BSA consists of a single break at a lower concentration compared to their CMC values in the buffer solution. This concentration corresponding to the saturation in interfacial tension is denoted by C1 and marks the concentration above which the aggregation of surfactants on the BSA backbone occurs [43]. At physiological pH, BSA has an overall negative charge [44]. The interaction between
proteins and ionic surfactants at the interface is dominated by the electrostatic interactions [45,46]. With increase in concentration of CTAB and C16 C4 C16 Br2 the available charges in the protein molecules are compensated by the oppositely charged surfactant ions, thus forming electroneutral complexes of enhanced surface activity compared to native protein [45,46]. The surface excess concentration ( max ) in mol m−2 at CMC or C1 and minimum surface area per molecule (Amin ) in nm2 at air/solution interface both in absence as well as presence of BSA were calculated from the equations [47] max = −
Amin =
1 lim 2.303nRT C→CMC/C1
1018 Nmax
d d log C
(1)
(2)
where C is the total surfactant concentration, N the Avogadro’s constant and R and T have their usual meanings. n is the number of species whose interfacial concentration changes with the change in bulk phase concentration of surfactant. Because of swamping amount of electrolyte used (60 mM buffer), n can be assumed to be unity for all the surfactant systems [43,46,48]. The variation of 1 ACMC /ACmin with the spacer length of the gemini surfactant is premin 1 sented in Fig. 2. Lower values of ACMC than the ACmin signify less min compact monolayer in presence of BSA. This indicates the presence of BSA–surfactant complexes at the air/solution interface [43]. Fig. 2 indicates that gemini surfactants, both in absence and presence of BSA, form more compact monolayer compared to single chain surfactants but the compactness decreases slightly with increase in the spacer length due to increase in head group area. However, the ACMC values of gemini surfactants were found much lower than the min reported ACMC values for gemini surfactants [49,31]. The discrepmin ancies in ACMC values of gemini surfactants are related to the value min of n in Eq. (1). The exact value of n has been found to be function of state of the surfactant in the solution below CMC, nature of the surfactant and concentration of the surfactant in the submicellar range [50,51]. Therefore, very small ACMC values in our case may min be due to cumulative effect of screening of electrostatic repulsion between the gemini head groups by dense ionic atmosphere furnished by 60 mM phosphate buffer, use of n = 1 instead of 2 or 3 and formation of premicellar aggregates [52,53] which induce abrupt decrease in surface tension near CMC.
Table 1 Critical micelle concentration (CMC) and the concentration corresponding to saturation in interfacial tension in presence of BSA (C1 ) at physiological pH and 25 ◦ C. Surfactant
CMC (mM)
C1 (mM)
CTAB C16 C4 C16 Br2 C16 C5 C16 Br2 C16 C6 C16 Br2
0.2300 0.0052 0.0058 0.0063
0.0890 0.0048 0.0050 0.0056
1 in absence and presence of BSA with the spacer length Fig. 2. Variation of ACMC /ACmin min of the gemini surfactants at physiological pH and 25 ◦ C.
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Fig. 3. Fluorescence spectra (excited at 350 nm) of BSA in native state and in presence of 0.1 mM CTAB, 0.1 mM C16 Cs C16 Br2 (s = 4, 5, 6) at physiological pH and 25 ◦ C.
Fig. 5. Fluorescence spectra (excited at 280 nm) of BSA in native state and in presence of 0.1 mM CTAB, 0.1 mM C16 Cs C16 Br2 (s = 4, 5, 6) at physiological pH and 25 ◦ C.
3.2. Aggregation studies
tryptophan residues get excited and are used to probe changes in their respective microenvironments and provide a picture of local change in the protein. At 280 nm both tryptophan as well as tyrosine residues get excited and provide a picture of global change in the protein. The change in the wavelength of maximum emission by excinm , is illustrated in Fig. 4 and indicate that tation at 280 nm, 280 max BSA exhibits a blue shift in presence of the surfactants. The blue shift increases with increase in the concentration of the surfactant and then attains a constant value at higher surfactant concentranm with increase in surfactant concentration tions. Decrease in 280 max indicates shift of fluorophores towards more hydrophobic environnm thereafter means no change in ment [55]. The constancy in 280 max the hydrophobicity and suggests saturation of BSA backbone by the micelles, in corroboration with the binding isotherms suggested for protein–surfactant interactions [14]. Although the decreasing nm is similar in both CTAB and C C C Br , but the trend of 280 16 s 16 2 max nm is more when the BSA interacts with rate of decrease of 280 max nm (Fig. 5) C16 Cs C16 Br2 than the CTAB. The rate of decrease of 280 max of BSA slows down as the of the spacer length of the gemini surfactant increases. These changes in the microenvironment of BSA are harmonious with the variation of C1 as a function of spacer length of gemini surfactants.
