Interaction of a Hydrophobic-Functionalized PAMAM Dendrimer with

May 5, 2014 - The interaction between a hydrophobic-functionalized PAMAM dendrimer (PAMAM-NH2-C12, 25%, G4) and bovine serum albumin (BSA) has been in...
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Interaction of a Hydrophobic-Functionalized PAMAM Dendrimer with Bovine Serum Albumin: Thermodynamic and Structural Changes Hong-Mei Zhang,† Kai Lou,†,‡ Jian Cao,*,† and Yan-Qing Wang*,† †

Institute of Applied Chemistry and Environmental Engineering, Yancheng Teachers University, Yancheng City, Jiangsu Province 224002, People’s Republic of China ‡ Chemistry and Chemical Engineering, Nanjing University of Technology, Nanjing City, Jiangsu Province 210009, People’s Republic of China S Supporting Information *

ABSTRACT: The interaction between a hydrophobic-functionalized PAMAM dendrimer (PAMAM-NH2-C12, 25%, G4) and bovine serum albumin (BSA) has been investigated by circular dichroism (CD), UV−vis, and fluorescence spectroscopic methods and molecular modeling. The analysis of the effects of dendrimer complexation on the stability and conformation of BSA indicated that the binding process of the hydrophobic-functionalized dendrimer with BSA induced the relatively large changes in secondary structure of protein. Thermal denaturation of BSA, when carried out in the presence of dendrimer, also indicated that this hydrophobic-functionalized dendrimer acted as a structure destabilizer for BSA. The hydrophobic, electrostatic, and hydrogen bonding forces played important roles in the complex formation. The putative binding site of PAMAM-NH2-C12 (25%) dendrimer on BSA was near to domain I and domain II. The effect of hydrophobic modification on the stability and structure of BSA would find useful information on the cytotoxicity of PAMAM dendrimer.

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INTRODUCTION As a class of monodisperse nanomaterials, polyamidoamine (PAMAM) dendrimers have been tested in clinical studies for various applications, including DNA transfection,1,2 gene and drug delivery,3−8 antibacterial agents, and so forth.9 Since PAMAM dendrimers are commercially available, widely used synthetic polymers, the studies on their biocompatibility and cytotoxicity have become an increasingly hot topic in biochemical and physiological research. So, precise knowledge related to the binding mechanism of PAMAM dendrimers with proteins is of great importance.10−12 Among thousands of proteins, serum albumins stand out because they play important roles in the blood circulatory system. For example, bovine serum albumin (BSA, Scheme 1A) presents an ideal model of serum carrier protein. First, BSA has 76% sequence identity with human serum albumin.13 Second, BSA acts as a carrier in the transport and distribution of various endogenous and exogenous ligands.14 In addition, BSA often changes some properties of the aliens binding to it.15 Therefore, BSA is one of the best studied models to understand the physicochemical basis of PAMAM dendrimer−protein interactions. From some studies on the cytotoxicity of PAMAM dendrimers, we know that the cationic charge and the hydrophobicity chains on the surface of them are the main facts that affect their toxicity, and the most popular of them is the cytotoxicity research of cationic PAMAM dendrimers.16 © 2014 American Chemical Society

However, seldom are studies about whether the dendrimer hydrophobicity is the possible factor determining cytotoxicity. Halets et al. have studied the effect of dendrimer hydrophobicity on their cytotoxicity.17 The results showed that the increase of dendrimer hydrophobicity induced the possibility of hemolysis. In addition, they made their points that the interactions of PAMAM dendrimers with blood proteins could significantly reduce the cytotoxicity of dendrimers toward red blood cells. However, to our knowledge, there are seldom studies exploring the nature of the interaction of the hydrophobic PAMAM dendrimer with BSA. Herein, we used PAMAM-NH2-C12 (25%) dendrimer (Scheme. 1 B) of the fourth generation bearing 25% acyl groups that replace the terminated −NH2 as the model of hydrophobic dendrimer to study the binding mechanism of it with BSA. Effects of the hydrophobic PAMAM dendrimer on the thermal stability and conformational structure of BSA were also studied to obtain the nature of their binding interactions.

