Role of hydrophobic and polar interactions for BSA–amphiphile composites

Chemistry and Physics of Lipids 164 (2011) 144–150

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Chemistry and Physics of Lipids journal homepage: www.elsevier.com/locate/chemphyslip

Role of hydrophobic and polar interactions for BSA–amphiphile composites Bimlesh Ojha, Gopal Das ∗ Department of Chemistry, Indian Institute of Technology Guwahati, Assam 781 039, India

a r t i c l e

i n f o

Article history: Received 5 July 2010 Received in revised form 27 August 2010 Accepted 17 December 2010 Available online 24 December 2010 Keywords: Protein BSA Amphiphile Hydrophobic UV–visible Time-resolved fluorescence

a b s t r a c t To evaluate the role of hydrophobic and electrostatic or other polar interactions for protein–ligand binding, we have studied the interactions of bovine serum albumin (BSA) with 2-alkylmalonic acid and 2alkylbenzimidazole amphiphiles having different head group and alkyl chain length. The binding affinity for the protein–amphiphile interactions is found to depend predominantly on the length of hydrocarbon chain, suggesting the crucial role of hydrophobic forces, supported by polar interactions at the protein surface. The BSA fluorescence exhibits appreciable hypsochromic shift along with a reduction in fluorescence intensity and mean lifetime upon binding with 2-alkylmalonic acid. UV–visible, steady state and time-resolved fluorescence measurements were performed to compare the effects of amphiphiles on BSA as a function of the amphiphiles head group and alkyl chain length. © 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction The marginal stability of the native globular conformation of proteins, which is a delicate balance of various interactions in the proteins, is affected by the pH, temperature and the addition of small molecules such as substrates, coenzymes, inhibitors and activators that bind specifically to the native state. 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. Extensive studies on the interactions of surfactants with globular proteins have been reported and reviewed (Jones and Brass, 1991). The interaction of proteins with amphiphiles/surfactants has received a great deal of interest for many years due to its application in a great variety of industrial, biological, and cosmetics systems (Dickinson, 1993; Jones, 1992; Turro et al., 1995; Takeda et al., 2002). Surfactants can be broadly classified into those which bind and initiate protein unfolding, i.e. denaturing surfactants and those that only bind leaving the tertiary structure of the protein intact. Commonly ionic surfactants such as sodium n-dodecyl sulphate generally denature proteins whereas non-ionic surfactants do not. There are, however, exceptions to this rule too (Jones et al., 1987; El-Sayert and Roberts, 1985). The fluorescence properties of the tryptophan residues in folded proteins vary widely (Eftink, 2000, 1994; Kronman and Holmes, 1971). The quantum yields vary from near 0 to 0.35, and the wavelength

∗ Corresponding author. Tel.: +91 361 2582313; fax: +91 361 2582349. E-mail address: [email protected] (G. Das). 0009-3084/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.chemphyslip.2010.12.004

where the quantum yield is maximal, max , varies from as low as 308 nm for buried tryptophan residues to near 350 nm for tryptophan residues that are largely exposed to solvent (Alston et al., 2004; Callis and Liu, 2004; Pan et al., 2006). A number of studies have focused on the multifunctional binding properties of bovine serum albumin (BSA) (Peters, 1985, 1988; Foster, 1977; Chakraborty et al., 2009) which binds a wide variety of molecules. The binding function is a means of transporting soluble substances between tissues and organs. Binding also functions as a protection against the toxic effects of the bound ligand. Binding studies with BSA find broad and significant applications in the area of rational drug design as many pharmaceuticals are rendered less effective or entirely ineffective by virtue of their interaction with BSA. BSA functions biologically as a carrier for fatty acid anions and other simple amphiphiles in a blood stream. It has a molecular weight of 66,411 g mol−1 and contains 583 amino acids in a single polypeptide chain. The protein contains 17 disulphide bridges and one free –SH group, which can cause it to form a covalently linked dimer. The protein is known to have a heart-shaped structure (N form) in solution with a net charge of −18 on its surface. The interior of the protein is almost hydrophobic, while both the charged amino acid residues and the apolar patches cover the interface (Vijai and Forster, 1967; Curry et al., 1998). Based on our previous work of amphiphile–protein interactions (Ojha and Das, 2010), here we have reported, the effects of hydrocarbon chain length and nature of the hydrophilic group on the photophysical response of BSA, using two different types of amphiphilic molecules viz. 2-alkylmalonic acid (amphiphile 1) and 2-alkylbenzimidazole (amphiphile 2) having different lengths of aliphatic chain (Scheme 1). In this work, we have employed UV–visible, steady-state and time-resolved fluores-