The change in the scattering intensities at 350 nm of BSA mainly arises from the change in number and/or size of BSA–surfactant aggregates [54]. Fig. 3 illustrates the effect of surfactant on the BSA–surfactant aggregation at a particular surfactant concentration (0.1 mM). In conformity with the earlier reports [25,29,30], gemini surfactants, because of their unique aggregation properties, interact efficiently with BSA than their single chain counterparts. It is clear from the Fig. 3 that the interaction of gemini surfactants with the BSA decreases with increase in the spacer length. These results are in agreement with the tensiometric results that the C1 increases with increase in spacer length of gemini surfactants. Lower C1 implies earlier aggregation of surfactant with BSA and hence large number or increased size of BSA–surfactant aggregates at a given surfactant concentration. 3.3. Intrinsic fluorescence The change in the wavelength of emission maximum (max ), a parameter sensitive to the protein conformations and fluorescence intensities at 340 nm by excitation at 280 and 295 nm can be used to examine the protein–surfactant interactions [55]. At 295 nm only
nm nm Fig. 4. Variation of 280 (left) and 295 (right) of BSA with the concentration of CTAB and C16 Cs C16 Br2 (s = 4, 5, 6) gemini surfactants at physiological pH and 25 ◦ C. max max
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The less significant blue shift at the excitation wavelength of 295 nm than at 280 nm is attributed to the fact that the tryptophan residues are buried in the inner core of BSA while the tyrosine residues are distributed throughout. Binding of surfactant to BSA increases the hydrophobic environment around the fluorophores resulting in decreased fluorescence intensity (FI). The variations nm , is found to folin the wavelength of emission maximum, 280 max low the fluorescence intensity (FI) changes faithfully. A decrease nm is observed as the FI decreases as shown in Fig. 5 for in 280 max 0.1 mM surfactant concentration. Gemini surfactants because of their strong hydrophobic microdomains are more effective as comnm of BSA. Like 280 nm , pared to CTAB in decreasing the FI and 280 max max FI intensity of BSA decreases as the spacer length of the gemini surnm and FI suggest decrease factant decreases. These changes in 280 max in hydrophobic environment around the fluorophores of BSA with increase in spacer length of the gemini surfactants. Decrease in hydrophobic environment is attributed to decrease in number of micelle like clusters around the fluorescent residues of BSA due to increase in C1 with the spacer length. 3.4. Far-UV circular dichroism Circular dichroism can be used to probe transitions in the secondary structure of the proteins. BSA exhibits two negative bands in the far-UV CD spectrum at 208 and 222 nm, characteristic of ˛-helical structure [56]. Alterations in ellipticity at 222 nm ( 222 ) are used to monitor the change in ˛-helical content of BSA [57] by CTAB and C16 C4 C16 Br2 . Fig. 6 shows typical far-UV CD spectra of native BSA and BSA in presence of 0.1 mM CTAB and 0.1 mM C16 Cs C16 Br2 (where s = 4, 5, 6) at physiological pH. It is clear from the figure that the CTAB as well as C16 Cs C16 Br2 denature BSA but the gemini surfactants are more effective than CTAB. These results are in conformity with previous results [25,29,30] and have been attributed to the unique characteristics of gemini surfactants. The denaturation of BSA by the ionic surfactants arises from the replacement of hydrophobic interaction in the protein by the electrostatic repulsion between the micelle like aggregates formed on the BSA [30]. Among the gemini surfactants, the denaturation of BSA increases in the direction of decreasing spacer length. As the spacer length of gemini surfactant increases, the charge density decreases due to decrease in the compactness of micelle and hence reduces the electrostatic repulsion between the micelle like aggregates responsible for the denaturation of BSA. Increase in C1 with increase in spacer length of gemini surfactants also decreases the
Fig. 7. Variation of ellipticity of BSA with the concentration CTAB and C16 Cs C16 Br2 (s = 4, 5, 6) at physiological pH and 25 ◦ C.
