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Study of protein–surfactant interaction using excited state proton transfer
Chemical Physics Letters 404 (2005) 341–345 www.elsevier.com/locate/cplett
Study of protein–surfactant interaction using excited state proton transfer Kalyanasis Sahu, Durba Roy, Sudip Kumar Mondal, Rana Karmakar, Kankan Bhattacharyya * Physical Chemistry Department, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700 032, India Received 18 October 2004; in final form 8 December 2004
Abstract Excited state proton transfer (ESPT) of pyranine (8-hydroxypyrene-1,3,6-trisulfonate) is studied in a lysozyme–cetyltrimethylammonium bromide (CTAB) complex using picosecond emission spectroscopy. The critical association concentration (CAC) of CTAB is found to be 0.4 mM. Using a kinetic analysis it is shown that deprotonation, recombination and dissociation of the geminate ion pair in the lysozyme–CTAB aggregate are faster than that in a CTAB micelle. 2005 Elsevier B.V. All rights reserved.
1. Introduction Interaction of a protein with a surfactant plays an important role in protein folding [1]. Recently, protein–surfactant interaction has been studied using steady state and time resolved fluorescence [3–5], dynamic light scattering [3], thermodynamic [7] and using a piezoelectric crystal [8]. Most of the protein–surfactant systems include oppositely charged protein and surfactant (e.g., positively charged lysozyme and anionic SDS). Lysozyme is a globular protein which carries a net positive charge of +8 in an aqueous solution at a pH 6.5 [9,10]. Interaction between a positively charged protein lysozyme and cationic surfactant cetyltrimethylammonium bromide (CTAB) is difficult because of electrostatic repulsion. However, a recent micro-calorimetric study reveals that hydrophobic interaction dominates over electrostatic repulsion resulting lysozyme–CTAB aggregates [7].
*
Corresponding author. Fax: +33 2473 2805. E-mail address: [email protected] (K. Bhattacharyya).
0009-2614/$ - see front matter 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.cplett.2005.01.113
Ground and excited state proton transfer of pyranine (8-hydroxypyrene-1,3,6-trisulfonate) [11–24] and other hydroxy-aromatic probes [24–31] have been widely studied in water [13–16], micelle [17,20,26–31], reverse micelle [18], liquid mixtures [22] and biomolecules [11,12,21]. pKa of pyranine decreases from 7.4 in the ground state to 0.4 in the excited state [16]. In bulk water, pyranine exhibits a weak emission band at 440 nm due to the protonated form (ROH, Scheme 1) and a 20 times stronger band at 510 nm due to the deprotonated form (RO). In bulk water, ESPT from pyranine to water occurs in 150 ps and hence, the ROH emission exhibits a decay of 150 ps and RO emission shows a rise of 150 ps and decay of 5500 ps [13,14,20]. The anionic probe pyranine binds very strongly to a cationic surfactant CTAB. Addition of CTAB to a pyranine solution at a concentration below the critical micellar concentration (CMC = 1 mM) results a turbid solution [17]. The emission intensity of pyranine (both ROH and RO) sharply diminishes upon addition of CTAB. This is ascribed to the formation of an association complex and precipitates arising from electrostatic attraction [17]. Similar quenching is also reported for
K. Sahu et al. / Chemical Physics Letters 404 (2005) 341–345
_
_
O3S
OH
O3S
_ SO3
Scheme 1. Structure of the protonated form of the pyranine, (ROH).
pyranine in the presence of n-alkylpyridinium ion [19] and for other anionic fluorescence probes in CTAB [32]. Above CMC, when CTAB micelles are formed the ESPT process of pyranine is found to be markedly retarded [17,20]. At a CTAB concentration (20 mM) much above CMC (1 mM) when almost all the pyranine molecules bind to CTAB micelles the intensity ratio of the ROH and RO is 1: 0.75 [20]. At a lower CTAB concentration a significant amount of pyranine remains in unbound form (free) and the pyranine emission consists of contributions from both free and micelle bound probes. Thus, at a low concentration of CTAB, RO emission intensity is higher than that of ROH emission (e.g., ROH:RO is 1:6 in 1.2 mM CTAB). In 20 mM CTAB, the ROH emission exhibits a decay of lifetime 2000 ps and the RO exhibits a rise time of 2000 ps and a decay of time constant 6500 ps [20]. Recently, we have shown that pyranine binds strongly to the cationic micelle CTAB [20]. Lysozyme is a positively charged protein, hence it is expected that pyranine should bind to the protein. A recent mass spectroscopic study reports the formation of non-covalent complex between the surface arginine residues of lysozyme and sulfonate receptors, e.g., pyrene-tri and tetrasulfonates which are structurally similar to pyranine [2]. In this work, we report on the excited state proton transfer (ESPT) of a probe pyranine (8-hydroxypyrene-1,3,6trisulfonate) in a lysozyme–CTAB complex.
