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Spectroscopic investigation of interaction between bovine serum albumin and amine-functionalized silicon quantum dots† Surajit Chatterjee and Tushar Kanti Mukherjee* We have investigated the dynamics and mechanistic details of the interaction between bovine serum albumin (BSA) and allylamine-capped silicon quantum dots (Si QDs) by means of fluorescence spectroscopy, circular dichroism (CD), and FTIR spectroscopy. The intrinsic fluorescence of BSA gets quenched in the presence of Si QDs due to ground-state complex formation. The binding stoichiometry and various thermodynamic parameters have been evaluated by using the van’t Hoff equation. It has been observed that the association process is driven by a favourable negative enthalpy change with an unfavorable negative entropy change. These results have been explained by considering specific hydrogen bonding interactions between amine moieties (–NH2) of Si QDs and carboxylate groups
Received 24th January 2014, Accepted 13th March 2014
(–COO) of aspartate (Asp) and glutamate (Glu) residues of BSA. Circular dichroism (CD) and FTIR
DOI: 10.1039/c4cp00372a
spectroscopy revealed nominal changes in the secondary structure of the adsorbed proteins due to partial unfolding of the native protein upon surface adsorption while the overall tertiary structure remains
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close to that of the native state.
1. Introduction With the recent advances in nanotechnology in the field of pharmaceutical and medical diagnostics, there is growing interest in studying the interaction of various nanoparticles (NPs) with the biological macromolecules.1–5 It is now well established that in contact with biological fluids these NPs are always surrounded by proteins, which is known as the corona.6–11 The corona formed on the NPs surface becomes the biologically significant entity and determines the ultimate cell–NP interactions. The physiochemical properties of these protein-coated NPs often differ significantly from those of the bare NPs, which may influence the cell uptake as well as its distribution. On the other hand, the adsorbed proteins may undergo conformational changes, which could lead to unwanted biological side effects. Therefore, it is essential to design a nanoparticle that would be truly specific to a given cell and cellular pathway. Specific targeting strategies of NPs involve the conjugation of NPs with various biofunctional moieties. However, the major drawback associated with these biofunctional moieties on the NPs surface is that they can hinder the membrane penetration.
Discipline of Chemistry, Indian Institute of Technology Indore, M-Block, IET-DAVV Campus, Khandwa Road, Indore-452017, M.P., India. E-mail: [email protected]; Tel: +91-731-2438738 † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c4cp00372a
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To date, various studies have been performed to understand the effect of various NPs (ZnO, Ag, Au, Si, etc.) on the structure and stability of a range of proteins.12–19 It has been shown that the structure and stability of the corona are strongly influenced by the size, surface charge and composition of the NPs. Recently, Lesniak et al. have reported that silica NPs exposed to cells in the absence of serum show stronger adhesion to the cell membrane with higher internalization efficiency in comparison to medium containing serum.8 However, it has also been proposed that stronger adhesion of the bare NPs to the cell membrane may lead to cell damage due to formation of a new kind of corona composed of proteins close to the cell membrane and cytoskeleton. In most of these studies the adsorbed proteins lose their native-like secondary structures due to either hydrophobic, electrostatic, van der Waals, or hydrogen bonding interactions with the NPs surface. Earlier, Chakrabarti and coworkers have shown that hydrophobic interactions between ZnO NPs and bovine serum albumin (BSA) have more deleterious effects on the secondary structure of the proteins compared to the electrostatic interaction between positively charged polyethyleneiminefunctionalized ZnO NPs and BSA.13 In the present work, we have investigated the effect of the amine-functionalized Si QDs on the structure and stability of bovine serum albumin. Serum albumins are the most abundant proteins in the blood plasma and their major physiological role is to carry various ligands to their respective target organs.20,21 Within the family of serum albumins, BSA is the most well studied protein
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which has a single polypeptide chain with a molecular weight of 66 kDa and consists of 583 amino acid residues and contains 76% sequence homology with Human Serum Albumin (HSA).22–29 The tertiary structure of BSA consists of three domains (I, II and III), and each domain contains two sub-domains (A and B). The intrinsic fluorescence of BSA is mainly due to two tryptophan (Trp) residues at positions 134 and 212 of the amino acid sequence. Trp 134 is located on the surface of the subdomain IB and more exposed to the surrounding environments compared to Trp 212 in the hydrophobic cavity of subdomain IIA. The majority of the secondary structure of BSA contains a-helices (B67%) with the remaining content consisting of b-sheets, turns and random coils. Being one of the most abundant blood plasma proteins, serum albumin forms the first layer of the long-lived ‘‘hard’’ corona on the NPs surface.7 Adsorption and subsequent structural dynamics of serum albumins on various NP surfaces have been studied in detail.7,9,13,17,19,30 Depending on the size and surface of NPs, one can broadly assign three distinct kinds of molecular interactions between the NPs surface and proteins, namely electrostatic, hydrophobic and hydrogen bonding interactions.12–14,31 Earlier, it has been proposed that both enthalpy- and entropy-driven adsorption processes involve electrostatic interactions, while entropy-driven adsorption involves hydrophobic interactions. The involvement of specific hydrogen bonding and van der Waals interactions has also been proposed for only the enthalpy-driven adsorption process. Quantum dots are semiconductor nanocrystals that have emerged as far better candidates for optoelectronic and biomedical applications than organic dyes.32–34 Their unique characteristics such as higher brightness, photostability and broad excitation with narrow emission band distinguish them from conventional organic dyes. As a consequence of these advantages, QDs find enormous applications in biomedical imaging as an optical marker. Extensive studies have been performed on Cd-based core–shell QDs in the past few decades. However, they have a significant drawback in biomedical applications due to their bigger size and cytotoxicity.35 Recently, silicon quantum dots (Si QDs) have emerged as a most promising candidate for biomedical imaging applications due to their smaller size and no adverse cytotoxic effects.36–42 Different types of hydrophilic molecules have been successfully attached as a capping layer on the Si QDs surface in order to disperse them in water.43–47 Although water dispersed Si QDs have successfully applied inside the cells as an optical marker, their labeling was found to be nonselective and nonspecific.48,49 One of the possible reason for this kind of nonspecific and nonselective cell labeling could be due to random aggregation inside the cell environments. Recently, we have shown that amine-functionalized Si QDs undergo random aggregation in the presence of a negatively charged water–surfactant interface similar to that encountered in a biological cell membrane.50 Earlier, Salvati et al. have shown that the specific targeting ability of transferring-functionalized silica NPs disappears in the presence of fetal bovine serum (FBS) due to the formation of the corona.6 It has been proposed that proteins in the biological media can shield transferring from binding to its targeted receptors on cells. Earlier, Tenzer et al.
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have also studied the effect of different sized amorphous silica NPs on the physicochemical properties of the human blood plasma corona by liquid chromatography mass spectrometry.7 They have identified around 125 proteins in the corona from the blood plasma. In the literature, there are no earlier reports on the effect of these amine-functionalized hydrophilic Si QDs on the structure and stability of the serum albumin proteins. This motivates us to investigate the effect of these hydrophilic Si QDs on the structure and stability of bovine serum albumin (BSA). The aim of the present study is twofold: first, we want to know the effect of these hydrophilic Si QDs on the structure and stability of the adsorbed BSA and second, the effect of surface amine moieties on the mechanistic aspects of BSA–QD interactions.
2. Experimental section 2.1.
Materials
Silicon tetrachloride (99%) and tetrahydrofuran (THF, 99.5%) were purchased from Merck (Germany). Allylamine (99%) was purchased from Spectrochem (India). Bovine serum albumin (BSA, Z99%, essentially fatty acid free and globulin free), tetraoctylammonium bromide (TOAB, 98%), chloroplatinic acid hexahydrate, and isopropyl alcohol (99%) were purchased from Sigma-Aldrich. Lithium aluminum hydride (LAH, 97%) and toluene (99%) were purchased from SD Fine chemicals (India). Milli-Q water was obtained from a Millipore water purifier system (Milli-Q integral). 2.2.