number of micelle like aggregates at a given surfactant concentration available for denaturation of BSA. Fig. 7 depicts the variation of denaturation of BSA as a function of concentration of CTAB and C16 Cs C16 Br2 . BSA denatures more and more with increase in surfactant concentration and then attains a constant value. From the inset of the figure, it is clearly shown that lower spacer gemini are more effective in denaturation of BSA, due to the above mentioned reasons. 4. Conclusions The study reveals that, compared to single chain surfactants, gemini surfactants interact strongly with BSA. Like other properties of gemini surfactants, their interaction with BSA is also dependent on the spacer length. Our multi-technique approach showed that the interaction of gemini surfactants with BSA decreases with increase in spacer length. The decrease in interaction has been attributed to increase in C1 with increase in spacer length/head group hydrophobicity of the gemini surfactants. Keeping in view the results presented, the present study may prove fruitful in selection of gemini surfactants for solubilizing agents to recover proteins from the inclusion bodies, efficient and inexpensive folding catalysts, skin care formulations as they act at lower concentrations compared to conventional surfactants. Acknowledgements We are thankful to Head, Department of Chemistry, University of Kashmir, for providing the laboratory facilities and his constant encouragement and inspiration. MAM (JRF) acknowledges the financial support [File No. 09/251(0021)/2008-EMR-I], from the Council of Scientific and Industrial Research, India. References
Fig. 6. Far-UV CD spectra of native BSA and in presence of 0.1 mM CTAB, 0.1 mM C16 C4 C16 Br2 at physiological pH and 25 ◦ C.
[1] K.P. Ananthapadmanabhan, in: E.D. Goddard, K.P. Ananthapadmanabhan (Eds.), Interactions of Surfactants with Polymers and Proteins, CRC Press, Inc., London, UK, 1993, Chapter 8. [2] J.A. Reynolds, C. Tanford, J. Biol. Chem. 245 (1970) 5161. [3] C. Tanford, J. Mol. Biol. 67 (1972) 59. [4] K. Shirahama, K. Tsujii, T. Takagi, J. Biochem. (Tokyo) 75 (1974) 309. [5] M.N. Jones, P. Manley, J. Chem. Soc. Faraday Trans. 1 75 (1979) 1736. [6] M.N. Jones, Biochem. J. 151 (1975) 109. [7] P.C. Griffiths, J.A. Roe, B.L. Bales, A.R. Pitt, A.M. Howe, Langmuir 16 (2000) 8248. [8] N.J. Turro, X.G. Lie, K.P. Ananthapadmanabhan, M. Aronson, Langmuir 11 (1995) 2525.
M.A. Mir et al. / Colloids and Surfaces B: Biointerfaces 77 (2010) 54–59 [9] D. Kelley, D.J. McClements, Food Hydrocolloids 17 (2003) 73. [10] A. Valstar, M. Almgren, W. Brown, Langmuir 16 (2000) 922. [11] C. Honda, H. Kamizono, K. Matsumoto, K. Endo, J. Colloid Interf. Sci. 278 (2004) 310. [12] S.F. Santos, D. Zanette, H. Fischer, R. Itri, J. Colloid Interf. Sci. 262 (2003) 400. [13] A. Wangsakan, P. Chinachoti, D.J. McClements, Langmuir 20 (2004) 3913. [14] A.V. Few, R.H. Ottewill, H.C. Parreira, Biochim. Biophys. Acta 18 (1955) 136. [15] Y. Nozaki, J.A. Reynolds, C. Tanford, J. Biol. Chem. 249 (1974) 4452. [16] M.N. Jones, Chem. Soc. Rev. 21 (1992) 