2. Experimental 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (pyranine, Fluka), lysozyme (from hen egg white, Fluka) and cetyltrimethylammonium bromide (CTAB, Aldrich) were used without further purification. The steady state absorption and emission spectra were recorded in a Shimadzu UV-2401 spectrophotometer and a Perkin–Elmer 44B spectrofluorimeter, respectively. For lifetime measurements, the samples were excited at 375 nm using a picosecond diode laser (IBH Nanoled-07) in an IBH Fluorocube apparatus. The emission was collected at a magic angle polarization using a Hamamatsu MCP photomultiplier (5000U-09). The time correlated single photon counting (TCSPC) setup
consists of an Ortec 9327 CFD and a Tennelec TC 863 TAC. The data were collected with a PCA3 card (Oxford) as a multi-channel analyzer. The typical FWHM of the system response using a liquid scatterer is about 80 ps. The fluorescence decays were deconvoluted using IBH DAS6 software.
3. Results 3.1. Effect of lysozyme and CTAB on steady state spectra of pyranine In bulk water, pyranine exhibits two major absorption peaks at 405 and 450 nm which are ascribed to the ROH and RO forms [20]. On addition of lysozyme to an aqueous solution the absorbance at 450 nm (due to RO) decreases markedly. On addition of CTAB to an aqueous solution containing pyranine and lysozyme no appreciable change occurs in the absorption spectrum. Fig. 1 shows steady state emission spectra of pyranine in bulk water and in lysozyme. When lysozyme (0.09 and 0.5 wt%) is added to an aqueous solution of pyranine the ROH emission intensity increases followed by a concomitant decrease of the RO emission intensity. The intensity ratio of ROH:RO emission is 1:8 and 1:7 in 0.09% and 0.5% lysozyme, respectively. This is significantly larger than the intensity ratio in bulk water (1:20) and suggests suppression of ESPT in lysozyme. The emission characteristics of pyranine in lysozyme– CTAB aggregate differ significantly from those in CTAB and lysozyme alone (Figs. 1 and 2). In a 0.09 wt% lysozyme solution, addition of CTAB below 0.6 mM results in marked reduction in the intensity of both the protonated and the deprotonated forms. This is similar to the CTAB induced suppression of emission reported earlier in absence of lysozyme
Emission Intensity (a.u.)
342
0
400
450
500 550 Wavelength (nm)
600
Fig. 1. Emission spectra of pyranine in (i) water ( ); (ii) 0.5 wt% lysozyme (—-); (iii) 1.2 mM CTAB (–Æ–); (iv) 0.5 wt% lysozyme and 0.6 mM CTAB (–ÆÆ–) and (v) 0.5 wt% lysozyme and 1.2 mM CTAB (- - - -) at kex = 375 nm.
K. Sahu et al. / Chemical Physics Letters 404 (2005) 341–345
343
higher concentration of 1 mM (i.e., CMC of CTAB). The variation of ROH and RO emission intensities with CTAB concentration in presence of 0.5 wt% lysozyme is compared in Fig. 3. The ROH:RO emission intensity ratio in a lysozyme–CTAB aggregate is 1:1.
IROH (a.u.)
3.2. Effect of CTAB and lysozyme on fluorescence decays of pyranine
0.0
0.5
1.0
1.5
2.0
[CTAB] (mM) Fig. 2. Plot of emission intensity of protonated form of pyranine (IROH) against CTAB concentration in (i) aqueous solution (n), (ii) 0.09 wt% lysozyme (j) and (iii) 0.5 wt% lysozyme (s).
Emission Intensity (a. u.)
