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Interaction of Bovine (BSA), Rabbit (RSA), and Porcine (PSA) Serum Albumins with Cationic Single-Chain/Gemini Surfactants: A Comparative Study Nuzhat Gull,† Priyankar Sen,‡ Rizwan Hasan Khan,‡ and Kabir-ud-Din*,† †
Department of Chemistry and ‡Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202 002, India Received May 8, 2009. Revised Manuscript Received June 10, 2009
The interactions among bovine, rabbit, and porcine serum albumins and single-chain cationic surfactant cetyltrimethylammonium bromide (CTAB) versus its gemini counterpart (designated as G4) have been studied. The studies were carried out in an aqueous medium at pH 7.0 using UV, intrinsic and extrinsic fluorescence spectroscopy, and far-UV circular dichroism techniques. The results indicate that compared to CTAB, G4 interacts strongly with the serum albumins, resulting in a significantly larger unfolding or decrease in R-helical content as reflected by the significantly larger decrease in ellipticity in the far-UV range. Unlike CTAB, a remarkable increase in the R-helical content of BSA at 625 μM G4 and at 250 μM G4 for RSA and PSA is observed. The appearance of conformational changes and saturation points in the proteins occurs at considerably lower [G4] compared to [CTAB]. The results obtained from the multi-technique approach are ascribed to the stronger forces in G4 owing to the presence of two charged headgroups and two hydrocarbon tails. Keeping the results in view, it is suggested that the gemini surfactants may be effectively used in the renaturation of proteins produced in genetically engineered cells via the artificial chaperone protocol and may also prove useful in drug delivery as solubilizing agents to recover proteins from insoluble inclusion bodies.
Introduction Proteins are the most abundant and versatile macromolecules in living systems and serve crucial functions in essentially all biological processes. Globular proteins are used as functional ingredients in health care and pharmaceutical products. They are known to catalyze biochemical reactions and to bind to other molecules and form molecular aggregates.1 The function of a protein is directly dependent on its 3D structure. The interaction of proteins with surfactants has been studied extensively2,3 ever since surfactants were found to be strong denaturants of water-soluble proteins.4 These interactions are important in a wide variety of industrial, biological, pharmaceutical, and cosmetic systems and also contribute to the understanding of the action of surfactants as solubilizing agents to recover proteins from inclusion bodies and in the renaturation of the proteins produced in the genetically engineered cells via the artificial chaperone protocol.5-13 *Corresponding author. E-mail:
[email protected]. (1) Food Emulsions; Sjoblom, J., Ed.; Marcel Dekker: New York, 1996; p 287. (2) Guo, H.; Zhao, N. M.; Chen, S. H.; Teixeira, J. Biopolymers 1990, 29, 335. (3) Tanford, C. The Hydrophobic Effect: Formation of Micelles and Biological Membranes, 2nd ed.; Wiley Interscience: New York, 1980; p 63. (4) Anson, M. L. Science 1939, 90, 256. (5) De, S.; Girigoswami, A.; Das, S. J. Colloid Interface Sci. 2005, 285, 562. (6) Vasilescu, M.; Angelescu, D.; Almgren, M.; Valstar, A. Langmuir 1999, 15, 2635. (7) Kamat, B. P.; Seetharamappa, J. J. Pharm. Biomed. Anal. 2004, 35, 655. (8) Khodagholi, F.; Eftekharzadeh, B.; Yazdanparast, R. Protein J. 2008, 27, 123. (9) Sulkowska, A. J. Mol. Struct. 2002, 614, 227. (10) Kelley, D.; McClements, D. J. Food Hydrocolloids 2003, 17, 73. (11) Sun, C.; Yang, J.; Wu, X.; Huang, X.; Wang, F.; Liu, S. Biophys. J. 2005, 88, 3518. (12) Protein-Based Surfactants; Nnanna, I. A., Xia, J., Eds.; Marcel Dekker: New York, 2001; p 261. (13) La Mesa, C. J. Colloid Interface Sci. 2005, 286, 148.