B. Ojha, G. Das / Chemistry and Physics of Lipids 164 (2011) 144–150

O

HO

H N R2

OH

R1 Amphiphile 1 Where R1 = C8H17 (1a) H (1b) 12 25 H (1c) 16 33

A

0.10

0.08

N Amphiphile 2 Where R2 = C4H9 (2a) C9H19 (2b) C11H23 (2c)

0.06

Absorbance

O

145

0.04

0.02

Scheme 1. Structures of the amphiphiles 1 and 2.

cence measurements in order to obtain information related to the binding mechanism of the probes to BSA such as binding modes, binding constants, binding sites and quenching rate constants as a function of the amphiphiles head group and alkyl chain length. The results may cast some light on the future study of interaction between amphiphiles and other proteins.

0 260

280

300

320

340

Wavelength (nm) 0.15

B

2. Experimental 2.1. Materials Commercially available diethyl malonate, o-phenylenediamine, aliphatic acids and alkyl halide were obtained from Merck, India and Sigma, USA. BSA was purchased from Fluka Germany and was used as supplied. Other chemicals were of reagent grade and used without further purification. A stock solution of BSA (10 ␮M) in 10 mM aqueous phosphate buffer of pH 7.0 was prepared, while the amphiphiles stock solution were prepared in DMSO because of their lower solubility in water. For interaction studies of amphiphiles with protein, a 3.0 mL aqueous solution of BSA (10 ␮M) was titrated with various concentrations of the amphiphiles ranging from 0 to 24 ␮M, where the total volume of DMSO did not exceed 15%. The presence of 15% DMSO induces no major BSA structural changes (Supporting information). Each solution was mixed thoroughly before spectral measurements at room temperature. 2.2. Methods The absorption spectra were recorded on a Perkin Elmer Lambda-25 UV–visible spectrophotometer using 10 mm path length quartz cuvettes in the range of 200–350 nm wavelengths, while fluorescence measurements were taken on a Carry eclipse spectrofluorometer using 10 mm path length quartz cuvettes with slit width of 5 nm by exciting the protein solution at 295 nm. An excitation wavelength of 295 nm was applied to selectively excite the tryptophan residues in protein. Background intensities of the buffer blanks in which BSA was omitted were subtracted from each sample spectrum to cancel out any contribution due to the solvent. Time-resolved intensity decays of the protein were measured using a Life Spec II spectrofluorimeter (Edinburgh instrument) at 298 K. The sample was excited by Pico-quant 290 nm light emitting diode (LED) and the decay was measured through a 50 ns time scale at a time resolution of 0.0122 ns/channel. The decay curves were analyzed by FAST software using discrete exponential method, provided by Edinburgh instrument. The generated curves for intensity decay were fitted in the functions of Eq. (1), where, ˛i is the initial intensity of the decay component i, having a lifetime  i , (Swaminathan et al., 1994). I(t) =



˛i exp

t i

(1)

Absorbance

0.1

0.05

0 260

280

300

320

340

Wavelength (nm) Fig. 1. UV–visible absorption spectra of BSA (3.0 ␮M) with increasing concentrations of (A) amphiphile 1a and (B) amphiphile 2a from 0 to 24 ␮M in phosphate buffer of pH 7.0.

2.3. Synthesis of amphiphiles 2-Alkylmalonic acid (amphiphile 1) and 2-alkylbenzimidazloes (amphiphile 2) were prepared by the following literature methods (Pool et al., 1937; Hui et al., 2007; Palou et al., 2005; Aggett and Timperley, 1969; Nokami et al., 1979). The structures of the amphiphiles were shown in Scheme 1. The purity of the compounds were checked by NMR, IR, melting point and were found to be in good agreement with the reported data (Supporting information). 3. Results and discussion 3.1. UV–visible spectra of BSA in the presence of the amphiphiles UV–visible absorption spectrum is a very simple and applicable method to explore the structural change and to know the complex formation in solution (Hu et al., 2004). Fig. 1 shows the UV–visible absorption spectra of BSA in the presence of different concentrations of the amphiphiles (1 and 2). Amphiphile 1 has no absorption under the presence experiment conditions. As can be seen from Fig. 1, free BSA has maxima at 279 nm and it increases with the addition of the amphiphiles (1 and 2), while free amphiphile 2 has two bands at 280 and 274 nm, showed gain in intensity upon com-

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B. Ojha, G. Das / Chemistry and Physics of Lipids 164 (2011) 144–150

400

A

300 250 200 150 100 50 0 400

C

345

Maximum EW (nm)

Intensity (a. u.)