Synthesis
Allylamine capped Si QDs were synthesized in reverse micelles according to literature (Scheme 1A).43 In brief, the hydrogenterminated Si QDs were synthesized under an argon atmosphere by reduction of SiCl4 by LAH. TOAB (1.5 g) was dispersed in dry toluene (100 mL) by stirring for 30 min. 92 mL of SiCl4 was added through a gastight syringe, followed by stirring for 1 h. Hydrogen-terminated Si QDs were then formed by adding 2 mL of 1 M LAH in THF. The mixture was left to react for 3 h. Anhydrous methanol (20 mL) was added to oxidize the excess LAH. Hydrophilic particles were then formed by modifying the surface Si–H bond by reaction with 100 mL of 0.1 M chloroplatinic acid hexahydrate in isopropanol as a catalyst and 2 mL of allylamine. The solution was then left to stir for 3 h. All solvents were then removed by rotary evaporation to produce
Scheme 1 (A) Synthesis of allylamine-capped Si QDs in TOAB micelles. (B) Schematic representation of the native BSA structure with relative positions of two Trp residues (yellow asterisks).
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a white dry powder consisting of mainly TOAB and Si QDs. The dry powder was then redispersed in 20 mL of distilled water and sonicated for 30 min. Allylamine-capped Si QDs were dissolved in water, and undissolved TOAB was then removed by successive filtration through a 0.22 mm membrane filter. On an average each Si QD contains around 24 allylamine molecules on the surface.51 The concentration of synthesized Si QDs solution was determined spectrophotometrically using the extinction coefficient at 260 nm (e260) of 2.6 105 M1 cm1.52 2.3.
Instrumentation
Absorption spectra were recorded in a quartz cuvette (10 10 mm) using a Varian UV-Vis spectrophotometer (Carry 100 Bio). PL spectra were recorded using a Fluoromax-4 spectrofluorimeter (HORIBA Jobin Yvon, model FM-100) with excitation and emission slit width at 4 nm. All measurements were performed at room temperature. For pKa determination of allylamine-capped Si QDs the solution pH was adjusted from 2 to 10 using a pH meter (Eutech Instruments). CD spectra of BSA were acquired by a Jasco J-815 CD spectropolarimeter using a quartz cell of 1 mm path length. Scans were made with a slit width of 1 mm and a speed of 20 nm min1. The far-UV CD spectra of BSA in the absence and presence of Si QDs were analyzed using CDNN software. AFM images were recorded on a cleaned glass coverslip using an AIST-NT microscope (model SmartSPM-1000). ATR-FTIR measurements were carried out in a Bruker spectrometer (Tensor-27). Fluorescence decays were recorded on a HORIBA Jobin Yvon picosecond time correlated single photon counting (TCSPC) spectrometer (model Fluorocube-01-NL). The samples were excited at 279 nm by a nanosecond diode laser (Deltadiode, Model: DD-280). The decays were collected using the emission polarizer at a magic angle of 54.71 using a photomultiplier tube (TBX-07C). The instrument response function (IRF, fwhm B1.2 ns) was recorded using a dilute scattering solution. The PL decays were analyzed using IBH DAS 6.0 software by the iterative reconvolution method, and the goodness of the fit was judged by a reduced w-square (w2) value. 2.4.
Sample preparation
All solutions were prepared in pH 7.4 phosphate buffer. For pKa estimation the pH of the aqueous solution was adjusted with either HCl or NaOH. For steady-state and time-resolved fluorescence experiments different concentrations of BSA samples were prepared by dissolving a required amount (MBSA = 66 000 g mol1) of BSA in pH 7.4 phosphate buffer. The final concentrations were determined spectrophotometrically using the extinction coefficient at 280 nm (e280) of 44 720 M1 cm1.20,53 For the AFM image the sample was spin coated with a spin-coater (Apex Instruments, Spin NXG-P1) on a clean glass cover slide. The cover slides were first cleaned with 2% Hellmanex III (Sigma-Aldrich) followed by piranha solution (3 : 1 concentrated sulfuric acid and 30% hydrogen peroxide). After each cleaning step the cover slides were repeatedly washed with Milli-Q water and methanol (Merck). Finally, the slides were dried in a hot oven at 100 1C. The QDs sample was spin-casted on washed cover slides at 2000 rpm for 3 min.
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3. Results and discussion 3.1.
Characterization of Si QDs
Fig. 1A shows the AFM image of synthesized allylamine-capped Si QDs. The size distribution of Si QDs is determined by analyzing 140 QDs from the AFM image. They show a mean diameter of 3.41 0.10 nm with a spherical shape (Fig. 1A). The attachment of allylamine moieties on the Si QDs surface is confirmed by FTIR spectroscopy (ESI,† Fig. S1). The characteristic peak at 1637 cm1 indicates the attachment of allylamine at the surface of Si QDs. The peaks at 1462 and 1261 cm1 are assigned to the vibrational scissoring and symmetric bending of Si–CH2. The peaks between 2500 and 3500 cm1 arise due to the symmetric and asymmetric vibrations of C–CH2 and C–NH2 moieties.50 These Si QDs show a continuous absorption between 200 and 500 nm with size dependent PL in the visible region. Excitation at 375 nm results in a luminescence band centered at 455 nm (Fig. 1B).
3.2.
Steady-state and time-resolved measurements
The peak position and quantum yield of the 455 nm PL band of Si QDs (lex = 375 nm) remain unchanged in the presence of BSA (ESI,† Fig. S2). However, significant changes have been observed for BSA emission in the presence of Si QDs. It is well known that the intrinsic fluorescence of BSA is mainly due to the presence of two Trp residues at position 134 and 212 in domain IB and IIA respectively (Scheme 1B). Any changes in the fluorescence quantum yield or the peak position directly indicate changes in polarity surrounding these two Trp residues due to unfolding of the native conformation. Therefore, the intrinsic fluorescence of BSA is used to monitor the binding of Si QDs to BSA. BSA in its native state in pH 7.4 phosphate buffer shows an intense fluorescence centered at 339 nm (lex = 280 nm). Fig. 2A displays the effect of increasing concentration of Si QDs on the intrinsic fluorescence of BSA. With gradual addition of Si QDs the intrinsic fluorescence of BSA gets quenched. The intrinsic fluorescence of BSA is quenched by 56% with a red shift of 3 nm in the presence of 1.50 mM Si QDs. These spectral changes clearly indicate the association of BSA with Si QDs and subsequent unfolding of the native state.
Fig. 1 (A) AFM image of synthesized allylamine-capped Si QDs. The inset shows the size distribution histogram. (B) Absorption (black line) and normalized PL (blue line) spectra (lex = 375 nm) of allylamine-capped Si QDs.
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Fig. 2 (A) Fluorescence spectra (lex = 280 nm) of 2 mM BSA in pH 7.4 phosphate buffer at 298 K with different concentrations of allylamine-capped Si QDs. (B) Steady-state Stern–Volmer plot for quenching of BSA fluorescence by Si QDs at 298 K. (C) Lifetime decay curves (lex = 279 nm) of 2 mM BSA in the absence and presence of 1.50 mM Si QDs. (D) Overlap of steady-state and time-resolved Stern–Volmer plots.