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Because of the hydrophobic and hydrophilic properties of the amino acids, a protein exhibits dualism that causes amphiphilic molecules to interact with it. The amphiphilic molecules chosen for the study of protein-surfactant interactions, in general, are ionic surfactants in view of their application in the area of membrane study.14,15 Cationic surfactants interact with proteins to a lesser extent compared to the anionics because of the smaller relevance of electrostatic interactions at the pH values of interest.16 However, the binding isotherms of both of the surfactants have been found to be similar.16,17 Whereas the single-chain surfactant/protein interaction has been extensively studied, the interaction of double-chain or gemini surfactants with proteins18-21 is an area that demands a thorough investigation. Gemini surfactants have stimulated considerable interest because of their unique aggregation properties in comparison to those of conventional single-chain surfactants. A gemini is a dimeric substance composed of two identical amphiphilic moieties covalently linked by a spacer group at or near the ionic headgroup.22 The spacer group increases the hydrophobicity of gemini surfactants and results in a critical micellar concentration (cmc) much lower than those of their ionic monomeric counterparts. (14) Helenius, A.; Simons, K. Biochim. Biophys. Acta 1975, 415, 29. (15) Biological Membranes; Findley, J. B. C., Evans, W. H., Eds.; IRL Press: Oxford, England, 1987; p 139. (16) Few, A. V.; Ottewill, R. H.; Parreira, H. C. Biochim. Biophys. Acta 1955, 18, 136. (17) Nozaki, Y.; Reynolds, J. A.; Tanford, C. J. Biol. Chem. 1974, 249, 4452. (18) Li, Y.; Wang, X.; Wang, Y. J. Phys. Chem. B 2006, 110, 8499. (19) Pi, Y.; Shang, Y.; Peng, C.; Liu, H.; Hu, Y.; Jiang, J. Biopolymers 2006, 83, 243. (20) Wu, D.; Xu, G.; Feng, Y.; Li, Y. Int. J. Biol. Macromol. 2007, 40, 345. (21) Wu, D.; Xu, G.; Sun, Y.; Zhang, H.; Mao, H.; Feng, Y. Biomacromolecules 2007, 8, 708. (22) Menger, F.; Littau, C. A. J. Am. Chem. Soc. 1993, 115, 10083.
Published on Web 07/09/2009
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This typical morphology imparts special characteristics to gemini surfactants such as a low Kraft temperature, low cmc, strong hydrophobic microdomain, and so forth.23-26 Referred to as second-generation surfactants, the geminis have shown promise in various potential areas of surfactant application and show stronger surface activity and better solubility, wetting, foaming, and lime soap dispersion capability than do the traditional surfactants. This study details a comparative interaction of CTAB [C16H33Nþ(CH3)3Br-] and its gemini counterpart bis(cetyldimethylammonium)butane dibromide (C16H33(CH3)2Nþ-(CH2)4Nþ(CH3)2C16H332Br-, G4) with bovine serum albumin (BSA), rabbit serum albumin (RSA), and porcine serum albumin (PSA). Each serum albumin consists of a globular protein synthesized by the liver in mammals corresponding to the most abundant protein in serum, accounting for more than 60% of the total globular protein in plasma, corresponding to a concentration of 42 g/L and providing about 80% of the osmotic pressure of the blood.27 Albumins have been used as model proteins for many diverse biophysical, biochemical, and physicochemical studies. These proteins are interesting because they bind to a variety of hydrophobic ligands such as fatty acids, lysolecithin, bilirubin, warfarin, tryptophan, steroids, anesthetics, and several dyes.28-31 They play an important role in the transport and deposition of a variety of endogenous and exogenous substances in the blood32 as a result of the existence of a limited number of binding regions of very different specificity.33 Serum albumins are also used in peritoneal dialysis in combating the harmful effect of antibiotics and as scavengers of toxic substances and free radicals.34