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MATERIALS AND METHODS

Materials. BSA (A1933, lyophilized powder, ≥98%) and PAMAMNH2-C12 (25%) G4 dendrimer (10 wt % in methanol) were Received: March 26, 2014 Revised: April 26, 2014 Published: May 5, 2014 5536

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Scheme 1. Structures of BSA (A) and PAMAM-NH2-C12 (25%) G4 Dendrimer

Figure 1. (A) Effect of PAMAM-NH2-C12 (25%) G4 dendrimer on the CD spectrum of BSA (3.0 μM). PAMAM-NH2-C12 (25%) G4 dendrimer concentrations from line 1 to line 7 were 0.0, 1.5, 3.0, 6.0, 15.0, 24.0, and 30.0 μM. (B) Plots of the percentages of the different structures of BSA in the absence and presence of PAMAM-NH2-C12 (25%) G4 dendrimer. purchased from Sigma-Aldrich Chemical. All other chemicals used were of analytical purity or higher. Experiments were carried out in 0.05 M phosphate-buffer (pH, 7.40). Methods. Circular Dichroism Experiments. CD measurements of BSA in the absence and presence of PAMAM dendrimer were carried out with a Chirascan spectrometer (Applied Photophyysics Ltd., Leatherhead, Surrey, U.K.) equipped with temperature control quantum for temperature control. The spectra were recorded from 190 to 260 nm with a scan speed of 50 nm/min and a bandwidth of 1 nm using 0.1 cm quartz cells. BSA concentration was kept constant (3.0 μM) while PAMAM-NH2-C12 (25%) G4 dendrimer concentration was varied from 0.0 μM to 30.0 μM. The changes in the contents of different secondary structure were calculated using CDNN software equipped with the spectrometer. Thermal scans of BSA in the absence and presence of PAMAMNH2-C12 (25%) G4 dendrimer were collected with a wavelength range from 190 to 260 nm in 0.1 cm path length cells. The temperature of thermal denaturation of BSA was varied from 20 to 90 °C in 5 °C steps, with 240 s increments. The Global Analysis Software was used to obtain the melting temperature (Tm) and the enthalpy changes at the melting temperature (ΔHm).18 UV−Vis Absorption and Fluorescence Measurements. The UV−vis absorption measurements were carried out at room temperature using a SPECORD S600 spectrophotometer (Jena, Germany). Fluorescence measurements were performed in a LS− 50B Spectrofluorimeter (Perkin-Elmer). The excitation wavelength was 280 nm and the emission spectra were recorded between 300 and 500 nm for steady-state fluorescence spectra. Synchronous fluorescence spectra of BSA in the absence and presence of PAMAMNH2-C12 (25%) G4 dendrimer were recorded at 15 or 60 nm. Resonance light scattering spectra (RLS) of BSA in the absence and presence of PAMAM-NH2-C12 (25%) G4 dendrimer were obtained by synchronous scanning (Δλ = 0 nm) with a wavelength range of

200−800 nm on the spectrofluorophotometer. The slit widths of all fluorescence experiments were 2.5 nm/2.5 nm. During the fluorescence experiments, the protein concentration in each sample was 3.0 μM while PAMAM-NH 2-C12 (25%) G4 dendrimer concentrations were varied from 0.0 to 12.0 μM. Molecular Modeling. In this report, Autodock tools (ADT) v1.5.4 and Autodock 4.2.3 program were used to perform the molecular modeling studies.19 The crystal structure of BSA (PDB ID 3V03) was taken from RCSB Protein Data Bank.20 In addition, the diameter of PAMAM-NH2-C12 (25%) G4 dendrimer is about 4.5 nm, which will lead to difficulty in the dock modeling as a big ligand. Therefore, we had the three-dimensional structure of PAMAM-NH2-C12 (25%) G2 dendrimer for PAMAM-NH2-C12 (25%) G4 dendrimer. The PAMAM-NH2-C12 (25%) G2 dendrimer was built and optimized using DFT/B3LYP/6-311G by Gaussian 09.21 During molecular modeling docking studies, we used a big grid box of 126 × 126 × 126 Å3 with 0.675 Å grid maps to provide enough space for the translational and rotational walk of ligand. The Lamarckian Genetic Algorithm was applied for minimization in the blind docking and the docking parameters used were as follows: the number of GA runs = 10; the GA population size = 150; the maximum number of energy evaluation = 2 500 000, and others used were default parameters. Finally, the best docking result was analyzed using the Molegro Molecular Viewer software.22

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RESULTS AND DISCUSSION Effect of PAMAM-NH2-C12 (25%) G4 Dendrimer on the Conformation and Thermal Stability of BSA. In the present work, circular dichroism (CD) spectra of BSA in the absence and presence of PAMAM-NH2-C12 (25%) G4 dendrimer were recorded. If PAMAM-NH2-C12 (25%) G4 dendrimer interacts with BSA, the binding interaction can alter 5537

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Figure 2. Far-UV CD spectra of the BSA (A) and BSA-PAMAM-NH2-C12 (25%) G4 dendrimer system (B) during thermal denaturation. c(BSA) = 3.0 μM, c(dendrimer) = 6.0 μM.