350

320

340 360 380 Wavelength (nm)

400

2

340

1a

335 330

1b

325

1c

320

0

5

10

15

20

25

B

Intensity (a. u.)

320 240 160 80 0

320

340

360

380

400

Wavelength (nm) Fig. 2. Fluorescence spectra of BSA (10 ␮M) with increasing concentration of (A) amphiphile 1a and (B) amphiphile 2a from 0 to 24 ␮M in phosphate buffer of pH 7.0 and (C) variation in fluorescence emission maxima of BSA as a function of amphiphiles concentration.

plexation with protein (Fig. 1B), along with a blue shift of maximum peak position from 280 to 275 nm and 274 to 269 nm, respectively. A reasonable explanation for the two evidences may come from the ground state complex formation between BSA and amphiphiles (Yue et al., 2008). 3.2. Quenching of intrinsic fluorescence of BSA by the amphiphiles Tryptophan (Trp) fluorescence is widely used as a tool to monitor changes in proteins structures and to make inferences regarding local structure and dynamics. There are two tryptophan residues in BSA: Trp-134 is in domain I in the 8th helix of D129-R144, which is well exposed, and Trp-212 is in domain II, in the 2nd helix of E206-F221, and is buried inside the protein structure (Sun and Yang, 2005). The fluorescence spectrum of BSA presents strong emission with maximum at 345 nm, when excited at 295 nm. Steady-state fluorescence studies show that tryptophan fluorescence decreases with increasing concentration of the amphiphiles. The changes in the tryptophan fluorescence intensity (Fig. 2) showed that the environment of tryptophan is highly affected during the interaction with the amphiphiles. Steady-state fluorescence experiments showed that the amphiphiles 1 (a–c) and 2 (a–c) can markedly influence the emission properties of BSA. The effect of amphiphiles on tryptophan fluorescence intensity is shown in Fig. 2 at pH 7.0 upon excitation at 295 nm. Excitation wavelength of 295 nm was taken to avoid the contribution from the tyrosine residues. As shown in

Fig. 2A, increasing concentration of the amphiphile 1a caused a progressive reduction of fluorescence intensity accompanied by a blue shift in max by 9, 18 and 25 nm for the amphiphile 1a, 1b and 1c, respectively, upon complexation with BSA (Fig. 2C). This blue-shift of emission maximum along with decrease in intensity indicates that interaction of BSA with amphiphile changes the environment of tryptophan residues in protein with probable increase in hydrophobicity in its vicinity and this phenomenon is seen when the protein is in either native or structurally disrupted states (Wu et al., 2007). Both shifts in wavelength and changes in fluorescence intensity of tryptophan residues in a protein are generally observed upon unfolding. The tryptophan emission of a native protein can be greater or smaller than the emission of a free tryptophan in aqueous solution. The emission maximum is usually shifted from shorter to longer wavelengths upon protein unfolding, which corresponds to the fluorescence maximum of tryptophan in aqueous solution. In a hydrophobic environment, such as in the interior of a folded protein, tryptophan emission occurs at shorter wavelengths (Wu et al., 2007). The apparent maximum emission wavelength of BSA at about 345 nm indicates that the tryptophan residues present in the native protein are on the surface and exposed to an aqueous environment (Lakowicz, 1999). The addition of amphiphiles 1 (a–c) results in a shift of max from 345 to 336, 326 and 320 nm. Since tryptophan emission occurs at shorter wavelength in a hydrophobic environment, the shift of max towards lower wavelength indicates the transfer of protein to a more hydrophobic environment consistent with