Experimental Section Materials. Bovine serum albumin (BSA, Sigma), rabbit serum albumin (RSA, Sigma), porcine serum albumin (PSA, Sigma), cetyltrimethylammonium bromide (CTAB, Sigma), and 1-anilino8-naphthalene sulfonic acid (ANS, Sigma) were used as received. Gemini G4 was synthesized and characterized as described elsewhere.35 The cmc in the 20 mM sodium phosphate buffer (pH 7) was determined to be 0.634 mM and 15 μM for CTAB and G4, respectively.36 All other reagents and buffer components used were of analytical grade. Doubly distilled water was used throughout the study. Stock solutions of BSA, RSA, PSA, CTAB, and G4 were prepared in 20 mM sodium phosphate buffer (pH 7) and utilized to prepare samples of the desired concentrations. The concentrations of the serum albumins were determined by measuring the absorbance of protein solutions at 280 nm on a Hitachi U-1500 spectrophotometer or alternatively by the method of Lowry et al.37 used for the measurements, which (23) Gemini Surfactants; Zana, R., Xia, J., Eds.; Marcel Dekker: New York, 2003. (24) Zana, R. Adv. Colloid Interface Sci. 2002, 97, 205. (25) Siddiqui, U. S.; Ghosh, G.; Kabir-ud-Din Langmuir 2006, 22, 9874. (26) Wettig, S. D.; Verrall, R. E.; Foldvari, M. Curr. Gene. Ther. 2008, 8, 9. (27) Peters Jr., T. All about Albumins: Biochemistry, Genetics and Medical Applications, Academic Press: San Diego CA, 1996; p 259. (28) Curry, S.; Mandelkow, H.; Brick, P.; Franks, N. Nat. Struct. Biol. 1998, 5, 827. (29) Narazaki, R.; Maruyama, T.; Otagiri, M. Biochim. Biophys. Acta 1997, 1338, 275. (30) Chadborn, N.; Bryant, J.; Bain, A. J.; O’Shea, P. Biophys. J. 1999, 76, 2198. (31) Reynolds, J. A.; Herbert, S.; Polet, H.; Steinhardt, J. Biochemistry 1967, 6, 937. (32) Kosa, T.; Maruyama, T.; Otagiri, M. Pharm. Res. 1997, 14, 1607. (33) Moreno, F.; Cortijo, M.; Gonzalez-Jimenez, J. Photochem. Photobiol. 1999, 69, 8. (34) Holt, M. E.; Ryall, M. E.; Campbell, A. K. Br. J. Exp. Pathol. 1984, 65, 231. (35) Kabir-ud-Din; Fatma, W.; Khan, Z. A.; Dar, A. A. J. Phys. Chem. B 2007, 111, 8860. (36) Gull, N.; Sen, P.; Khan, R. H.; Kabir-ud-Din J. Biochem. 2009, 145, 67. (37) Lowry, O. H.; Rosenbrough, N. J.; Farr, A. L.; Randall, R. J. J. Biol. Chem. 1951, 193, 265.
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was 1 g/dm3 for BSA and RSA and 0.6 g/dm3 for PSA. In the Lowry method, the phenolic group of tyrosine residues in a protein will produce a blue-purple color, with a maximum absorption in the region of 660 nm wavelength, with the FolinCiocalteau reagent consisting of sodium tungstate, molybdate, and phosphate. The molar concentration of the ANS used was 50 times that of the protein and was determined by measuring the absorbance at 350 nm. The pH measurements were carried out on an ELICO digital pH meter (model LI610). Turbidity Measurements. The turbidity of the surfactant/ protein solutions was monitored by UV absorbance at 350 nm using a Hitachi U-1500 spectrophotometer. A cuvette with a 1 cm path length was used. The measurements were carried out at 25 °C. A reference sample containing buffer and the detergent was measured, and the absorbance observed was negligible. CD Measurements. 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 of 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 of 200-250 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 a response time of 1s. Each spectrum is the average of four scans. The results are expressed in terms of mean residue molar ellipticity, θ, expressed in units of deg cm2 dmol-1. Data Analysis. The secondary structure was estimated from spectra between 200 and 240 nm using a K2d CD secondary structure server.38 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 emission spectra were recorded in the range of 300-400 nm. For extrinsic fluorescence measurements in the ANS binding studies, the excitation was set at 380 nm, and the emission spectra were taken in the range of 400-600 nm or at a fixed wavelength of 470 nm.