[17]. The variation of ROH emission intensity against CTAB concentration is shown in Fig. 2. At a CTAB concentration >0.6 mM, both ROH and RO emission increases sharply. Above 0.6 mM CTAB, lysozyme–CTAB complexes are formed in which ESPT of pyranine is severely suppressed. This concentration may be considered as the critical association concentration (CAC) of the lysozyme–CTAB system at 0.09 wt% lysozyme. From Fig. 2 it is evident that in 0.5 wt% lysozyme the break in the ROH emission intensity (i.e., CAC) occurs at a CTAB concentration of 0.4 mM. Thus, the CAC of CTAB depends slightly on the concentration of the lysozyme. It is also clearly seen that in the absence of lysozyme the break occurs at a
In 0.09 wt% lysozyme, fluorescence decay of the protonated form of pyranine contains contribution of both free and bound probes (Table 1). The decay at 415 nm is fitted to a tri-exponential with time constants of a bulk water like component of 150 ps (65%) and additional slow components of 700 ps (25%) and 3800 ps (10%). The 700 ps rise time may be ascribed to probes (pyranine) bound to the protein, lysozyme. The emission of RO (550 nm) when fitted to a tri-exponential, exhibits a bulk water like rise time of 150 ps and a slower rise time of 700 ± 50 ps and a decay of time constant 6000 ps. In 0.5 wt% lysozyme solution the decay characteristics of pyranine (Figs. 4 and 5, Table 1) are almost identical to those in 0.09 wt% protein. In a lysozyme–CTAB aggregate (i.e., at [CTAB] > CAC), the fluorescence decays of pyranine differ significantly from those in bulk water and lysozyme solution. The most important feature of the fluorescence decays of pyranine in a lysozyme–CTAB aggregate is the emergence of a very slow component of decay at 415 nm (Fig. 4) and of rise at 550 nm (Fig. 5) at a CTAB concentration above CAC (but below CMC) (Table 1). In a 0.5 wt% lysozyme solution at 0.6 mM CTAB, the decay at 415 nm is fitted to a tri-exponential with components of 100 ps (25%), 750 ps (15%) and 1500 ps (45%) (Fig. 4 and Table 1). The RO decay displays two rise times of 250 ps and 1200 ps with relative contribution of 2.4:1. This is followed by a decay of 5000 ps (Fig. 5 and Table 1). In the absence of lysozyme, at 1-1.2 mM CTAB, very few probe molecules remain bound to CTAB. As a result the relative contribution of micelle-like slow component of decay at 415 nm and rise at 550 nm is very small and the fluorescence decays are dominated by water like components (Table 1). However, in the presence of lysozyme, even in 1.2 mM CTAB the slow components become very prominent. For instance in the case of the rise at 550 nm, in the absence of lysozyme the amplitude of the water like fast component is 6 times larger compared to the slow rise at 1.2 mM CTAB. However, in 0.09 wt% lysozyme, at 1.2 mM CTAB the slow rise of 2000 ps is 10 times larger than the water like fast rise while in 0.5 wt% lysozyme even at 1 mM CTAB the slow rise is 16 time larger (Table 1).
4. Discussion
0.0
0.5
1.0 1.5 [CTAB] (mM)
2.0
Fig. 3. Plot of emission intensity of protonated (j) and deprotonated (s) form of pyranine against CTAB concentration in 0.5 wt% lysozyme.
The results on ESPT of pyranine in lysozyme–CTAB complex, is consistent with the necklace model of a protein (or polymer)–surfactant aggregate [3–6,33,34]. According to this model, a protein (or polymer)–surfactant aggregate resembles a necklace with spherical micelles as beads and macromolecular chains as the
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K. Sahu et al. / Chemical Physics Letters 404 (2005) 341–345
Table 1 Decay parameters of the emission from the protonated (ROH, at 415 nm) and the deprotonated (RO at 550 nm) forms of pyranine in different systems Lysozyme (wt%)
CTAB (mM)
0.00
0.0 1.0 1.2 0.0 1.0 1.2 0.0 0.6 1.0
0.09
0.50
a b
RO emissiona
ROH emissiona s1 (ps)
s2 (ps)
s3 (ps)
Rise times (ps) (X:Y)b
Decay times (ps)
150(90%) 100(83%) 100(40%) 150(75%) 100(30%) 100(25%) 150(75%) 100(25%) 100(20%)
850(10%) 350(15%) 100(45%) 700(15%) 550(35%) 600(20%) 750(15%) 750(30%) 1100(25%)
– 1000(2%) 1400(15%) 3800(10%) 1400(35%) 1800(55%) 3800(10%) 1500(45%) 2000(55%)
150 130 200,900 (6:1) 150,700 (3.5:1) 250,1400 (2.5:1) 300,2000 (1:10) 150,750 (3.5:1) 250,1200 (2.4:1) 300,2000 (1:16)
5500 1000,4000 5000 6000 5500 6000 6000 5000 6000
±10%. Ratio of amplitudes of short and long rise components.
104
Counts
103
(iv) (ii)
102
(i)
(iii)
1
10
100
0
2
4 6 Time (ns)
8
10
Fig. 4. Fluorescence decays of ROH (kem = 415 nm) in: (i) 1.0 mM CTAB; (ii) 0.5 wt% lysozyme; (iii) 0.5 wt% lysozyme and 0.6 mM CTAB and (iv) 0.5 wt% lysozyme and 1.0 mM CTAB.