°C while the content of β-sheet and Random coil increase. In addition, it can also be seen from Figure 2A-2 that the changes in the second structural content against temperature is quite sharp when the temperature is beyond 55 °C, indicating that high temperature can easily unfold the α-helix structure of BSA. The critical temperature region of BSA is around 55 °C. Beyond this region in the thermal denatureation, the structural changes of the protein often become irreversible.25,26 In order to insight into the unfolding transition, the melting temperature (Tm) and the enthalpy changes at the melting temperature (ΔHm) were also obtained by using Global Analysis Software. The values Tm and ΔHm of BSA were (63.9 ± 0.1) °C and (120.7 ± 2.3) kJ·M−1, respectively. Compared with the thermal denaturation process of BSA in the absence of PAMAM-NH2C12 (25%) G4 dendrimer, the change trend in the same process of BSA−dendrimer system showed some differences (Figure 2B). First, when the temperature was beyond 45 °C, the structural change of BSA became sharp. Second, the values of Tm (56.1 ± 0.1 °C) and ΔHm (150.0 ± 1.3 kJ·M−1) were also different. The values of Tm was reduced by 7.8 °C in the presence of PAMAM-NH2-C12 (25%) G4 dendrimer. This result implied that the thermal stability of BSA decreased and PAMAM-NH2-C12 (25%) G4 dendrimer acted as a structure destabilizer. The ordering of water molecules around exposed the hydrophobic groups of folded protein play an important role in the thermal change of protein.27 The binding of PAMAM-NH2-C12 (25%) G4 dendrimer to BSA maybe induce the loss of ordered water coating the peptide of BSA, especially the α-helix structure of BSA. In addition, this binding interaction maybe cause the distance changes of the −NH and −CO groups of the backbone of BSA. Due to these reasons, PAMAM-NH2-C12 (25%) G4 dendrimer induced the changes of the thermal denaturation process of BSA.

the secondary or tertiary structure of BSA. First, Figure 1 shows the far-UV CD spectra of BSA in the absence and presence of different concentrations of PAMAM-NH2-C12 (25%) G4 dendrimer. As shown in line 1 of Figure 1A, the spectrum of BSA has two negative bands at 208 and 222 nm, characteristic of the α-helical structure. With the increase of PAMAM-NH2C12 (25%) G4 dendrimer concentrations, the CD signal of BSA decreased remarkably. These CD spectra showed that the binding interaction induced the relatively large changes in secondary structure and probably some changes in the tertiary structure of BSA.23 the conversion of α-helix to other structure existed. The percentages of α-helix, β-sheet, β-turn, and random coil in BSA with the increase of dendrimer concentrations are plotted in Figure 1B. In the absence of dendrimer, we found 64.5% of α-helix, 6.9% of β-sheet, 12.7% of β-turn, and 15.8% of random coil, which are close to previously reported values.24 When PAMAM-NH2-C12 (25%) G4 dendrimer was added in the protein solution, the content of α-helix decreased to reach 21.4% (Figure 1B). The protein α-helix conformation partly changed into β-sheet and Random coil. The decrease in percent of α-helix in BSA induced by PAMAM-NH2-C12 (25%) G4 dendrimer is remarkably bigger than those of other PAMAM dendrimers.12 Very strong changes in the protein structures from α-helix to β-sheet and Random coil clearly indicated that the hydrophobic PAMAM dendrimer induced the denaturation and unfolding of BSA. CD experiment was also performed to explore the effect of PAMAM-NH2-C12 (25%) G4 dendrimer on the thermal unfolding of BSA. The decreasing percent of α-helix in BSA induced by PAMAM-NH2-C12 (25%) G4 dendrimer is like the effect of temperature on the secondary structure of BSA (Figure 2A). The temperature-dependent CD measurements are shown in Figure 2. As Figure 2A-1,2 shows, when the temperature increased, the content of α-helix decreased to 32.6% at about 79 5538

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Figure 3. CD spectra of BSA (3.0 μM) in the absence (A) and presence (B) of PAMAM-NH2-C12 (25%) G4 dendrimer (6.0 μM) at different conditions.