Results and Discussion Aggregation Study. The comparative effect of CTAB/G4 on the UV absorbance of BSA at 350 nm is illustrated in Figure 1. The turbidity (absorbance of BSA at 350 nm) of the protein remained unchanged in the presence of 0-40 mM CTAB, but in the presence of only 145 μM G4, it jumped to 0.6 and then dropped to 0.2 around 1000 μM. The changes in absorbance arise mainly from the change in the number and size of the aggregates formed as a result of the protein-surfactant interaction.39 The observed absorbance changes are thus associated with the formation and redissolution of protein-surfactant complexes. Thus, the results suggest that the BSA-G4 interactions are stronger than the interactions of the protein with CTAB. The very small change in the absorbance of the BSA-CTAB mixed solutions compared to the native protein is attributed to the fact that conventional cationic surfactants exhibit a small tendency to (38) Andrade, M. A.; Chacon, P.; Merelo, J. J.; Moran, F. Protein Eng. 1993, 6, 383. (39) Xia, J.; Zhang, H.; Rigsbee, D. R.; Dubin, P. L.; Shaikh, T. Macromolecules 1993, 26, 2759.
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Figure 1. Aggregation study. Variations in the absorbance of BSA at pH 7 observed at 350 nm with varying concentrations of surfactants (i.e., CTAB (b) and G4 (O)) (T=25 °C). (Inset) Variations in the absorbance of BSA at pH 7 observed at 350 nm with varying concentrations of G4 (O).
interact with proteins.2,40 Similar results were exhibited by RSA and PSA and hence are not shown. To gain insight into the interactions and to interpret them meaningfully, CD and fluorescence spectroscopy are employed. Far-UV CD. Far-UV CD spectroscopy can be used to probe transitions in the secondary structure of the proteins. The CD spectra of the native albumins exhibit two negative bands in the far-UV region at 208 and 222 nm, characteristic of the R-helical structure.41 Surfactant does not contribute to the CD signal in the range of 200-250 nm, thus the observed CD is solely due to the peptide bonds of protein. Alterations of ellipticity at 222 nm (-θ222 nm) are a useful probe for visualizing varying R-helical content.42 Figure 2A illustrates the variations in -θ222 nm with varying CTAB concentrations. The proteins show a decrease in -θ222 nm values with increasing surfactant concentration, suggesting decreased R-helical content and the resulting unfolding of the proteins. The R-helical content attains an almost constant value at a surfactant concentration of 1000 μM for RSA and PSA and 3000 μM CTAB for BSA. Variations in the ellipticity (-θ222 nm) with varying [G4] are given in Figure 2B. The ellipticity and hence the R-helical content decrease with increasing [G4], undergo a significant increase at 625 μM G4 for BSA and 250 μM G4 for RSA, and attain constant values thereafter. Although a fluctuating trend is observed in the ellipticity values of PSA, an increase in molar ellipticity after it undergoes a certain decrease is observed in this protein as well. The differences observed between the albumins are ascribed to the differences in their hydrophobicity values (130.7, 158.6, and 165.8 for BSA, RSA, and PSA, respectively) as calculated by a ProtScale tool devised by Abraham and Leo.43 Figures 3-5 show the typical far-UV-CD spectra of BSA, RSA, and PSA scanned in the wavelength range of 200-250 nm in the absence and presence of CTAB/G4. The variation of the percentage of R-helical content of BSA and RSA (Table 1) with varying CTAB/G4 concentration as determined by K2d is found to follow the same pattern as that of -MRE at 222 nm. The R-helical content decreases from 62% for native BSA to 43% as [CTAB] reaches 3000 μM and remains more or less constant thereafter. In the case of RSA, the helical content decreases from 61 to 45% as the surfactant concentration reaches 1500 μM and does not register any significant change (40) Interaction of Surfactants with Polymers and Proteins; Goddard, E. D., Ananthapadmanabhan, K. P., Eds.; CRC Press: London, 1993; p 319. (41) Carbin, J.; Methlot, N.; Wang, H. H.; Baenziger, J. E.; Blanton, M. P. J. Biol. Chem. 1998, 273, 771. (42) Chen, R. F. Arch. Biochem. Biophys. 1974, 160, 106. (43) Abraham, D. J.; Leo, A. J. Protein Struct., Funct., Genet. 1987, 2, 130.