B. Ojha, G. Das / Chemistry and Physics of Lipids 164 (2011) 144–150

4

147

A

1c

the binding of amphiphiles near the tryptophan sites of BSA (Scheme 2). The emission maximum of tryptophan can vary greatly between proteins and its emission spectrum should reflect the average environment of the tryptophan. The variations in tryptophan emissions are due to the structure of the protein. For tryptophan in completely apolar environment a blue shifted emission was observed (Wu et al., 2007). Such behavior of BSA emission upon complexation with amphiphiles 1 are expected due to the increase in hydrophobic environment around the fluorophore, i.e. tryptophan residues as shown in Scheme 2 (Wu et al., 2007; Lakowicz, 1999). Thus, it can be concluded that the addition of amphiphiles 1 increase the hydrophobicity around the tryptophan residues results in the blue shift of the emission spectrum. Whereas for amphiphiles 2, no spectral shift was observed for the emission spectra of BSA upon protein–amphiphile complexation (Fig. 2B and 2C), indicating that Trp-134 was not exposed to any polarity change. The emission of tryptophan remains at 345 nm, and the quenching may arise from amphiphile interactions on overall BSA conformation via the hydrophobic interactions with tryptophan residues (Scheme 2). The fluorescence quenching efficiency of BSA seems to be depend on the length of hydrocarbon chain of the amphiphiles, with increase in alkyl chain length, the extent of quenching of tryptophan fluorescence increases, suggesting the probable role hydrophobic interactions via the hydrophobic regions of the amphiphiles. The increase in restriction around the environment of tryptophan residues in protein is also shown in Fig. 2c, where emission maximum is plotted as a function of the amphiphiles (1 and 2) concentration. The difference in the way of interactions of the amphiphiles 1 and 2 with the albumin molecule is not surprising because it is expected that the protein–amphiphile interactions will be strongly influenced by the amphiphilic nature; that is, the hydrocarbon chain length and the charge or nature of the hydrophilic group (Wu et al., 2007). Both amphiphiles 1 and 2 are a single-chain molecule having a strong hydrophobic microdomain. In 1 the polar part is malonic acid, while in 2 the polar part comprises of benzimidazole, suggesting that the head group in 1 is more hydrophilic than 2. It is believed that, due to the repulsion with net negative charge on the protein surface, the amphiphiles 1, result ultimately in perturbation of the interchain hydrophobic interactions of the protein (Takizawa et al., 1995; Hirai et al., 1995). The complexformation processes in solution are clearly evidenced for the BSA with amphiphiles, as the steady-state fluorescence spectra in Fig. 2 suggest. 3.3. Quenching constant parameters for BSA–amphiphile composites A gradual quenching of fluorescence intensity of tryptophan residues in BSA with increasing concentration of amphiphiles at pH 7.0 is shown in Fig. 2. Hence, it is possible to estimate the quenching of tryptophan fluorescence in the presence of the amphiphiles using the Stern–Volmer equation. On the basis of relationship between quenching of excited states and quencher concentration,

2.4

1b 1a

1.6

0.8

0

5

10

15

20

25

B

1.6

2c 1.4

2b

Fo/F

Scheme 2. Schematic model showing the effect of amphiphiles 1 and 2 on the environment of tryptophan (Trp) residues in protein.

Fo/F

3.2

1.2

2a 1 0

5

10

15

20

25

Fig. 3. Stern–Volmer plots for the quenching of BSA fluorescence by (A) amphiphile 1 and (B) amphiphile 2 at room temperature.

the Stern–Volmer equation is given by Eq. (2) (Lakowicz, 1999) Fo = 1 + Kq o [Q ] = 1 + KSV [Q ] F

(2)

Here, Fo and F are the relative fluorescence intensity in the absence and presence of quencher; Kq is the bimolecular quenching rate constant;  o is the average lifetime of the fluorophore in the absence of quencher, [Q] is the concentration of the quencher, respectively, and KSV represents the Stern–Volmer quenching constant which measures the efficiency of quenching. Fig. 3 shows the Stern–Volmer plots of BSA–amphiphile composites at pH 7.0. It shows that the Stern–Volmer plots are linear and does not change from linearity with increasing concentration of the amphiphiles, revealing the occurrence of a single type of quenching, either static or dynamic. In Table 1, the binding constants obtained using the Stern–Volmer method for different amphiphile-BSA composites are listed. The plots with larger slopes indicate large amount of quench-

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Table 1 Quenching constants parameters for BSA–amphiphile composites.