Table 1. Percentages of α-Helix Structures of BSA at Different Temperature temperature

α-helix %

BSA

19.14 °C 79.42 °C 20.00 °C (recovered from 79.42 °C)

64.5 32.6 51.0

BSA + dendrimer c(PAMAM-NH2-C12 (25%) G4 dendrimer) = 6.0 μM

20.32 °C 79.55 °C 20.00 °C (recovered from 79.55 °C)

35.4 14.5 15.4

system

hydrophobic-functionalized PAMAM dendrimer not only has protonated amines, but also bears 25% acyl groups. So PAMAM-NH2-C12 (25%) G4 dendrimer is an amphiphilic macromolecule. In the ordinary course of events, the pKa values of the primary amines on the out shell of PAMAM dendrimer are between 7 and 9, while tertiary amines moieties with pKa values between 3 and 6.28,29 At pH = 7.4, the primary amines of PAMAM-NH2-C12 (25%) G4 dendrimer are expected to become partly protonated, while BSA (pI = 4.9) has a number of negatively charged surfaces groups at its surface. Therefore, electrostatic interactions between the protonated −NH3+ of PAMAM dendrimer and the −COO− groups in BSA are first involved in their binding interaction. Second, hydrophobic force between the acyl groups and hydrophobic peptides should not be excluded. Third, the unprotonated amines of PAMAM dendrimer could form hydrogen bonding with the groups in BSA. The possible binding forces push the formation of complex between PAMAM dendrimer and BSA. During the unfolding process of BSA α-helix with the increase of temperature, the disruption of the helical structures enhances the cross-linking function between the hydrophobic or hydrophilic groups in the complex between PAMAM dendrimer and BSA, which results in disrupting the apolar contacts and decreasing the reversibility of the unfolding process when the protein is cooled from high temperature to 20 °C. In a word, the existence of PAMAM-NH2-C12 (25%) G4 dendrimer decreased the reversibility of the unfolding of BSA. PAMAM-NH2-C12 (25%) G4 dendrimer acted as a structure destabilizer for BSA by the complex formation between PAMAM-NH2-C12 (25%) G4 dendrimer and BSA. Changes in the Fluorescence Spectroscopic Properties of BSA in the Presence of PAMAM-NH2-C12 (25%) G4 Dendrimer. To get more insight into the nature of binding interaction of PAMAM-NH2-C12 (25%) G4 dendrimer with BSA, the fluorescence spectral method was also used. There are 18 tyrosine residues (Tyr) and 2 tryptophan (Trp) residues in

From the above CD analysis, we found that PAMAM-NH2C12 (25%) G4 dendrimer induced the denaturation and unfolding of BSA and affected the thermal denaturation process of protein. This result indicated the complex formation between PAMAM-NH2-C12 (25%) G4 dendrimer and BSA. The formation of this complex can affect the reversibility of the unfolding process when the protein is cooled from high temperature to 20 °C. It can be seen from Figure 3A and Table 1 that the percentage of BSA α-helix changed from 64.5% to 35.4% when we heated the protein from 19.14 to 79.42 °C. The helicity of 35.4% indicated that 206 amino acid residues (583 total residues × 0.354) are still maintained α-helix at 79.42 °C.27 In addition, BSA can only recover 51.0% of its α-helix when we cooled the protein from 79.42 to 20.00 °C. This result implied that the structures of 91 amino acid residues (585 total residues × (0.510−0.354)) recovered from unfolding to α-helix and about 80% (297/helical 376 residues) of the original helices still have α-helix when we cooled the protein from 79.42 to 20.00 °C. However, the percentage of BSA α-helix changed from 35.4% to 14.5% when we heated the protein from 20.32 to 79.55 °C in the presence of PAMAM-NH2-C12 (25%) G4 dendrimer (Figure 3B, Table. 1). This meant that only 84 amino acid residues (583 total residues × 0.145) are still maintained α-helix at 79.55 °C in the presence of dendrimer. Compared with the percentage of BSA α-helix structures in the absence of dendrimer, BSA can only recover 15.4% of its αhelix when the temperature were recovered from 79.55 to 20.00 °C. This implied that only 51 amino acid residues (583 total residues × (0.154−0.145)) recovered from unfolding to αhelix. BSA has three domains and the stable helical structures appear likely to be formed within these three domains.27 These stable helical structures of BSA begin to be disrupted by PAMAM-NH2-C12 (25%) G4 dendrimer while BSA loses the reversibility of the structural change in the thermal denaturation. Considering a question in the molecular structure of PAMAM-NH2-C12 (25%) G4 dendrimer, we found that this 5539

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Figure 4. (A) Effects of PAMAM-NH2-C12 (25%) G4 dendrimer on the fluorescence spectra of BSA (3.0 μM) (298 K, pH 7.40, λex = 280 nm). c(PAMAM-NH2-C12 (25%) G4 dendrimer)/μM, curves (from top to bottom): 0.0, 0.75, 1.5, 2.25, 3.0, 4.5, 6.0, 7.5, 9.0, 10.5, and 12.0. (B) Inset showing the absorption spectra of PAMAM-NH2-C12 (25%) G4 dendrimer (3.0 μM). (C, D) The synchronous fluorescence spectra of 3.0 μM BSA in the absence and presence of PAMAM-NH2-C12 (25%) G4 dendrimer in buffer pH 7.4 at 298 K, (C) Δλ = 15 nm, (D) Δλ = 60 nm. c(PAMAMNH2-C12 (25%) G4 dendrimer)/μM, curves (from top to bottom): 0.0, 0.75, 1.5, 2.25, 3.0, 4.5, 6.0, 7.5, 9.0, 10.5, and 12.0.