10 4
3 2 ROH X d6 6 þ7 4 RO H 5 ¼ 4 k PT dt 0 RO 2
Counts
10 3 10 2
(iv)
10 1
(i)
10
We now discuss a kinetic model to analyze the result. As shown in Scheme 2, the ESPT process in pyranine consists of the following steps: (a) ultrafast deprotonation (rate constant, kPT) to form the geminate ion pair followed by (b) recombination of the geminate ion pair (rate constant, krec) and (c) dissociation of the geminate ion pair into solvent separated ion pair (rate constant, kdiss). The solvent separated ion pair may be converted back into geminate ion pair with a rate kP[H+]. At the pH (6.5) used in this work kP[H+] is negligible. kROH, and kRO denote the total decay rates (radiative and non-radiative other than proton transfer) in the excited state of ROH and RO. The time evolution of different species may be expressed in terms of the following coupled differential equations [31]: k rec Y k diss
3 2 3 0 ROH 7 6 7 0 5 4 RO Hþ 5; Z RO
ð1Þ
0
0
4
8 12 Time (ns)
16
Fig. 5. Fluorescence decays of RO (kem = 550 nm) in: (i) 1.0 mM CTAB; (ii) 0.5 wt% lysozyme; (iii) 0.5 wt% lysozyme and 0.6 mM CTAB and (iv) 0.5 wt% lysozyme and 1.0 mM CTAB.
thread. In such an aggregate, the protein segments reduce two unfavorable interactions in a surfactant aggregate (micelle), viz., electrostatic repulsion among the head groups and residual contact of the first few peripheral carbon atoms of the hydrocarbon chains with water. Thus, fewer number of surfactant molecules (aggregation number) are required for the formation of the micelles in the presence of a protein [6,34]. It is evident that the formation of such an aggregate involving a cationic surfactant (CTAB) and a positively charged protein (lysozyme) in spite of electrostatic repulsion indicates that the interaction of CTAB and lysozyme is predominantly hydrophobic in nature [7,8].
where X ¼ k PT þ k ROH k PT ; Y ¼ k rec þ k diss þ k RO ; Z ¼ k RO : According to this equation decay of the protonated (ROH) and deprotonated (RO) form are tri-exponential. From the amplitude and time constant of decay individual rate constants are obtained for CTAB and
(ROH)* + H2O kROH ROH
kPT krec
(RO-*….H3O+) kRORO-
kdiss +
kp[H ]w
RO-* + H3O+ kRORO-
Scheme 2. Kinetic scheme depicting the proton transfer process in pyranine.
K. Sahu et al. / Chemical Physics Letters 404 (2005) 341–345 Table 2 Rate constants of deprotonation (kPT), recombination (krec) and dissociation (kdiss) of geminate ion pair of pyranine at 293 K System
kPT · 103 (ps1)
krec · 103 (ps1)
kdiss · 103 (ps1)
20 mM CTAB 0.50 wt% lysozyme + 1.0 mM CTAB 0.09 wt% lysozyme + 1.2 mM CTAB Watera Methanol–watera (1.3:1)
1.00 5.00
0.14 4.20
1.00 1.60
6.30
2.80
2.40
8.00 0.80
7.20 3.20
– –
a
From Ref. [22].
lysozyme–CTAB complex. The rate constants are given in Table 2. In Table 2, we have also included the rate constants obtained using a more rigorous model based on generalized Smoluchowski theory [22–24]. It is readily seen that in a CTAB micelle the rate constant of deprotonation (kPT) is smaller than that in water but is close to that in a methanol–water mixture[22]. The recombination rate (krec) in the geminate ion pair in a micelle is found to be much smaller than that in water or methanol. In a lysozyme–CTAB complex both the deprotonation and the recombination process is found to be much faster than those in a CTAB micelle.
5. Conclusion In this work, we demonstrate that ESPT of pyranine may be used to study the interaction between lysozyme and CTAB. Lysozyme and CTAB forms a supra-molecular assembly by hydrophobic interaction, in which the proton transfer rate of pyranine is markedly retarded. This is attributed to rigidity of the water hydrogen bond network and slow solvation. The rate constant for deprotonation, recombination, and dissociation of geminate ion pair inside the lysozyme–CTAB aggregate is faster than that in a CTAB micelle. This shows that the microenvironment of pyranine in the lysozyme– CTAB aggregate is different from that in CTAB micelle.
Acknowledgments Thanks are due to Department of Science and Technology (DST), India (Project Number: IR/I1/CF-01/ 2002) and to Council of Scientific and Industrial Research (CSIR) for generous research grants. K.S., D.R., S.K.M. and R.K. thank CSIR for awarding fellowships. We thank Mr. P. Sen for useful discussions.