The mechanism of observed fluorescence quenching could be due to either static, dynamic or a combination of both these processes. To reveal the mechanism and the extent of quenching, we have used the Stern–Volmer (SV) equation which can be expressed as: F0 ¼ 1 þ KSV ½Q ¼ 1 þ kq t0 ½Q F
(1)
where F0 and F are the fluorescence intensities in the absence and presence of a quencher, respectively. KSV is the Stern–Volmer (SV) constant and [Q] is the molar concentration of quencher. kq is the bimolecular quenching rate constant and t0 is the fluorescence lifetime of the fluorophore in the absence of a quencher. The SV constant and quenching rate constant can be evaluated from a plot of F0/F against [Q]. Fig. 2B shows the steady-state SV plot of the BSA–QDs conjugate at 298 K. The plot is linear in nature which indicates that either the static or dynamic type of quenching mechanism is responsible for the observed fluorescence quenching. The SV constant is evaluated from the slope of the plot and at 298 K it has a value of 8.23 105 M1. The bimolecular quenching rate constant has been calculated by using t0 = 5.6 ns of BSA (Table 1) to be 1.46 1014 M1 s1 at 298 k which is four orders of magnitude higher than that for a purely dynamic quenching
Table 1 Fluorescence lifetime components of BSA (lex = 279 nm) in the absence and presence of different concentrations of Si QDs in pH 7.4 phosphate buffer
Sample
t1 (ns) a1
BSA 2 mM 3.60 BSA 2 mM + 1 mM Si QDs 3.80 BSA 2 mM + 1.5 mM Si QDs 3.50
t2 (ns) a2
0.39 6.80 0.43 6.80 0.38 6.60
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hti (ns)
w2
0.61 5.60 0.04 1.03 0.57 5.56 0.01 1.06 0.62 5.45 0.02 1.09
mechanism.54,55 Hence, in the present system the observed fluorescence quenching of BSA by Si QDs might be due to static ground state complex formation and not due to collisional encounter. A similar kind of static quenching mechanism has been proposed earlier for other protein–NP complexes.12–14,17 Time-resolved lifetime measurements have been performed to investigate the dynamics of the BSA–QDs complex. Fig. 2C displays the typical lifetime decays of BSA in the absence and presence of 1.50 mM Si QDs. In the absence of Si QDs, BSA shows a biexponential decay curve with an average lifetime of 5.60 0.04 ns and having lifetime components of 3.60 ns and 6.80 ns with the relative abundance of 39% and 61%, respectively. A similar kind of biexponential fluorescence decay of BSA has been observed earlier and assigned to the presence of two Trp residues at different conformational states in two distinct chemical environments.29 The decay curve of BSA remains unaltered in the presence of different concentrations of Si QDs. The fluorescence decay of BSA remains biexponential in the presence of 1.50 mM Si QDs with an average lifetime of 5.45 0.02 ns having lifetime components of 3.50 ns and 6.60 ns with the relative abundance of 38% and 62%, respectively. Although the average lifetime of BSA does not change significantly in the presence of Si QDs, it is important to note the small change observed in average lifetime of BSA from 5.60 ns to 5.45 ns. This nominal decrease in lifetime in the presence of the highest concentration of Si QDs used in the present study could be due to the local conformational alterations of BSA upon adsorption. A time-resolved Stern–Volmer plot was generated using the Stern–Volmer equation P with average lifetime hti = aiti and can be expressed as follows: ht0 i ¼ 1 þ KSV ½Q hti
(2)
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where ht0i and hti are the average fluorescence lifetimes of BSA in the absence and presence of a quencher, respectively. Fig. 2D shows the overlap of steady-state and time-resolved Stern–Volmer plots for the BSA–QDs complex. The time-resolved Stern–Volmer plot is parallel to the x-axis indicating a static quenching mechanism. Based on these results it is hard to determine which Trp of BSA gets quenched upon adsorption. However, the two Trp residues (Trp 134 and 212) of BSA located at two separate domains in two distinct chemical environments. Trp 134 is located at the surface of the protein in subdomain IB while Trp 212 is located in the hydrophobic cavity of subdomain IIA. Hydrophobic interactions will lead to binding of Si QDs at the hydrophobic binding pocket at subdomain IIA near Trp 212 while electrostatic and hydrogen bonding interactions will lead to binding near the Trp 134 residue. Therefore, the nature of the interactions between Si QDs and BSA will allow us to determine which Trp gets quenched during the binding process. To address this fundamental question we calculated various thermodynamic parameters, which have been discussed in the next section. 3.3.
Effect of temperature and thermodynamics of binding
In order to investigate the effect of temperature on the BSA–QDs association process we have evaluated Ksv at different temperatures ranging from 298 K to 318 K. Fig. 3A displays the Stern– Volmer plots for the fluorescence quenching of BSA by Si QDs at
four different temperatures. All the plots are linear indicating a single type of quenching mechanism. The calculated SV constants at different temperatures are listed in Table 2. The gradual decrease of the SV constant with increase in temperature indicates ground state complex formation between BSA and Si QDs resulting in static quenching. The binding constant (Kb) and the number of binding sites (n) were determined using the Scatchard equation, which can be expressed as follows: ðF 0 F Þ log (3) ¼ log Kb þ n log½Q F where Kb is the binding constant of the protein–ligand complex, and n is the number of binding sites. The plot of log[(F0 F)/F] against log[Q] should yield a straight line with an intercept equal to log Kb and a slope equal to n. Fig. 3B shows the plot of log[(F0 F)/F] against log[Si QDs] at four different temperatures. All the plots are linear and the estimated binding constants and the number of binding sites are listed in Table 2. The estimated binding constant for the BSA–QDs system is 3.40 0.08 106 M1 at 298 K, similar to those previously obtained for different NP–protein complexes.12–14,17,30 The binding constant for the BSA–QDs complex decreases with increase in temperature, indicating the static nature of the complex. The number of binding sites i.e. the number of QDs per BSA is found to be nearly 1. The driving force for the formation of a ground state complex between Si QDs and BSA could be due to various molecular interactions namely, electrostatic, hydrophobic, hydrogen bond, or van der Waals interactions.31 For a simple two-state model of equilibrium as in the present system the thermodynamic parameters (enthalpy change, entropy change, and Gibbs free energy change) can be calculated from the van’t Hoff equation which can be expressed as follows: ln Kb ¼
DS DH R RT
(4)
where Kb is the binding constant, R is the gas constant, DS is the standard entropy change and DH is the standard enthalpy change during the BSA–QDs association reaction at temperature T. The changes in enthalpy (DH) and entropy (DS) of the association process between BSA and Si QDs have been evaluated by plotting ln Kb against 1/T for four different temperatures (Fig. 3C). The Gibbs free energy change (DG) during the binding process can be estimated from the equation: DG = DH TDS
Fig. 3 (A) Steady-state Stern–Volmer plots at four different temperatures. (B) Plots showing the effect of temperature on the binding constant between BSA and Si QDs. (C) The van’t Hoff plot for the BSA–Si QDs system. The equation of the fitted line is ln Kb = 5508.79/T 3.42 (R2 = 0.999).