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Figure 2. Far-UV-CD study. (A) Change in ellipticity values of BSA ((), RSA (O), and PSA (b) at pH 7 with increasing [CTAB] (T=25 °C). (B) Change in ellipticity values of BSA ((), RSA (O), and PSA (b) at pH 7 with increasing [G4] (T = 25 °C).
Figure 3. Far-UV-CD spectra of BSA. (A) Far-UV-CD spectra of BSA at pH 7 in the native state (1) and in the presence of 725 (2), 1500 (3), 3000 (4), and 14 500 μM (5) CTAB. (B) Far-UV-CD spectra of BSA at pH 7 in the native state (1) and in the presence of 62.5 (2), 250 (3), 625 (4), 935 (5), and 1250 μM G4 (6). Langmuir 2009, 25(19), 11686–11691
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Article Table 1. r-Helix Percentage of Bovine Serum Albumin (BSA) and Rabbit Serum Albumin (RSA) in the Absence and Presence of Detergents at Points of Maximum Unfolding and Refolding R-helix (%) BSA 62 (native) 43 (3000 μM [CTAB]) 30 (250 μM [G4]) 43 (625 μM [G4]) RSA 61 (native) 45 (1500 μM [CTAB]) 24 (125 μM [G4]) 45 (250 μM [G4])
Figure 4. Far-UV-CD spectra of RSA. (A) Far-UV-CD spectra of RSA at pH 7 in the native state (1) and in the presence of 300 (2), 600 (3), 1500 (4), 4300 (5), and 7250 μM CTAB (6). (B) Far-UVCD spectra of RSA at pH 7 in the native state (1) and in the presence of 19 (2), 16.5 (3),125 (4),250 (5),625 (6), and 1250 μM G4 (7).