345
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Study of protein–surfactant interaction using excited state proton transfer Kalyanasis Sahu, Durba Roy, Sudip Kumar Mondal, Rana Karmakar, Kankan Bhattacharyya * Physical Chemistry Department, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700 032, India Received 18 October 2004; in final form 8 December 2004
Abstract Excited state proton transfer (ESPT) of pyranine (8-hydroxypyrene-1,3,6-trisulfonate) is studied in a lysozyme–cetyltrimethylammonium bromide (CTAB) complex using picosecond emission spectroscopy. The critical association concentration (CAC) of CTAB is found to be 0.4 mM. Using a kinetic analysis it is shown that deprotonation, recombination and dissociation of the geminate ion pair in the lysozyme–CTAB aggregate are faster than that in a CTAB micelle. 2005 Elsevier B.V. All rights reserved.
1. Introduction Interaction of a protein with a surfactant plays an important role in protein folding [1]. Recently, protein–surfactant interaction has been studied using steady state and time resolved fluorescence [3–5], dynamic light scattering [3], thermodynamic [7] and using a piezoelectric crystal [8]. Most of the protein–surfactant systems include oppositely charged protein and surfactant (e.g., positively charged lysozyme and anionic SDS). Lysozyme is a globular protein which carries a net positive charge of +8 in an aqueous solution at a pH 6.5 [9,10]. Interaction between a positively charged protein lysozyme and cationic surfactant cetyltrimethylammonium bromide (CTAB) is difficult because of electrostatic repulsion. However, a recent micro-calorimetric study reveals that hydrophobic interaction dominates over electrostatic repulsion resulting lysozyme–CTAB aggregates [7].
*
Corresponding author. Fax: +33 2473 2805. E-mail address: [email protected] (K. Bhattacharyya).
0009-2614/$ - see front matter 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.cplett.2005.01.113
Ground and excited state proton transfer of pyranine (8-hydroxypyrene-1,3,6-trisulfonate) [11–24] and other hydroxy-aromatic probes [24–31] have been widely studied in water [13–16], micelle [17,20,26–31], reverse micelle [18], liquid mixtures [22] and biomolecules [11,12,21]. pKa of pyranine decreases from 7.4 in the ground state to 0.4 in the excited state [16]. In bulk water, pyranine exhibits a weak emission band at 440 nm due to the protonated form (ROH, Scheme 1) and a 20 times stronger band at 510 nm due to the deprotonated form (RO). In bulk water, ESPT from pyranine to water occurs in 150 ps and hence, the ROH emission exhibits a decay of 150 ps and RO emission shows a rise of 150 ps and decay of 5500 ps [13,14,20]. The anionic probe pyranine binds very strongly to a cationic surfactant CTAB. Addition of CTAB to a pyranine solution at a concentration below the critical micellar concentration (CMC = 1 mM) results a turbid solution [17]. The emission intensity of pyranine (both ROH and RO) sharply diminishes upon addition of CTAB. This is ascribed to the formation of an association complex and precipitates arising from electrostatic attraction [17]. Similar quenching is also reported for
K. Sahu et al. / Chemical Physics Letters 404 (2005) 341–345
_
_
O3S
OH
O3S
_ SO3
Scheme 1. Structure of the protonated form of the pyranine, (ROH).
pyranine in the presence of n-alkylpyridinium ion [19] and for other anionic fluorescence probes in CTAB [32]. Above CMC, when CTAB micelles are formed the ESPT process of pyranine is found to be markedly retarded [17,20]. At a CTAB concentration (20 mM) much above CMC (1 mM) when almost all the pyranine molecules bind to CTAB micelles the intensity ratio of the ROH and RO is 1: 0.75 [20]. At a lower CTAB concentration a significant amount of pyranine remains in unbound form (free) and the pyranine emission consists of contributions from both free and micelle bound probes. Thus, at a low concentration of CTAB, RO emission intensity is higher than that of ROH emission (e.g., ROH:RO is 1:6 in 1.2 mM CTAB). In 20 mM CTAB, the ROH emission exhibits a decay of lifetime 2000 ps and the RO exhibits a rise time of 2000 ps and a decay of time constant 6500 ps [20]. Recently, we have shown that pyranine binds strongly to the cationic micelle CTAB [20]. Lysozyme is a positively charged protein, hence it is expected that pyranine should bind to the protein. A recent mass spectroscopic study reports the formation of non-covalent complex between the surface arginine residues of lysozyme and sulfonate receptors, e.g., pyrene-tri and tetrasulfonates which are structurally similar to pyranine [2]. In this work, we report on the excited state proton transfer (ESPT) of a probe pyranine (8-hydroxypyrene-1,3,6trisulfonate) in a lysozyme–CTAB complex.