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(5)
The calculated thermodynamic parameters for the present system are listed in Table 2. The association reaction is found to have a favorable enthalpy change (DH o 0), which is compensated by an unfavorable entropy loss (DS o 0), giving rise to an overall negative free energy change. Earlier, based on the magnitude and sign of the thermodynamic parameters various groups have proposed different mechanisms for the association of various ligands with proteins. In general, association processes triggered by hydrophobic interactions between proteins and ligands proceed with a large positive
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Table 2 Stern–Volmer quenching constant (KSV), binding constants (Kb), the number of binding sites (n) and thermodynamic parameters for the BSA–Si QDs complex
T (K)
KSV (105 M1)
Kb (106 M1)
298 308 313 318
8.23 7.74 7.18 6.69
3.40 2.10 1.47 1.05
0.08 0.04 0.06 0.05
n 1.10 1.07 1.06 1.04
0.01 0.01 0.01 0.01
entropy change with a positive enthalpy change while processes triggered by electrostatic interactions proceed with a positive entropy change with a small positive or negative enthalpy change.31 However, our present thermodynamic parameters are not correlated with either of these two molecular interactions. A similar kind of negative enthalpy change with a negative entropy change was observed earlier in many association processes and has been assigned to involvement of specific hydrogen bonding interactions.31 Here, it is important to note that the magnitude and sign of the thermodynamic parameters strongly depend on the surface charge and size of the associating species. Therefore a closer look at the surface properties of BSA and Si QDs is required to propose a mechanism of their association process based on the thermodynamic parameters. The isoelectric point (pI) of BSA is 4.7 at 25 1C and hence at physiological pH (pH = 7.4) the carboxylic groups (–COOH) will remain in deprotonated form (–COO) with a net negative charge on them. Out of the three domains of BSA, domain I contains the majority of the negatively charged aspartate (Asp) and glutamate (Glu) residues followed by domain II. Domain III of BSA is neutral.56 On the other hand the estimated pKa of the
R2
DG (kJ mol1)
0.999 0.999 0.999 0.999
37.31 37.02 36.88 36.74
DH (kJ mol1)
DS (J mol1 K1)
45.78 0.27
28.42
ammonium ions (–NH3+) on the Si QDs surface is 5.7 (ESI,† Fig. S3). Hence, at pH 7.4 these surface amine moieties on Si QDs will remain unprotonated (–NH2) which is in agreement with our earlier observation.50 By considering the charges of BSA and Si QDs at pH 7.4 and the estimated thermodynamic parameters, we propose that the most probable mechanism of their association process involves specific hydrogen bonding interactions between negatively charged –COO groups of aspartate (Asp) and glutamate (Glu) residues of BSA and surface amine groups of Si QDs (Scheme 2). Here it is important to mention that both domains I and II of BSA are involved in hydrogen bond formation with the Si QDs surface. Although only surface exposed –COO groups of BSA from domains I and II form hydrogen bonds with Si QDs, we cannot rule out the conformational changes around the Trp 212 at the hydrophobic cavity of subdomain IIA which may contribute to the observed fluorescence quenching. Hence, the observed Trp quenching in the earlier section might be due to both the Trp residues (Trp 134 and Trp 212) of BSA. Whereas the unfavorable negative entropy changes during the association process are due to the conformational restriction of both proteins and Si QDs upon binding.
Scheme 2 Schematic representation of the association process of BSA with different surface funtionalized NPs or the QD surface driven by either electrostatic, hydrophobic or hydrogen bonding interactions. Different types of interactions proceed with characteristic changes in thermodynamic parameters and lead to different extent of structural destabilisation of the native conformation of protein. The present proposed model is highlighted in the scheme.
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3.4.
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Secondary structure of BSA in the presence of Si QDs
The far-UV CD and FTIR spectroscopy was used to monitor the changes in the secondary structure of BSA upon adsorption onto the Si QDs surface. Fig. 4A shows the CD spectra of BSA with increasing concentration of Si QDs. Native BSA at pH 7.4 shows two characteristic minima at 208 and 222 nm in the CD spectrum with 65% a-helix and 6.5% b-sheet content (Table 3). Notably, the secondary structure of the native protein does not change significantly in the presence of highest concentration of Si QDs used in this study. The secondary structure analysis of BSA in the presence of 1.50 mM Si QDs shows that the a-helix content decreases to 61.2% while the b-sheet content increases to 7.7% (Table 3). However, the shape and peak positions remain the same in the presence of Si QDs. These spectral changes demonstrate marginal alteration (5.8% loss of helicity) in the secondary structure of adsorbed BSA. Earlier, Vertegel et al. have reported significant structural destabilization (61–80% loss of helicity) and loss of enzymatic activity of positively charged lysozyme (pI = 11) on negatively charged silica NPs (pI = 3) at physiological pH.16 Similarly, Shang et al. have studied the structural perturbation and unfolding of positively charged ribonuclease A (pI = 9.4) on negatively charged silica NPs.15 From these earlier studies it is clear that strong electrostatic interactions between protein and NPs result in significant structural alteration of the native conformation due to coulombic
Fig. 4 (A) The far-UV CD spectra of BSA (2 mM) in the absence and presence of different concentrations of Si QDs. (B) FTIR spectra of BSA (black line) and BSA in the presence of 1.50 mM allylamine-capped Si QDs (red line).
Table 3 Secondary structure analysis of BSA in the absence and presence of different concentrations of Si QDs
Sample BSA BSA BSA BSA
2 2 2 2
mM mM + 0.60 mM Si QDs mM + 1.13 mM Si QDs mM + 1.50 mM Si QDs
a-Helix
b-Sheet
Turn
Random coil
65.0 64.1 62.1 61.2
6.5 6.8 7.3 7.7
12.3 12.4 12.7 12.9
16.2 16.7 18.0 18.2
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attractions. The structure and stability of BSA on various NP surfaces have also been explored in great detail. Recently, it has been reported that the structural destabilization of BSA on bare hydrophobic ZnO NPs was greater than polyethyleneiminefunctionalized ZnO NPs.13 However, in the present system the observed nominal changes in the secondary structure of the adsorbed BSA on the Si QDs surface is due to the specific hydrogen bonding interactions between surface exposed –COO groups of BSA and –NH2 moieties of allylamine-capped Si QDs (Scheme 2). Therefore it is evident from our present results and literature that the adsorbed protein undergoes a maximum amount of structural destabilization for association processes driven by hydrophobic interactions followed by electrostatic and hydrogen bond induced processes (i.e. hydrophobic Z electrostatic 4 hydrogen bonding interactions). The adsorption and subsequent structural alterations of BSA on the Si QDs surface have been further confirmed by FTIR spectroscopy. The amide I and amide II bands of proteins in the region of 1600–1700 cm1 and 1500–1600 cm1 respectively provide useful information about their conformation in native and unfolded states.12–14 Fig. 4B shows the FTIR spectra of BSA in the absence and presence of Si QDs. In the absence of Si QDs, BSA shows two peaks at 1648 cm1 and 1547 cm1 assigned to amide I and amide II, respectively. However, in the presence of Si QDs, the amide I band shifted to 1649 cm1 while the amide II band shifted to 1549 cm1 with decreased intensity. Similar spectral changes in amide I and amide II bands of BSA have been observed earlier.13 The reduced intensity with a shift in amide I and amide II band positions clearly indicates the adsorption of BSA on the Si QDs surface and subsequent alteration in its local conformation. Earlier, it has been proposed that hydrophobic as well as electrostatic interactions between proteins and NPs have more deleterious effects on the structure and activity of proteins compared to other types of interactions. However, unfolding and loss of activity of the adsorbed proteins could be of potential risk for the cells which may trigger a sequence of adverse reactions. Serum albumin, being the most abundant plasma protein, forms the first layer of the corona on the NPs surface. Hence, it is of utmost importance to know the structure and stability of the plasma protein corona on the NPs or QDs surface to understand their physicochemical effects inside biological cell environments. Based on our present results we propose a model where specific hydrogen bonding interactions between surface exposed –COO groups of aspartate (Asp) and glutamate (Glu) residues of BSA and amine moieties (–NH2) of Si QDs drive the association process. We believe that the observed minor alteration of the secondary structure of adsorbed BSA is mainly due to the partial unfolding of domain I as well as domain II. However, BSA retains its overall tertiary structure upon surface adsorption.
4. Conclusion In this paper, we have demonstrated the dynamics and mechanistic details of BSA adsorption on the amine-functionalized Si
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QDs surface. The adsorption process is mainly due to ground state complex formation which leads to static fluorescence quenching. The extent of fluorescence quenching decreases with an increase in temperature which further highlights the static mechanism of fluorescence quenching. The estimated thermodynamic parameters suggest the involvement of specific hydrogen bonding interactions between surface amine groups (–NH2) of Si QDs and –COO groups of BSA. CD and FTIR spectra revealed nominal alteration in the secondary structure of adsorbed BSA on the Si QDs surface.
Acknowledgements The authors thank IIT Indore for proving the infrastructure, experimental facilities, and financial support. This work is supported by Council of Scientific and Industrial Research grant no. 01(2695)/12/EMR-II. The authors also thank the reviewers for their constructive comments.