Figure 5. Far-UV-CD spectra of PSA. (A) Far-UV-CD spectra of PSA at pH 7 in the native state (1) and in the presence of 600 (2), 3000 (3), and 7250 μM CTAB (4). (B) Far-UV-CD spectra of PSA at pH 7 in the native state (1) and in the presence of 12.5 (2), 19 (3), 125 (4), 250 (5), and 625 μM G4 (6). Langmuir 2009, 25(19), 11686–11691
afterward. The R-helical content when G4 is added to BSA decreases from 62% for native protein to 30% at 250 μM G4. The R-helical content rises to 43%, registering a 13% increase at a G4 concentration of 625 μM. In the case of RSA, the R-helical content decreases from 61 to 24% at 125 μM G4, rises to 45% at 250 μM surfactant concentration, and attains a constant value thereafter. The present investigation indicates that the albumins undergo unfolding at lower [G4] compared to [CTAB]. A significant rise in ellipticity/R-helical content is observed before the helicity attains a constant value when the protein is treated with G4 (at 625 μM for BSA and 250 μM for RSA), whereas the decrease in ellipticity is directly followed by a constant value in the case of CTAB. On the basis of these results, we conclude that the binding of the surfactant to the protein leads to unfolding by both CTAB and G4 up to a particular surfactant concentration. The more pronounced unfolding detected in G4 compared to that in CTAB is associated with the fact that the electrostatic and hydrophobic forces that control this phenomenon17 are more significant in the case of the gemini surfactant. That the conformational changes are observed at considerably lower concentrations is again attributed to the chemistry of the gemini surfactants. The increased R-helical content observed at high [G4] is attributed to the stronger hydrophobic interaction between the protein and surfactant molecules owing to the structural transition of micelles to a stronger hydrophobic core in comparison to that of CTAB.11 The self-association of the surfactant molecules in the aqueous media is very cooperative and generally starts with the formation of roughly spherical micelles. At higher surfactant concentrations, nonspherical micelles, such as the rod-shaped, are formed.44 We have characterized the micelles formed at different concentrations of CTAB/G4, and it was established that the micellization in the case of G4 occurs at very low surfactant concentrations and the micelles formed have a stronger hydrophobic core compared to CTAB.35 The hydrophobic interactions, as a result of the stronger hydrophobic microdomain of the gemini surfactant, are enhanced to such an extent that they do not only limit the unfolding but also compress or refold the protein as indicated by increased R-helical content. The constant ellipticity values observed at high surfactant concentrations are attributed to the fact that the protein backbone at such concentrations is saturated by micelles and further addition does not bring about any alteration in the secondary structure.16,17 Intrinsic Fluorescence. Fluorescence spectroscopy is widely employed to study proteins and peptides. Aromatic amino acids tryptophan, tyrosine, and phenylalanine offer intrinsic fluorescence probes of protein conformation, dynamics, and intermolecular interactions. The changes in the wavelength of the emission maximum (λmax), a parameter sensitive to protein conformation, and fluorescence intensities at 340 nm by excitation at 280/295 nm can be used to examine protein-surfactant interactions.45 At 295 nm, only tryptophan residues get excited whereas at 280 nm both tryptophan and tyrosine residues are excited. (44) Israelachvili, J. N. Intermolecular and Surface Forces, 2nd ed.; Academic Press: San Diego, CA, 1998. (45) Deep, S.; Ahluwalia, J. C. Phys. Chem. Chem. Phys. 2001, 3, 4583.
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Figure 6. Intrinsic fluorescence with CTAB and G4. Changes in the maximum emission wavelengths of BSA ((), RSA (O), and PSA (b) albumins at pH 7 in the presence of CTAB (A) and G4 (B) at an excitation wavelength of 280 nm (T = 25 °C).
Tyrosine residues have a much lower quantum yield than tryptophan, and in proteins where both tyrosine and tryptophan residues are present, tyrosine is usually quenched by tryptophan residues. The variations exhibited at 295 nm are therefore found to be similar to that at 280 nm and hence are not shown. The variations in the wavelength of the emission maximum (λmax), as plotted in Figure 6A, indicate that all three proteins exhibit a blue shift (of 10 to 20 nm) with increasing [CTAB] assuming a constant value. λmax decreases with increasing surfactant concentration as the fluorescence emission shows a shift of fluorophores toward a nonpolar environment.45 At still higher surfactant concentration, the protein backbone is saturated with the micelles, and further addition of the surfactant does not alter its environment.16,17 With the increase in surfactant concentration, the shift of fluorophores toward a nonpolar environment can be possible in two cases: (1) internalization of fluorophores toward the core of the protein, which is possible when proteins get stabilized, and (2) hydrophobic interaction between the nonpolar moieties of detergents and exposed fluorophores, which is possible during unfolding. The secondary structure studies clearly show the unfolding of proteins in the presence of detergents (Figure 2), but the stabilization of the tertiary structure cannot be possible along with the disruption of the secondary structure. Therefore, case 2 seems to be more feasible. Figure 6B depicts the variation in λmax as G4 interacts with serum albumins. As in the case of CTAB, a blue shift, attributed to the creation of a hydrophobic environment by the addition of surfactant, is observed in all three proteins, but the rate of decrease of the wavelength is more significant when the protein interacts with G4. λmax also attains a constant value, signaling the 11690 DOI: 10.1021/la901639h
Figure 7. Extrinsic fluorescence. (A) Fluorescence intensity at 470 nm of ANS-bound BSA ((), RSA (O), and PSA (b) at pH 7 in the presence of CTAB when excited at 380 nm (T = 25 °C). (B) Fluorescence intensity at 470 nm of ANS-bound BSA ((), RSA (O), and PSA (b) at pH 7 in the presence of G4 when excited at 380 nm (T = 25 °C).