A

Kq (M−1 s−1 )

KA (M−1 )

n

BSA + 1a BSA + 1b BSA + 1c BSA + 2a BSA + 2b BSA + 2c

9.58 × 103 17.9 × 103 38.1 × 103 4.05 × 103 16.2 × 103 26.2 × 103

1.92 × 1012 3.58 × 1012 7.62 × 1012 0.81 × 1012 3.24 × 1012 5.25 × 1012

5.89 × 103 19.1 × 103 38.2 × 103 5.71 × 103 8.40 × 103 13.0 × 103

1.08 1.00 1.02 0.85 1.20 1.23

ing and can be used to calculate the bimolecular quenching constant (Kq ) using Eq. (2). Since the maximum value of Kq for diffusion controlled quenching process (Lakowicz, 1999) with biopolymer is about 2.0 × 1010 M−1 s−1 , the high value of quenching rate constants imply that the quenching is static in nature (Table 1). The larger value of Kq for the BSA–amphiphile 1 composites compared to that for the BSA–amphiphile 2 again indicates less quenching in the latter. Static quenching arises only from the formation of complex between BSA and amphiphiles. The changes in the fluorescence parameters (Fig. 2) show that the environment of tryptophan is highly affected during the interaction with the amphiphiles 1 and 2. The blue-shift of max and the increase in KSV suggests strong interaction between BSA and amphiphiles 1 and comparatively weaker interaction takes place between BSA and amphiphiles 2. For the static quenching process, under the assumptions that BSA has the same and independent binding sites, Eq. (3) was used for the determination of binding constant or association constant (KA ) and the number of binding sites (n) (Bi et al., 2004). log

 (F − F)  o F

= log KA + n log[Q ]

(3)

Binding constant and the number of binding sites can be obtained by plotting log[(Fo − F)/F] versus log[amphiphile] as shown in Fig. 4. From the slope of the linear fitting plots, number of binding sites (n) and from the intercept (log KA ), the binding constants (KA ), were calculated and tabulated in Table 1. From Table 1, the values of n indicate that in each case the probe is located at only one binding site and the association constants obtained for the BSA–amphiphile composites suggest lower binding affinity compared to other strong protein–ligand complexes with binding constants ranging from 106 to 108 M−1 . However, lower binding constants (103 –105 M−1 ) were recently reported for several other protein–ligand complexes (Lakowicz, 1999; He et al., 2005; Dufour and Dangles, 2005; Nsoukpoe-Kossi et al., 2007). 3.4. Fluorescence lifetime decay of BSA in the presence of the amphiphiles Fluorescence lifetime serves as a sensitive parameter for exploring the local environment around a fluorophore and it is sensitive to excited-state interactions (Das et al., 2006). It also contributes to the understanding of interactions between probe and proteins (Mallick et al., 2005). The fluorescence lifetime of the tryptophan residues in protein is a complex function of its interactions with the local environment and the solvent. To explain the variation of large blue shift and increase in the KSV values of BSA–amphiphile 1 composites with the variation of alkyl chain length, timeresolved fluorescence study was further applied to investigate the quenching mechanism of BSA fluorescence in the presence of the amphiphiles. There are two lifetime components in native BSA, one ( 1 = 5.8 ± 0.2 ns) contributing 75% of the total fluorescence and the other ( 2 = 1.2 ± 0.2 ns) contributing the rest of the fluorescence (Patel et al., 1999). The one with a longer lifetime was assigned to Trp-134 and the other was assigned to Trp-212. The relative contribution of each component was dependent on the folding/unfolding conditions due to the protein’s multiple local configurations and

0.4

log[(Fo-F)/F]

KSV (M−1 )

1c

0

1b 1a

-0.4

-0.8 0.6

0.8

1

1.2

1.4

log [amphiphile] B -0.4

log[(Fo-F)/F]

Sample

2c

-0.8

2b

-1.2

2a

-1.6

0.6

0.8

1

1.2

1.4

log [amphiphile] Fig. 4. Plot of log[(Fo − F)/F] versus log[amphiphile] for (A) BSA–amphiphile 1 and (B) BSA–amphiphile 2 composites at room temperature.

changes in the extent of solvent accessibility. Thus, the information of the protein conformational behavior can be obtained from the emission spectra of tryptophan in the bioconjugates. The fluorescence lifetime decay of BSA (10 ␮M) in the absence and presence of amphiphiles (24 ␮M) are shown in Fig. 5, while the fluorescence lifetimes and various statistical parameters used to check the goodness of fit are given in Table 2. The best fits for BSA were obtained using a bi-exponential function, which is typical for tryptophan in proteins. The two time constants of BSA alone in buffer are found to be 6.01 and 1.22 ns, which is in good agreement with the above-mentioned values. But in the presence of amphiphiles 1 fluorescence lifetime of the tryptophan residues decreased significantly (Fig. 5A), compared to amphiphiles 2 (Fig. 5B) where no significant change has been observed (Table 2). Instead of placing too much importance on the magnitude of individual decay constants for such bi-exponential decays, we chose to use the mean fluorescence lifetime ( m ) defined by Eq. (4) as an important parameter for exploiting the behavior of BSA bound