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Cite this: Phys. Chem. Chem. Phys., 2014, 16, 8400
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Spectroscopic investigation of interaction between bovine serum albumin and amine-functionalized silicon quantum dots† Surajit Chatterjee and Tushar Kanti Mukherjee* We have investigated the dynamics and mechanistic details of the interaction between bovine serum albumin (BSA) and allylamine-capped silicon quantum dots (Si QDs) by means of fluorescence spectroscopy, circular dichroism (CD), and FTIR spectroscopy. The intrinsic fluorescence of BSA gets quenched in the presence of Si QDs due to ground-state complex formation. The binding stoichiometry and various thermodynamic parameters have been evaluated by using the van’t Hoff equation. It has been observed that the association process is driven by a favourable negative enthalpy change with an unfavorable negative entropy change. These results have been explained by considering specific hydrogen bonding interactions between amine moieties (–NH2) of Si QDs and carboxylate groups
Received 24th January 2014, Accepted 13th March 2014
(–COO) of aspartate (Asp) and glutamate (Glu) residues of BSA. Circular dichroism (CD) and FTIR
DOI: 10.1039/c4cp00372a
spectroscopy revealed nominal changes in the secondary structure of the adsorbed proteins due to partial unfolding of the native protein upon surface adsorption while the overall tertiary structure remains
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close to that of the native state.
1. Introduction With the recent advances in nanotechnology in the field of pharmaceutical and medical diagnostics, there is growing interest in studying the interaction of various nanoparticles (NPs) with the biological macromolecules.1–5 It is now well established that in contact with biological fluids these NPs are always surrounded by proteins, which is known as the corona.6–11 The corona formed on the NPs surface becomes the biologically significant entity and determines the ultimate cell–NP interactions. The physiochemical properties of these protein-coated NPs often differ significantly from those of the bare NPs, which may influence the cell uptake as well as its distribution. On the other hand, the adsorbed proteins may undergo conformational changes, which could lead to unwanted biological side effects. Therefore, it is essential to design a nanoparticle that would be truly specific to a given cell and cellular pathway. Specific targeting strategies of NPs involve the conjugation of NPs with various biofunctional moieties. However, the major drawback associated with these biofunctional moieties on the NPs surface is that they can hinder the membrane penetration.
Discipline of Chemistry, Indian Institute of Technology Indore, M-Block, IET-DAVV Campus, Khandwa Road, Indore-452017, M.P., India. E-mail: [email protected]; Tel: +91-731-2438738 † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c4cp00372a
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To date, various studies have been performed to understand the effect of various NPs (ZnO, Ag, Au, Si, etc.) on the structure and stability of a range of proteins.12–19 It has been shown that the structure and stability of the corona are strongly influenced by the size, surface charge and composition of the NPs. Recently, Lesniak et al. have reported that silica NPs exposed to cells in the absence of serum show stronger adhesion to the cell membrane with higher internalization efficiency in comparison to medium containing serum.8 However, it has also been proposed that stronger adhesion of the bare NPs to the cell membrane may lead to cell damage due to formation of a new kind of corona composed of proteins close to the cell membrane and cytoskeleton. In most of these studies the adsorbed proteins lose their native-like secondary structures due to either hydrophobic, electrostatic, van der Waals, or hydrogen bonding interactions with the NPs surface. Earlier, Chakrabarti and coworkers have shown that hydrophobic interactions between ZnO NPs and bovine serum albumin (BSA) have more deleterious effects on the secondary structure of the proteins compared to the electrostatic interaction between positively charged polyethyleneiminefunctionalized ZnO NPs and BSA.13 In the present work, we have investigated the effect of the amine-functionalized Si QDs on the structure and stability of bovine serum albumin. Serum albumins are the most abundant proteins in the blood plasma and their major physiological role is to carry various ligands to their respective target organs.20,21 Within the family of serum albumins, BSA is the most well studied protein
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which has a single polypeptide chain with a molecular weight of 66 kDa and consists of 583 amino acid residues and contains 76% sequence homology with Human Serum Albumin (HSA).22–29 The tertiary structure of BSA consists of three domains (I, II and III), and each domain contains two sub-domains (A and B). The intrinsic fluorescence of BSA is mainly due to two tryptophan (Trp) residues at positions 134 and 212 of the amino acid sequence. Trp 134 is located on the surface of the subdomain IB and more exposed to the surrounding environments compared to Trp 212 in the hydrophobic cavity of subdomain IIA. The majority of the secondary structure of BSA contains a-helices (B67%) with the remaining content consisting of b-sheets, turns and random coils. Being one of the most abundant blood plasma proteins, serum albumin forms the first layer of the long-lived ‘‘hard’’ corona on the NPs surface.7 Adsorption and subsequent structural dynamics of serum albumins on various NP surfaces have been studied in detail.7,9,13,17,19,30 Depending on the size and surface of NPs, one can broadly assign three distinct kinds of molecular interactions between the NPs surface and proteins, namely electrostatic, hydrophobic and hydrogen bonding interactions.12–14,31 Earlier, it has been proposed that both enthalpy- and entropy-driven adsorption processes involve electrostatic interactions, while entropy-driven adsorption involves hydrophobic interactions. The involvement of specific hydrogen bonding and van der Waals interactions has also been proposed for only the enthalpy-driven adsorption process. Quantum dots are semiconductor nanocrystals that have emerged as far better candidates for optoelectronic and biomedical applications than organic dyes.32–34 Their unique characteristics such as higher brightness, photostability and broad excitation with narrow emission band distinguish them from conventional organic dyes. As a consequence of these advantages, QDs find enormous applications in biomedical imaging as an optical marker. Extensive studies have been performed on Cd-based core–shell QDs in the past few decades. However, they have a significant drawback in biomedical applications due to their bigger size and cytotoxicity.35 Recently, silicon quantum dots (Si QDs) have emerged as a most promising candidate for biomedical imaging applications due to their smaller size and no adverse cytotoxic effects.36–42 Different types of hydrophilic molecules have been successfully attached as a capping layer on the Si QDs surface in order to disperse them in water.43–47 Although water dispersed Si QDs have successfully applied inside the cells as an optical marker, their labeling was found to be nonselective and nonspecific.48,49 One of the possible reason for this kind of nonspecific and nonselective cell labeling could be due to random aggregation inside the cell environments. Recently, we have shown that amine-functionalized Si QDs undergo random aggregation in the presence of a negatively charged water–surfactant interface similar to that encountered in a biological cell membrane.50 Earlier, Salvati et al. have shown that the specific targeting ability of transferring-functionalized silica NPs disappears in the presence of fetal bovine serum (FBS) due to the formation of the corona.6 It has been proposed that proteins in the biological media can shield transferring from binding to its targeted receptors on cells. Earlier, Tenzer et al.
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have also studied the effect of different sized amorphous silica NPs on the physicochemical properties of the human blood plasma corona by liquid chromatography mass spectrometry.7 They have identified around 125 proteins in the corona from the blood plasma. In the literature, there are no earlier reports on the effect of these amine-functionalized hydrophilic Si QDs on the structure and stability of the serum albumin proteins. This motivates us to investigate the effect of these hydrophilic Si QDs on the structure and stability of bovine serum albumin (BSA). The aim of the present study is twofold: first, we want to know the effect of these hydrophilic Si QDs on the structure and stability of the adsorbed BSA and second, the effect of surface amine moieties on the mechanistic aspects of BSA–QD interactions.
2. Experimental section 2.1.
Materials
Silicon tetrachloride (99%) and tetrahydrofuran (THF, 99.5%) were purchased from Merck (Germany). Allylamine (99%) was purchased from Spectrochem (India). Bovine serum albumin (BSA, Z99%, essentially fatty acid free and globulin free), tetraoctylammonium bromide (TOAB, 98%), chloroplatinic acid hexahydrate, and isopropyl alcohol (99%) were purchased from Sigma-Aldrich. Lithium aluminum hydride (LAH, 97%) and toluene (99%) were purchased from SD Fine chemicals (India). Milli-Q water was obtained from a Millipore water purifier system (Milli-Q integral). 2.2.