saturation of the polypeptide backbone by micelles, at a significantly lower surfactant concentration in the case of G4 compared to its single-chain counterpart (i.e., CTAB). The results clearly show that the variations in the normalized RFI as well as λmax observed in protein/G4 are more significant than the protein/CTAB variations. The changes resulting from the protein-surfactant interactions also culminate or reach the saturation point at a much lower concentration in the case of the gemini surfactant than for its single-chain counterpart. The results are in agreement with the far-UV-CD studies and reveal that protein has a stronger affinity for the gemini surfactant than for its single-chain counterpart. It is associated with the special characteristics possessed by the gemini surfactant that enhance the electrostatic interactions, responsible for protein-surfactant interactions at low surfactant concentrations, between the protein and two ionic head groups as well as hydrophobic interactions that dominate the interactions in the higher surfactant concentration range. Extrinsic Fluorescence. 1-Anilino-8-naphthalenesulfonate (ANS) is a widely used fluorescent probe known to bind to hydrophobic patches in proteins.46 Figure 7A depicts the changes in the normalized RFI at 470 nm at an excitation wavelength of 380 nm with increasing [CTAB]. The proteins exhibit a 50-60% decrease in ANS binding with increasing surfactant concentration of up to 3000 μM and then assume a constant value. Although the (46) Stryer, L. J. Mol. Biol. 1965, 13, 482.
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unfolding of proteins increases the number of exposed hydrophobic patches, the surfactant molecules seem to compete with ANS molecules to be seated in the hydrophobic patches, resulting in less binding of ANS. It assumes a constant value around a surfactant concentration of 3000 μM that interestingly coincides with the R-helical content. Like the far-UV-CD results and the intrinsic fluorescence studies, ANS binding also follows the binding isotherm, suggesting that the protein undergoes conformational changes only up to a particular surfactant concentration, after which the protein backbone is saturated with micelles and further addition of the surfactant does not bring about any significant change.16,17 The variation in normalized RFI at 470 nm with increasing [G4] is illustrated in Figure 7B. The RFI, like that in case of CTAB, decreases up to a particular surfactant concentration in both BSA and RSA and is associated with the decreased R-helical content due to the unfolding of the protein. Unlike CTAB, the minimum, when the proteins are treated with G4, is followed by 13.6 and 38.6% rises in normalized RFI in BSA and RSA, respectively. The increase in RFI is associated with the increase in the R-helical content as illustrated by the far-UV-CD results. On further addition of surfactant, no significant change occurs, and RFI assumes an almost constant value as the protein backbone is saturated with micelles. Matulis and Lovrien47 concluded that there are at least two modes of ANS binding to BSA. In the first mode, where five sites were found, ANS is inside the hydrophobic cavity far away from water molecules, and these ANS molecules fluoresce. Among them, three binding sites are within the Foster radius limit close to Trp-214 and one within that of Trp-134.48 The easy displacement of ANS molecules by CTAB and G4 molecules (Figure 7A,B) shows that the detergent molecules may have binding sites in close proximity to the tryptophanyl residues of the proteins. ANS binding, along with the other spectroscopic tools used, makes it clear that G4 is more efficient than its conventional counterpart. Both the electrostatic interactions that control the protein-surfactant interactions at low surfactant concentration and the hydrophobic forces responsible for the conformational changes that the proteins undergo at high surfactant concentrations are more significant in the case of the gemini