127. [17] F.M. Menger, C.A. Littau, J. Am. Chem. Soc. 113 (1991) 1451. [18] M.J. Rosen, Chemtech 23 (1993) 30. [19] F.M. Menger, C.A. Littau, J. Am. Chem. Soc. 115 (1993) 10083. [20] R. Zana, J. Xia, Gemini Surfactants, Marcel Dekker, New York, 2004. [21] R. Zana, J. Xia, Gemini Surfactants, Marcel Dekker, New York, 2003. [22] R. Zana, Adv. Colloid Interf. Sci. 97 (2002) 205. [23] U.S. Siddiqui, G. Ghosh, Kabir-ud-Din, Langmuir 22 (2006) 9874. [24] S.D. Wettig, R.E. Verrall, M. Foldvari, Curr. Gene Ther. 8 (2008) 9. [25] Y. Li, X. Wang, Y. Wang, J. Phys. Chem. B 110 (2006) 8499. [26] Y. Pi, Y. Shang, C. Peng, H. Liu, Y. Hu, J. Jiang, Biopolymers 83 (2006) 243. [27] D. Wu, G. Xu, Y. Feng, Y. Li, Int. J. Biol. Macromol. 40 (2007) 345. [28] D. Wu, G. Xu, Y. Sun, H. Zhang, H. Mao, Y. Feng, Biomacromolecules 8 (2007) 708. [29] N. Gull, P. Sen, R.H. Khan, Kabir-ud-Din, J. Biochem. 145 (2009) 67. [30] N. Gull, P. Sen, R.H. Khan, Kabir-ud-Din, Langmuir 25 (2009) 11686. [31] R. Zana, J. Colloid Interf. Sci. 248 (2002) 203. [32] D. Danino, Y. Talmon, R. Zana, Langmuir 11 (1995) 1448. [33] S.K. Hait, S.P. Moulik, Curr. Sci. 82 (May (9)) (2002). [34] M.J. Rosen, D.J. Tracy, J. Surfactants Deterg. 1 (October (4)) (1998). [35] T.J. Peters, in: T.J. Peter (Ed.), All about Albumin Biochemistry, Genetics, and Medical Applications, Academic Press, San Diego, CA, 1996.
59
[36] C. Giancola, C. De Sena, D. Fessas, G. Graziano, G. Barone, Int. J. Biol. Macromol. 20 (1997) 193. [37] S. Magdassi, Y. Vinetsky, P. Relkin, Colloids Surf. B 6 (1996) 353. [38] Kabir-ud-Din, W. Fatma, Z.A. Khan, A.A. Dar, J. Phys. Chem. B 111 (2007) 8860. [39] K. Kabir-ud-Din, M.S. Sheikh, A.A. Dar, J. Colloid Interf. Sci. 333 (2009) 605. [40] A.A. Dar, G.M. Rather, A.R. Das, J. Phys. Chem. B 111 (2007) 3122. [41] De. Soma, V.K. Aswal, S. Prem, G.S. Bhattacharya, J. Phys. Chem. 100 (1996) 11664. [42] Kabir-ud-Din, U.S. Siddiqui, S. Kumar, A.A. Dar, Colloid Polym. Sci. 284 (2006) 807. [43] T. Chakraborty, I. Chakraborty, S.P. Moulik, S. Ghosh, Langmuir 25 (2009) 3062. [44] C. Ted Lee, K.A. Smith, T.A. Hatton, Biochemistry 44 (2005) 524. [45] R. Miller, V.B. Fainerman, A.V. Makievski, J. Kragel, D.O. Grigoriev, N. Kazakov, O.V. Sinyachenko, Adv. Colloid Interf. Sci. 86 (2000) 39. [46] V.B. Fainerman, S.A. Zholob, M.E. Leser, M. Michel, R. Miller, J. Phys. Chem. 108 (2004) 16780. [47] Q. Zhou, M.J. Rosen, Langmuir 19 (2003) 4555. [48] D.K. Chattoraj, R.P. Pal, Indian J. Chem. 10 (1972) 410. [49] E. Alami, G. Beinert, P. Marie, R. Zana, Langmuir 9 (1993) 1465. [50] Z.X. Li, C.C. Dong, R.K. Thomas, Langmuir 15 (1999) 4392. [51] Z.X. Li, C.C. Dong, J.B. Wang, R.K. Thomas, Langmuir 18 (2002) 6614. [52] L.D. Song, M.J. Rosen, Langmuir 12 (1996) 1149. [53] J.H. Mathias, M.J. Rosen, L. Davenport, Langmuir 17 (2001) 6148. [54] J. Xia, H. Zhang, D.R. Rigsbee, P.L. Dubin, T. Shaikh, Macromolecules 26 (1993) 2759. [55] S. Deep, J.C. Ahluwalia, Phys. Chem. Chem. Phys. 3 (2001) 4583. [56] J. Carbin, N. Methlot, H.H. Wang, J.E. Baenziger, M.P. Blanton, J. Biol. Chem. 273 (1998) 771. [57] R.F. Chen, Arch. Biochem. Biophys. 160 (1974) 106.