2. Experimental 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (pyranine, Fluka), lysozyme (from hen egg white, Fluka) and cetyltrimethylammonium bromide (CTAB, Aldrich) were used without further purification. The steady state absorption and emission spectra were recorded in a Shimadzu UV-2401 spectrophotometer and a Perkin–Elmer 44B spectrofluorimeter, respectively. For lifetime measurements, the samples were excited at 375 nm using a picosecond diode laser (IBH Nanoled-07) in an IBH Fluorocube apparatus. The emission was collected at a magic angle polarization using a Hamamatsu MCP photomultiplier (5000U-09). The time correlated single photon counting (TCSPC) setup
consists of an Ortec 9327 CFD and a Tennelec TC 863 TAC. The data were collected with a PCA3 card (Oxford) as a multi-channel analyzer. The typical FWHM of the system response using a liquid scatterer is about 80 ps. The fluorescence decays were deconvoluted using IBH DAS6 software.
3. Results 3.1. Effect of lysozyme and CTAB on steady state spectra of pyranine In bulk water, pyranine exhibits two major absorption peaks at 405 and 450 nm which are ascribed to the ROH and RO forms [20]. On addition of lysozyme to an aqueous solution the absorbance at 450 nm (due to RO) decreases markedly. On addition of CTAB to an aqueous solution containing pyranine and lysozyme no appreciable change occurs in the absorption spectrum. Fig. 1 shows steady state emission spectra of pyranine in bulk water and in lysozyme. When lysozyme (0.09 and 0.5 wt%) is added to an aqueous solution of pyranine the ROH emission intensity increases followed by a concomitant decrease of the RO emission intensity. The intensity ratio of ROH:RO emission is 1:8 and 1:7 in 0.09% and 0.5% lysozyme, respectively. This is significantly larger than the intensity ratio in bulk water (1:20) and suggests suppression of ESPT in lysozyme. The emission characteristics of pyranine in lysozyme– CTAB aggregate differ significantly from those in CTAB and lysozyme alone (Figs. 1 and 2). In a 0.09 wt% lysozyme solution, addition of CTAB below 0.6 mM results in marked reduction in the intensity of both the protonated and the deprotonated forms. This is similar to the CTAB induced suppression of emission reported earlier in absence of lysozyme
Emission Intensity (a.u.)
342
0
400
450
500 550 Wavelength (nm)
600
Fig. 1. Emission spectra of pyranine in (i) water ( ); (ii) 0.5 wt% lysozyme (—-); (iii) 1.2 mM CTAB (–Æ–); (iv) 0.5 wt% lysozyme and 0.6 mM CTAB (–ÆÆ–) and (v) 0.5 wt% lysozyme and 1.2 mM CTAB (- - - -) at kex = 375 nm.
K. Sahu et al. / Chemical Physics Letters 404 (2005) 341–345
343
higher concentration of 1 mM (i.e., CMC of CTAB). The variation of ROH and RO emission intensities with CTAB concentration in presence of 0.5 wt% lysozyme is compared in Fig. 3. The ROH:RO emission intensity ratio in a lysozyme–CTAB aggregate is 1:1.
IROH (a.u.)
3.2. Effect of CTAB and lysozyme on fluorescence decays of pyranine
0.0
0.5
1.0
1.5
2.0
[CTAB] (mM) Fig. 2. Plot of emission intensity of protonated form of pyranine (IROH) against CTAB concentration in (i) aqueous solution (n), (ii) 0.09 wt% lysozyme (j) and (iii) 0.5 wt% lysozyme (s).
Emission Intensity (a. u.)