B. Ojha, G. Das / Chemistry and Physics of Lipids 164 (2011) 144–150

much, where  m has decreased from 5.05 to 5.04, 4.90 and 4.85 for amphiphiles 2a, 2b and 2c, respectively (Table 2). The results showed clear consequences of significant interactions between the amphiphiles 1 and BSA compared to amphiphiles 2. It is also clear from the fluorescence lifetime measurement that the decay of BSA is faster with increase in chain length of the amphiphiles, showing the same trend as observed in the steady state fluorescence measurements

1000 A

100

 ˛i i m = i

Counts

2

i

10

5 1 1

5

10

15

20

25

30

35

40

Time (ns) 1000

149

B

˛i

(4)

From lifetime data of the tryptophan in BSA, the degree of exposure of the probe to aqueous phase can be predicted. The closer are the lifetime values in the amphiphilic environment and the aqueous environment, the greater is the degree of exposure of the probe to the aqueous environment. Table 2 reveals that all of the lifetime components of BSA in the presence of amphiphiles are lower than the corresponding values in pure BSA, indicates that in native BSA the polarity of the microenvironment is higher than that in the amphiphilic environment; that is, the probe molecule resides in the more apolar region in BSA–amphiphile composites than that in native BSA in an aqueous buffer environment, resulting in a concomitant decrease in the lifetime values. 4. Conclusions

100

Counts

2

10 1

5

1 5

10

15

20

25

30

35

40

Time (ns) Fig. 5. Time-resolved fluorescence decays of BSA (10 ␮M) in the presence of 24 ␮M of (A) amphiphile 1 and (B) amphiphile 2 in phosphate buffer of pH 7.0; where Trace 1: IRF, Trace 2: BSA alone and Traces 3–5 are BSA in the presence of amphiphile a, b and c. ex = 290 nm, and the emission wavelengths were chosen according to the steady-state fluorescence data.

to amphiphiles (Mallick et al., 2005). A decrease of mean lifetime ( m ) from 5.05 to 4.34, 3.01 and 2.90 ns results due to the addition of amphiphiles 1a, 1b and 1c, respectively, while in the presence of amphiphile 2, the mean lifetime of BSA were not affected

b

Acknowledgements This work was supported by CSIR and DST through Grant 01-2235/08/EMR-II and SR/S1/IC-01/2008, New Delhi, India. B.O. thanks IIT Guwahati for the fellowship and CIF IIT Guwahati for providing instrument facility. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.chemphyslip.2010.12.004.

Table 2 Fluorescence lifetime () of BSAa and BSA–amphiphileb composites.

a

The mechanism of binding of amphiphiles 1 and 2 to BSA was investigated using UV–visible, steady-state and lifetime fluorescence measurements. Quenching of BSA fluorescence was found to be a function of the amphiphiles head group and alkyl chain length. On the basis of our spectroscopy data on BSA–amphiphile composites, different binding patterns were observed for amphiphiles 1 and 2. The study showed that BSA has a high binding affinity for the amphiphiles 1 than the amphiphiles 2. The results also indicate a static quenching mechanism operating in the both the composites. The binding constant of protein–amphiphile complexes calculated from fluorescence data shows moderate amphiphile–protein interaction. The number of binding sites of both complexes was also estimated to be one which indicates that both probes bind only at one site. The fluorescence results showed that the interaction of amphiphile changes the environment of tryptophan residues in BSA.

Sample

max (nm)

 1 (ns)

 2 (ns)

˛1

˛2

 m (ns)

2

References

Native BSA BSA + 1a BSA + 1b BSA + 1c BSA + 2a BSA + 2b BSA + 2c

345 336 327 320 345 345 345

6.01 5.32 4.67 4.30 6.00 5.82 5.80

1.22 1.24 1.20 1.07 1.25 1.05 1.00

0.016 0.016 0.012 0.013 0.016 0.015 0.016

0.004 0.005 0.011 0.010 0.004 0.005 0.004

5.05 4.34 3.01 2.90 5.04 4.90 4.85

1.001 1.001 1.000 1.003 1.002 1.006 1.003

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