Synthesis
Allylamine capped Si QDs were synthesized in reverse micelles according to literature (Scheme 1A).43 In brief, the hydrogenterminated Si QDs were synthesized under an argon atmosphere by reduction of SiCl4 by LAH. TOAB (1.5 g) was dispersed in dry toluene (100 mL) by stirring for 30 min. 92 mL of SiCl4 was added through a gastight syringe, followed by stirring for 1 h. Hydrogen-terminated Si QDs were then formed by adding 2 mL of 1 M LAH in THF. The mixture was left to react for 3 h. Anhydrous methanol (20 mL) was added to oxidize the excess LAH. Hydrophilic particles were then formed by modifying the surface Si–H bond by reaction with 100 mL of 0.1 M chloroplatinic acid hexahydrate in isopropanol as a catalyst and 2 mL of allylamine. The solution was then left to stir for 3 h. All solvents were then removed by rotary evaporation to produce
Scheme 1 (A) Synthesis of allylamine-capped Si QDs in TOAB micelles. (B) Schematic representation of the native BSA structure with relative positions of two Trp residues (yellow asterisks).
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a white dry powder consisting of mainly TOAB and Si QDs. The dry powder was then redispersed in 20 mL of distilled water and sonicated for 30 min. Allylamine-capped Si QDs were dissolved in water, and undissolved TOAB was then removed by successive filtration through a 0.22 mm membrane filter. On an average each Si QD contains around 24 allylamine molecules on the surface.51 The concentration of synthesized Si QDs solution was determined spectrophotometrically using the extinction coefficient at 260 nm (e260) of 2.6 105 M1 cm1.52 2.3.
Instrumentation
Absorption spectra were recorded in a quartz cuvette (10 10 mm) using a Varian UV-Vis spectrophotometer (Carry 100 Bio). PL spectra were recorded using a Fluoromax-4 spectrofluorimeter (HORIBA Jobin Yvon, model FM-100) with excitation and emission slit width at 4 nm. All measurements were performed at room temperature. For pKa determination of allylamine-capped Si QDs the solution pH was adjusted from 2 to 10 using a pH meter (Eutech Instruments). CD spectra of BSA were acquired by a Jasco J-815 CD spectropolarimeter using a quartz cell of 1 mm path length. Scans were made with a slit width of 1 mm and a speed of 20 nm min1. The far-UV CD spectra of BSA in the absence and presence of Si QDs were analyzed using CDNN software. AFM images were recorded on a cleaned glass coverslip using an AIST-NT microscope (model SmartSPM-1000). ATR-FTIR measurements were carried out in a Bruker spectrometer (Tensor-27). Fluorescence decays were recorded on a HORIBA Jobin Yvon picosecond time correlated single photon counting (TCSPC) spectrometer (model Fluorocube-01-NL). The samples were excited at 279 nm by a nanosecond diode laser (Deltadiode, Model: DD-280). The decays were collected using the emission polarizer at a magic angle of 54.71 using a photomultiplier tube (TBX-07C). The instrument response function (IRF, fwhm B1.2 ns) was recorded using a dilute scattering solution. The PL decays were analyzed using IBH DAS 6.0 software by the iterative reconvolution method, and the goodness of the fit was judged by a reduced w-square (w2) value. 2.4.
Sample preparation
All solutions were prepared in pH 7.4 phosphate buffer. For pKa estimation the pH of the aqueous solution was adjusted with either HCl or NaOH. For steady-state and time-resolved fluorescence experiments different concentrations of BSA samples were prepared by dissolving a required amount (MBSA = 66 000 g mol1) of BSA in pH 7.4 phosphate buffer. The final concentrations were determined spectrophotometrically using the extinction coefficient at 280 nm (e280) of 44 720 M1 cm1.20,53 For the AFM image the sample was spin coated with a spin-coater (Apex Instruments, Spin NXG-P1) on a clean glass cover slide. The cover slides were first cleaned with 2% Hellmanex III (Sigma-Aldrich) followed by piranha solution (3 : 1 concentrated sulfuric acid and 30% hydrogen peroxide). After each cleaning step the cover slides were repeatedly washed with Milli-Q water and methanol (Merck). Finally, the slides were dried in a hot oven at 100 1C. The QDs sample was spin-casted on washed cover slides at 2000 rpm for 3 min.
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3. Results and discussion 3.1.
Characterization of Si QDs
Fig. 1A shows the AFM image of synthesized allylamine-capped Si QDs. The size distribution of Si QDs is determined by analyzing 140 QDs from the AFM image. They show a mean diameter of 3.41 0.10 nm with a spherical shape (Fig. 1A). The attachment of allylamine moieties on the Si QDs surface is confirmed by FTIR spectroscopy (ESI,† Fig. S1). The characteristic peak at 1637 cm1 indicates the attachment of allylamine at the surface of Si QDs. The peaks at 1462 and 1261 cm1 are assigned to the vibrational scissoring and symmetric bending of Si–CH2. The peaks between 2500 and 3500 cm1 arise due to the symmetric and asymmetric vibrations of C–CH2 and C–NH2 moieties.50 These Si QDs show a continuous absorption between 200 and 500 nm with size dependent PL in the visible region. Excitation at 375 nm results in a luminescence band centered at 455 nm (Fig. 1B).
3.2.
Steady-state and time-resolved measurements
The peak position and quantum yield of the 455 nm PL band of Si QDs (lex = 375 nm) remain unchanged in the presence of BSA (ESI,† Fig. S2). However, significant changes have been observed for BSA emission in the presence of Si QDs. It is well known that the intrinsic fluorescence of BSA is mainly due to the presence of two Trp residues at position 134 and 212 in domain IB and IIA respectively (Scheme 1B). Any changes in the fluorescence quantum yield or the peak position directly indicate changes in polarity surrounding these two Trp residues due to unfolding of the native conformation. Therefore, the intrinsic fluorescence of BSA is used to monitor the binding of Si QDs to BSA. BSA in its native state in pH 7.4 phosphate buffer shows an intense fluorescence centered at 339 nm (lex = 280 nm). Fig. 2A displays the effect of increasing concentration of Si QDs on the intrinsic fluorescence of BSA. With gradual addition of Si QDs the intrinsic fluorescence of BSA gets quenched. The intrinsic fluorescence of BSA is quenched by 56% with a red shift of 3 nm in the presence of 1.50 mM Si QDs. These spectral changes clearly indicate the association of BSA with Si QDs and subsequent unfolding of the native state.
Fig. 1 (A) AFM image of synthesized allylamine-capped Si QDs. The inset shows the size distribution histogram. (B) Absorption (black line) and normalized PL (blue line) spectra (lex = 375 nm) of allylamine-capped Si QDs.
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Fig. 2 (A) Fluorescence spectra (lex = 280 nm) of 2 mM BSA in pH 7.4 phosphate buffer at 298 K with different concentrations of allylamine-capped Si QDs. (B) Steady-state Stern–Volmer plot for quenching of BSA fluorescence by Si QDs at 298 K. (C) Lifetime decay curves (lex = 279 nm) of 2 mM BSA in the absence and presence of 1.50 mM Si QDs. (D) Overlap of steady-state and time-resolved Stern–Volmer plots.