[17]. The variation of ROH emission intensity against CTAB concentration is shown in Fig. 2. At a CTAB concentration >0.6 mM, both ROH and RO emission increases sharply. Above 0.6 mM CTAB, lysozyme–CTAB complexes are formed in which ESPT of pyranine is severely suppressed. This concentration may be considered as the critical association concentration (CAC) of the lysozyme–CTAB system at 0.09 wt% lysozyme. From Fig. 2 it is evident that in 0.5 wt% lysozyme the break in the ROH emission intensity (i.e., CAC) occurs at a CTAB concentration of 0.4 mM. Thus, the CAC of CTAB depends slightly on the concentration of the lysozyme. It is also clearly seen that in the absence of lysozyme the break occurs at a
In 0.09 wt% lysozyme, fluorescence decay of the protonated form of pyranine contains contribution of both free and bound probes (Table 1). The decay at 415 nm is fitted to a tri-exponential with time constants of a bulk water like component of 150 ps (65%) and additional slow components of 700 ps (25%) and 3800 ps (10%). The 700 ps rise time may be ascribed to probes (pyranine) bound to the protein, lysozyme. The emission of RO (550 nm) when fitted to a tri-exponential, exhibits a bulk water like rise time of 150 ps and a slower rise time of 700 ± 50 ps and a decay of time constant 6000 ps. In 0.5 wt% lysozyme solution the decay characteristics of pyranine (Figs. 4 and 5, Table 1) are almost identical to those in 0.09 wt% protein. In a lysozyme–CTAB aggregate (i.e., at [CTAB] > CAC), the fluorescence decays of pyranine differ significantly from those in bulk water and lysozyme solution. The most important feature of the fluorescence decays of pyranine in a lysozyme–CTAB aggregate is the emergence of a very slow component of decay at 415 nm (Fig. 4) and of rise at 550 nm (Fig. 5) at a CTAB concentration above CAC (but below CMC) (Table 1). In a 0.5 wt% lysozyme solution at 0.6 mM CTAB, the decay at 415 nm is fitted to a tri-exponential with components of 100 ps (25%), 750 ps (15%) and 1500 ps (45%) (Fig. 4 and Table 1). The RO decay displays two rise times of 250 ps and 1200 ps with relative contribution of 2.4:1. This is followed by a decay of 5000 ps (Fig. 5 and Table 1). In the absence of lysozyme, at 1-1.2 mM CTAB, very few probe molecules remain bound to CTAB. As a result the relative contribution of micelle-like slow component of decay at 415 nm and rise at 550 nm is very small and the fluorescence decays are dominated by water like components (Table 1). However, in the presence of lysozyme, even in 1.2 mM CTAB the slow components become very prominent. For instance in the case of the rise at 550 nm, in the absence of lysozyme the amplitude of the water like fast component is 6 times larger compared to the slow rise at 1.2 mM CTAB. However, in 0.09 wt% lysozyme, at 1.2 mM CTAB the slow rise of 2000 ps is 10 times larger than the water like fast rise while in 0.5 wt% lysozyme even at 1 mM CTAB the slow rise is 16 time larger (Table 1).
4. Discussion
0.0
0.5
1.0 1.5 [CTAB] (mM)
2.0
Fig. 3. Plot of emission intensity of protonated (j) and deprotonated (s) form of pyranine against CTAB concentration in 0.5 wt% lysozyme.
The results on ESPT of pyranine in lysozyme–CTAB complex, is consistent with the necklace model of a protein (or polymer)–surfactant aggregate [3–6,33,34]. According to this model, a protein (or polymer)–surfactant aggregate resembles a necklace with spherical micelles as beads and macromolecular chains as the
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K. Sahu et al. / Chemical Physics Letters 404 (2005) 341–345
Table 1 Decay parameters of the emission from the protonated (ROH, at 415 nm) and the deprotonated (RO at 550 nm) forms of pyranine in different systems Lysozyme (wt%)
CTAB (mM)
0.00
0.0 1.0 1.2 0.0 1.0 1.2 0.0 0.6 1.0
0.09
0.50
a b
RO emissiona
ROH emissiona s1 (ps)
s2 (ps)
s3 (ps)
Rise times (ps) (X:Y)b
Decay times (ps)
150(90%) 100(83%) 100(40%) 150(75%) 100(30%) 100(25%) 150(75%) 100(25%) 100(20%)
850(10%) 350(15%) 100(45%) 700(15%) 550(35%) 600(20%) 750(15%) 750(30%) 1100(25%)
– 1000(2%) 1400(15%) 3800(10%) 1400(35%) 1800(55%) 3800(10%) 1500(45%) 2000(55%)
150 130 200,900 (6:1) 150,700 (3.5:1) 250,1400 (2.5:1) 300,2000 (1:10) 150,750 (3.5:1) 250,1200 (2.4:1) 300,2000 (1:16)
5500 1000,4000 5000 6000 5500 6000 6000 5000 6000
±10%. Ratio of amplitudes of short and long rise components.
104
Counts
103
(iv) (ii)
102
(i)
(iii)
1
10
100
0
2
4 6 Time (ns)
8
10
Fig. 4. Fluorescence decays of ROH (kem = 415 nm) in: (i) 1.0 mM CTAB; (ii) 0.5 wt% lysozyme; (iii) 0.5 wt% lysozyme and 0.6 mM CTAB and (iv) 0.5 wt% lysozyme and 1.0 mM CTAB.