surfactant. Also, the saturation point is reached earlier when the proteins are treated with G4 instead of CTAB. In light of our investigation, we suggest that a logical extension of the present work would be to exploit the gemini surfactants in the refolding of the proteins via the artificial chaperone protocol and in drug delivery. The expansion of the genomic database has necessitated the large-scale production of recombinant proteins. Although the refinement of genetic engineering has made the heterologous expression of proteins routine, such proteins must be folded from a chemically denatured state where misfolding and aggregation pose serious problems. Biological systems have sophisticated machinery, the chaperone proteins, for encouraging folding by (47) (48) (49) (50)
Matulis, D.; Lovrien, R. Biophys. J. 1998, 74, 422. Togashi, D. M.; Ryder, A. G. J. Fluoresc. 2008, 18, 519. Harlt, F. U. Nature 1996, 381, 571. Chandler, D. Nature 2002, 417, 491.
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discouraging aggregation.49 Surfactants are efficient folding aids and have been proven to be effective with several proteins.48,50 A method, called artificial chaperone-assisted refolding, involving the sequential introduction of a detergent and cyclodextrin and promoting the assembly of protein’s native conformation was proposed by Rozema and Gellman.51 In the first step of this approach, the detergent forms a complex with the denatured protein preventing aggregation whereas in the second step cyclodextrin selectively binds the detergent, stripping it from the protein that is then able to refold. Cationic detergent CTAB was found to efficiently refold xylanase used from alkalophilic thermophilic bacillus.52 Because the gemini surfactant has more affinity for the protein than does its conventional counterpart, it may be used as an efficient and inexpensive folding catalyst that acts like a chaperone and helps in the optimization of the refolding process. The gemini surfactant, because of stronger electrostatic and hydrophobic forces, may capture the denatured protein at much lower concentration than will the single-chain surfactant in the first step of the artificial chaperone protocol and hence will inhibit aggregation.
Conclusions The interaction between single-chain surfactant CTAB and its gemini counterpart with a spacer chain length of four (G4) with serum albumins is tracked using well-recognized spectroscopic techniques. The results presented clearly indicate that a significantly lower concentration of G4 in comparison to that of CTAB shows the same unfolding of albumins. The proteins thus undergo more significant unfolding when treated with G4, and unlike the situation for CTAB, refolding is also observed at higher G4 concentrations. The results are attributed to the enhanced electrostatic and hydrophobic interactions associated with the architecture of the gemini surfactant. On the basis of the present investigation, it is suggested that the gemini surfactants can be utilized to facilitate the refolding of proteins via the artificial chaperone protocol and also may prove fruitful in drug delivery.
Abbreviations BSA, bovine serum albumin; RSA, rabbit serum albumin; PSA, porcine serum albumin; ANS, 1-anilino-8-naphthalene sulfonic acid; CTAB, cetyltrimethylammonium bromide; G4, bis(cetyldimethylammonium)butane dibromide; and CD, circular dichroism. Acknowledgment. Financial assistance from the Council of Scientific and Industrial Research (CSIR), Government of India (to N.G.) and the Department of Biotechnology (DBT), Government of India (to P.S.) in the form of a research fellowship is acknowledged. We are thankful to Aligarh Muslim University for the use of their facilities. (51) Rozema, D.; Gellman, S. H. J. Am. Chem. Soc. 1995, 117, 2373. Rozema, D.; Gellman, S. H. J. Biol. Chem. 1996, 271, 3478. Rozema, D.; Gellman, S. H. Biochemistry 1996, 35, 15760. (52) Nath, D.; Rao, M. Eur. J. Biochem. 2001, 268, 5471.
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