The mechanism of observed fluorescence quenching could be due to either static, dynamic or a combination of both these processes. To reveal the mechanism and the extent of quenching, we have used the Stern–Volmer (SV) equation which can be expressed as: F0 ¼ 1 þ KSV ½Q ¼ 1 þ kq t0 ½Q F
(1)
where F0 and F are the fluorescence intensities in the absence and presence of a quencher, respectively. KSV is the Stern–Volmer (SV) constant and [Q] is the molar concentration of quencher. kq is the bimolecular quenching rate constant and t0 is the fluorescence lifetime of the fluorophore in the absence of a quencher. The SV constant and quenching rate constant can be evaluated from a plot of F0/F against [Q]. Fig. 2B shows the steady-state SV plot of the BSA–QDs conjugate at 298 K. The plot is linear in nature which indicates that either the static or dynamic type of quenching mechanism is responsible for the observed fluorescence quenching. The SV constant is evaluated from the slope of the plot and at 298 K it has a value of 8.23 105 M1. The bimolecular quenching rate constant has been calculated by using t0 = 5.6 ns of BSA (Table 1) to be 1.46 1014 M1 s1 at 298 k which is four orders of magnitude higher than that for a purely dynamic quenching
Table 1 Fluorescence lifetime components of BSA (lex = 279 nm) in the absence and presence of different concentrations of Si QDs in pH 7.4 phosphate buffer
Sample
t1 (ns) a1
BSA 2 mM 3.60 BSA 2 mM + 1 mM Si QDs 3.80 BSA 2 mM + 1.5 mM Si QDs 3.50
t2 (ns) a2
0.39 6.80 0.43 6.80 0.38 6.60
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hti (ns)
w2
0.61 5.60 0.04 1.03 0.57 5.56 0.01 1.06 0.62 5.45 0.02 1.09
mechanism.54,55 Hence, in the present system the observed fluorescence quenching of BSA by Si QDs might be due to static ground state complex formation and not due to collisional encounter. A similar kind of static quenching mechanism has been proposed earlier for other protein–NP complexes.12–14,17 Time-resolved lifetime measurements have been performed to investigate the dynamics of the BSA–QDs complex. Fig. 2C displays the typical lifetime decays of BSA in the absence and presence of 1.50 mM Si QDs. In the absence of Si QDs, BSA shows a biexponential decay curve with an average lifetime of 5.60 0.04 ns and having lifetime components of 3.60 ns and 6.80 ns with the relative abundance of 39% and 61%, respectively. A similar kind of biexponential fluorescence decay of BSA has been observed earlier and assigned to the presence of two Trp residues at different conformational states in two distinct chemical environments.29 The decay curve of BSA remains unaltered in the presence of different concentrations of Si QDs. The fluorescence decay of BSA remains biexponential in the presence of 1.50 mM Si QDs with an average lifetime of 5.45 0.02 ns having lifetime components of 3.50 ns and 6.60 ns with the relative abundance of 38% and 62%, respectively. Although the average lifetime of BSA does not change significantly in the presence of Si QDs, it is important to note the small change observed in average lifetime of BSA from 5.60 ns to 5.45 ns. This nominal decrease in lifetime in the presence of the highest concentration of Si QDs used in the present study could be due to the local conformational alterations of BSA upon adsorption. A time-resolved Stern–Volmer plot was generated using the Stern–Volmer equation P with average lifetime hti = aiti and can be expressed as follows: ht0 i ¼ 1 þ KSV ½Q hti
(2)
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where ht0i and hti are the average fluorescence lifetimes of BSA in the absence and presence of a quencher, respectively. Fig. 2D shows the overlap of steady-state and time-resolved Stern–Volmer plots for the BSA–QDs complex. The time-resolved Stern–Volmer plot is parallel to the x-axis indicating a static quenching mechanism. Based on these results it is hard to determine which Trp of BSA gets quenched upon adsorption. However, the two Trp residues (Trp 134 and 212) of BSA located at two separate domains in two distinct chemical environments. Trp 134 is located at the surface of the protein in subdomain IB while Trp 212 is located in the hydrophobic cavity of subdomain IIA. Hydrophobic interactions will lead to binding of Si QDs at the hydrophobic binding pocket at subdomain IIA near Trp 212 while electrostatic and hydrogen bonding interactions will lead to binding near the Trp 134 residue. Therefore, the nature of the interactions between Si QDs and BSA will allow us to determine which Trp gets quenched during the binding process. To address this fundamental question we calculated various thermodynamic parameters, which have been discussed in the next section. 3.3.
Effect of temperature and thermodynamics of binding
In order to investigate the effect of temperature on the BSA–QDs association process we have evaluated Ksv at different temperatures ranging from 298 K to 318 K. Fig. 3A displays the Stern– Volmer plots for the fluorescence quenching of BSA by Si QDs at
four different temperatures. All the plots are linear indicating a single type of quenching mechanism. The calculated SV constants at different temperatures are listed in Table 2. The gradual decrease of the SV constant with increase in temperature indicates ground state complex formation between BSA and Si QDs resulting in static quenching. The binding constant (Kb) and the number of binding sites (n) were determined using the Scatchard equation, which can be expressed as follows: ðF 0 F Þ log (3) ¼ log Kb þ n log½Q F where Kb is the binding constant of the protein–ligand complex, and n is the number of binding sites. The plot of log[(F0 F)/F] against log[Q] should yield a straight line with an intercept equal to log Kb and a slope equal to n. Fig. 3B shows the plot of log[(F0 F)/F] against log[Si QDs] at four different temperatures. All the plots are linear and the estimated binding constants and the number of binding sites are listed in Table 2. The estimated binding constant for the BSA–QDs system is 3.40 0.08 106 M1 at 298 K, similar to those previously obtained for different NP–protein complexes.12–14,17,30 The binding constant for the BSA–QDs complex decreases with increase in temperature, indicating the static nature of the complex. The number of binding sites i.e. the number of QDs per BSA is found to be nearly 1. The driving force for the formation of a ground state complex between Si QDs and BSA could be due to various molecular interactions namely, electrostatic, hydrophobic, hydrogen bond, or van der Waals interactions.31 For a simple two-state model of equilibrium as in the present system the thermodynamic parameters (enthalpy change, entropy change, and Gibbs free energy change) can be calculated from the van’t Hoff equation which can be expressed as follows: ln Kb ¼
DS DH R RT
(4)
where Kb is the binding constant, R is the gas constant, DS is the standard entropy change and DH is the standard enthalpy change during the BSA–QDs association reaction at temperature T. The changes in enthalpy (DH) and entropy (DS) of the association process between BSA and Si QDs have been evaluated by plotting ln Kb against 1/T for four different temperatures (Fig. 3C). The Gibbs free energy change (DG) during the binding process can be estimated from the equation: DG = DH TDS
Fig. 3 (A) Steady-state Stern–Volmer plots at four different temperatures. (B) Plots showing the effect of temperature on the binding constant between BSA and Si QDs. (C) The van’t Hoff plot for the BSA–Si QDs system. The equation of the fitted line is ln Kb = 5508.79/T 3.42 (R2 = 0.999).