10 4
3 2 ROH X d6 6 þ7 4 RO H 5 ¼ 4 k PT dt 0 RO 2
Counts
10 3 10 2
(iv)
10 1
(i)
10
We now discuss a kinetic model to analyze the result. As shown in Scheme 2, the ESPT process in pyranine consists of the following steps: (a) ultrafast deprotonation (rate constant, kPT) to form the geminate ion pair followed by (b) recombination of the geminate ion pair (rate constant, krec) and (c) dissociation of the geminate ion pair into solvent separated ion pair (rate constant, kdiss). The solvent separated ion pair may be converted back into geminate ion pair with a rate kP[H+]. At the pH (6.5) used in this work kP[H+] is negligible. kROH, and kRO denote the total decay rates (radiative and non-radiative other than proton transfer) in the excited state of ROH and RO. The time evolution of different species may be expressed in terms of the following coupled differential equations [31]: k rec Y k diss
3 2 3 0 ROH 7 6 7 0 5 4 RO Hþ 5; Z RO
ð1Þ
0
0
4
8 12 Time (ns)
16
Fig. 5. Fluorescence decays of RO (kem = 550 nm) in: (i) 1.0 mM CTAB; (ii) 0.5 wt% lysozyme; (iii) 0.5 wt% lysozyme and 0.6 mM CTAB and (iv) 0.5 wt% lysozyme and 1.0 mM CTAB.
thread. In such an aggregate, the protein segments reduce two unfavorable interactions in a surfactant aggregate (micelle), viz., electrostatic repulsion among the head groups and residual contact of the first few peripheral carbon atoms of the hydrocarbon chains with water. Thus, fewer number of surfactant molecules (aggregation number) are required for the formation of the micelles in the presence of a protein [6,34]. It is evident that the formation of such an aggregate involving a cationic surfactant (CTAB) and a positively charged protein (lysozyme) in spite of electrostatic repulsion indicates that the interaction of CTAB and lysozyme is predominantly hydrophobic in nature [7,8].
where X ¼ k PT þ k ROH k PT ; Y ¼ k rec þ k diss þ k RO ; Z ¼ k RO : According to this equation decay of the protonated (ROH) and deprotonated (RO) form are tri-exponential. From the amplitude and time constant of decay individual rate constants are obtained for CTAB and
(ROH)* + H2O kROH ROH
kPT krec
(RO-*….H3O+) kRORO-
kdiss +
kp[H ]w
RO-* + H3O+ kRORO-
Scheme 2. Kinetic scheme depicting the proton transfer process in pyranine.
K. Sahu et al. / Chemical Physics Letters 404 (2005) 341–345 Table 2 Rate constants of deprotonation (kPT), recombination (krec) and dissociation (kdiss) of geminate ion pair of pyranine at 293 K System
kPT · 103 (ps1)
krec · 103 (ps1)
kdiss · 103 (ps1)
20 mM CTAB 0.50 wt% lysozyme + 1.0 mM CTAB 0.09 wt% lysozyme + 1.2 mM CTAB Watera Methanol–watera (1.3:1)
1.00 5.00
0.14 4.20
1.00 1.60
6.30
2.80
2.40
8.00 0.80
7.20 3.20
– –
a
From Ref. [22].
lysozyme–CTAB complex. The rate constants are given in Table 2. In Table 2, we have also included the rate constants obtained using a more rigorous model based on generalized Smoluchowski theory [22–24]. It is readily seen that in a CTAB micelle the rate constant of deprotonation (kPT) is smaller than that in water but is close to that in a methanol–water mixture[22]. The recombination rate (krec) in the geminate ion pair in a micelle is found to be much smaller than that in water or methanol. In a lysozyme–CTAB complex both the deprotonation and the recombination process is found to be much faster than those in a CTAB micelle.
5. Conclusion In this work, we demonstrate that ESPT of pyranine may be used to study the interaction between lysozyme and CTAB. Lysozyme and CTAB forms a supra-molecular assembly by hydrophobic interaction, in which the proton transfer rate of pyranine is markedly retarded. This is attributed to rigidity of the water hydrogen bond network and slow solvation. The rate constant for deprotonation, recombination, and dissociation of geminate ion pair inside the lysozyme–CTAB aggregate is faster than that in a CTAB micelle. This shows that the microenvironment of pyranine in the lysozyme– CTAB aggregate is different from that in CTAB micelle.
Acknowledgments Thanks are due to Department of Science and Technology (DST), India (Project Number: IR/I1/CF-01/ 2002) and to Council of Scientific and Industrial Research (CSIR) for generous research grants. K.S., D.R., S.K.M. and R.K. thank CSIR for awarding fellowships. We thank Mr. P. Sen for useful discussions.
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