8404 | Phys. Chem. Chem. Phys., 2014, 16, 8400--8408
(5)
The calculated thermodynamic parameters for the present system are listed in Table 2. The association reaction is found to have a favorable enthalpy change (DH o 0), which is compensated by an unfavorable entropy loss (DS o 0), giving rise to an overall negative free energy change. Earlier, based on the magnitude and sign of the thermodynamic parameters various groups have proposed different mechanisms for the association of various ligands with proteins. In general, association processes triggered by hydrophobic interactions between proteins and ligands proceed with a large positive
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Table 2 Stern–Volmer quenching constant (KSV), binding constants (Kb), the number of binding sites (n) and thermodynamic parameters for the BSA–Si QDs complex
T (K)
KSV (105 M1)
Kb (106 M1)
298 308 313 318
8.23 7.74 7.18 6.69
3.40 2.10 1.47 1.05
0.08 0.04 0.06 0.05
n 1.10 1.07 1.06 1.04
0.01 0.01 0.01 0.01
entropy change with a positive enthalpy change while processes triggered by electrostatic interactions proceed with a positive entropy change with a small positive or negative enthalpy change.31 However, our present thermodynamic parameters are not correlated with either of these two molecular interactions. A similar kind of negative enthalpy change with a negative entropy change was observed earlier in many association processes and has been assigned to involvement of specific hydrogen bonding interactions.31 Here, it is important to note that the magnitude and sign of the thermodynamic parameters strongly depend on the surface charge and size of the associating species. Therefore a closer look at the surface properties of BSA and Si QDs is required to propose a mechanism of their association process based on the thermodynamic parameters. The isoelectric point (pI) of BSA is 4.7 at 25 1C and hence at physiological pH (pH = 7.4) the carboxylic groups (–COOH) will remain in deprotonated form (–COO) with a net negative charge on them. Out of the three domains of BSA, domain I contains the majority of the negatively charged aspartate (Asp) and glutamate (Glu) residues followed by domain II. Domain III of BSA is neutral.56 On the other hand the estimated pKa of the
R2
DG (kJ mol1)
0.999 0.999 0.999 0.999
37.31 37.02 36.88 36.74
DH (kJ mol1)
DS (J mol1 K1)
45.78 0.27
28.42
ammonium ions (–NH3+) on the Si QDs surface is 5.7 (ESI,† Fig. S3). Hence, at pH 7.4 these surface amine moieties on Si QDs will remain unprotonated (–NH2) which is in agreement with our earlier observation.50 By considering the charges of BSA and Si QDs at pH 7.4 and the estimated thermodynamic parameters, we propose that the most probable mechanism of their association process involves specific hydrogen bonding interactions between negatively charged –COO groups of aspartate (Asp) and glutamate (Glu) residues of BSA and surface amine groups of Si QDs (Scheme 2). Here it is important to mention that both domains I and II of BSA are involved in hydrogen bond formation with the Si QDs surface. Although only surface exposed –COO groups of BSA from domains I and II form hydrogen bonds with Si QDs, we cannot rule out the conformational changes around the Trp 212 at the hydrophobic cavity of subdomain IIA which may contribute to the observed fluorescence quenching. Hence, the observed Trp quenching in the earlier section might be due to both the Trp residues (Trp 134 and Trp 212) of BSA. Whereas the unfavorable negative entropy changes during the association process are due to the conformational restriction of both proteins and Si QDs upon binding.
Scheme 2 Schematic representation of the association process of BSA with different surface funtionalized NPs or the QD surface driven by either electrostatic, hydrophobic or hydrogen bonding interactions. Different types of interactions proceed with characteristic changes in thermodynamic parameters and lead to different extent of structural destabilisation of the native conformation of protein. The present proposed model is highlighted in the scheme.
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Secondary structure of BSA in the presence of Si QDs
The far-UV CD and FTIR spectroscopy was used to monitor the changes in the secondary structure of BSA upon adsorption onto the Si QDs surface. Fig. 4A shows the CD spectra of BSA with increasing concentration of Si QDs. Native BSA at pH 7.4 shows two characteristic minima at 208 and 222 nm in the CD spectrum with 65% a-helix and 6.5% b-sheet content (Table 3). Notably, the secondary structure of the native protein does not change significantly in the presence of highest concentration of Si QDs used in this study. The secondary structure analysis of BSA in the presence of 1.50 mM Si QDs shows that the a-helix content decreases to 61.2% while the b-sheet content increases to 7.7% (Table 3). However, the shape and peak positions remain the same in the presence of Si QDs. These spectral changes demonstrate marginal alteration (5.8% loss of helicity) in the secondary structure of adsorbed BSA. Earlier, Vertegel et al. have reported significant structural destabilization (61–80% loss of helicity) and loss of enzymatic activity of positively charged lysozyme (pI = 11) on negatively charged silica NPs (pI = 3) at physiological pH.16 Similarly, Shang et al. have studied the structural perturbation and unfolding of positively charged ribonuclease A (pI = 9.4) on negatively charged silica NPs.15 From these earlier studies it is clear that strong electrostatic interactions between protein and NPs result in significant structural alteration of the native conformation due to coulombic
Fig. 4 (A) The far-UV CD spectra of BSA (2 mM) in the absence and presence of different concentrations of Si QDs. (B) FTIR spectra of BSA (black line) and BSA in the presence of 1.50 mM allylamine-capped Si QDs (red line).
Table 3 Secondary structure analysis of BSA in the absence and presence of different concentrations of Si QDs
Sample BSA BSA BSA BSA
2 2 2 2
mM mM + 0.60 mM Si QDs mM + 1.13 mM Si QDs mM + 1.50 mM Si QDs
a-Helix
b-Sheet
Turn
Random coil
65.0 64.1 62.1 61.2
6.5 6.8 7.3 7.7
12.3 12.4 12.7 12.9
16.2 16.7 18.0 18.2
8406 | Phys. Chem. Chem. Phys., 2014, 16, 8400--8408
attractions. The structure and stability of BSA on various NP surfaces have also been explored in great detail. Recently, it has been reported that the structural destabilization of BSA on bare hydrophobic ZnO NPs was greater than polyethyleneiminefunctionalized ZnO NPs.13 However, in the present system the observed nominal changes in the secondary structure of the adsorbed BSA on the Si QDs surface is due to the specific hydrogen bonding interactions between surface exposed –COO groups of BSA and –NH2 moieties of allylamine-capped Si QDs (Scheme 2). Therefore it is evident from our present results and literature that the adsorbed protein undergoes a maximum amount of structural destabilization for association processes driven by hydrophobic interactions followed by electrostatic and hydrogen bond induced processes (i.e. hydrophobic Z electrostatic 4 hydrogen bonding interactions). The adsorption and subsequent structural alterations of BSA on the Si QDs surface have been further confirmed by FTIR spectroscopy. The amide I and amide II bands of proteins in the region of 1600–1700 cm1 and 1500–1600 cm1 respectively provide useful information about their conformation in native and unfolded states.12–14 Fig. 4B shows the FTIR spectra of BSA in the absence and presence of Si QDs. In the absence of Si QDs, BSA shows two peaks at 1648 cm1 and 1547 cm1 assigned to amide I and amide II, respectively. However, in the presence of Si QDs, the amide I band shifted to 1649 cm1 while the amide II band shifted to 1549 cm1 with decreased intensity. Similar spectral changes in amide I and amide II bands of BSA have been observed earlier.13 The reduced intensity with a shift in amide I and amide II band positions clearly indicates the adsorption of BSA on the Si QDs surface and subsequent alteration in its local conformation. Earlier, it has been proposed that hydrophobic as well as electrostatic interactions between proteins and NPs have more deleterious effects on the structure and activity of proteins compared to other types of interactions. However, unfolding and loss of activity of the adsorbed proteins could be of potential risk for the cells which may trigger a sequence of adverse reactions. Serum albumin, being the most abundant plasma protein, forms the first layer of the corona on the NPs surface. Hence, it is of utmost importance to know the structure and stability of the plasma protein corona on the NPs or QDs surface to understand their physicochemical effects inside biological cell environments. Based on our present results we propose a model where specific hydrogen bonding interactions between surface exposed –COO groups of aspartate (Asp) and glutamate (Glu) residues of BSA and amine moieties (–NH2) of Si QDs drive the association process. We believe that the observed minor alteration of the secondary structure of adsorbed BSA is mainly due to the partial unfolding of domain I as well as domain II. However, BSA retains its overall tertiary structure upon surface adsorption.
4. Conclusion In this paper, we have demonstrated the dynamics and mechanistic details of BSA adsorption on the amine-functionalized Si
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QDs surface. The adsorption process is mainly due to ground state complex formation which leads to static fluorescence quenching. The extent of fluorescence quenching decreases with an increase in temperature which further highlights the static mechanism of fluorescence quenching. The estimated thermodynamic parameters suggest the involvement of specific hydrogen bonding interactions between surface amine groups (–NH2) of Si QDs and –COO groups of BSA. CD and FTIR spectra revealed nominal alteration in the secondary structure of adsorbed BSA on the Si QDs surface.
Acknowledgements The authors thank IIT Indore for proving the infrastructure, experimental facilities, and financial support. This work is supported by Council of Scientific and Industrial Research grant no. 01(2695)/12/EMR-II. The authors also thank the reviewers for their constructive comments.
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