BSA. The intrinsic fluorescence emission of these residues could give some information about their binding interactions. Especially the two Trp residues, Trp-213 is located in the largest hydrophobic cavity of the subdomain IIA, whereas Trp134 is located on the surface of the subdomain IB.15 If the binding site of BSA with PAMAM-NH2-C12 (25%) G4 dendrimer nears the above domain of the protein, tryptophan fluorescence quenching induced by PAMAM-NH2-C12 (25%) G4 dendrimer can be used to analyze the formation of complex between PAMAM dendrimer and BSA. The effect of PAMAM dendrimer on the fluorescence intensity of BSA is shown in Figure 4. The intensity of the characteristic broad emission band at 345 nm decreased regularly with the increasing concentration of PAMAM dendrimer, which indicated that the binding occurred in close to Trp-213 or Trp-134. In addition, the interaction did change the emission λmax from 345 to 336 nm during the complexation, indicating that Trp 213 or Trp-134 was exposed to a more hydrophobic environment. Herein, synchronous fluorescence spectra can give specifical explanation about the effects of PAMAM dendrimer on the molecular environment of Trp residues and Tyr residues, respectively.30 The changes of polarity around Trp or Tyr residues can be indicated by the position of maximum emission wavelength in synchronous fluorescence spectra. The Synchronous fluorescence spectra of 3.0 μM BSA in the absence and presence of PAMAM-NH2-C12 (25%) G4 dendrimer are shown in Figure 4C, D. It was observed from Figure 4C, D that the position of maximum emission wavelength of Trp residues was obviously blue-shifted, which indicated an increase in the hydrophobicity around Trp

residues. The molecular environment of Tyr residues did not change obviously. Bearing 25% acyl groups, PAMAM-NH2-C12 (25%) G4 dendrimer is an amphiphilic macromolecule. In addition, the hydrophobic PAMAM dendrimer induced the strong denaturation and unfolding of BSA on the bases of CD experimental data. A lot of α-helix of BSA was changed to βsheet and random coil. The secondary structural rearrangement of BSA induced by PAMAM-NH2-C12 (25%) G4 dendrimer can made Trp residues locate in a more hydrophobic environment. As the unfolding of BSA and the formation of complex between BSA and PAMAM-NH2-C12 (25%) G4 dendrimer, the volume of complex might be bigger than that of only BSA. The RLS spectra of BSA, BSA- PAMAM-NH2-C12 (25%) G4 dendrimer complex and PAMAM-NH2-C12 (25%) G4 dendrimer are recorded synchronous scanning from 200 to 800 nm with Δλ = 0 nm. The results are shown in Figure S1 in the Supporting Information. Upon addition of PAMAM-NH2C12 (25%) G4 dendrimer to BSA solution, a remarkably increased RLS was observed, implying that there were interactions between PAMAM-NH2-C12 (25%) G4 dendrimer and BSA and the size of the newly formed complex may be bigger than that of BSA or PAMAM-NH2-C12 (25%) G4 dendrimer, since the production of RLS is correlated with the particle dimension of the formed aggregate in solution that dominates the RLS intensity.31 The hydrophobic groups of PAMAM-NH2-C12 (25%) G4 dendrimer probably play a crosslinking function between BSA and dendrimer and play an important role in unfolding BSA and changing the microenvironments of Trp residues. 5540

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PAMAM molecule, the distance between the later PAMAM molecule and binding site on BSA become great. Therefore, the Stern−Volmer plots had two regression curves. In addition, it can be found that the slopes of the Stern−Volmer plots decreased with increasing temperature, which indicated the ground state complex formation between PAMAM-NH2-C12 (25%) G4 dendrimer and BSA. Compared with other PAMAM dendrimers,11,37 the values of Ksv of PAMAM-NH2-C12 (25%) G4 dendrimer with BSA are obviously bigger (Table S1). This result implied that the 25% acyl groups in PAMAM-NH2-C12 (25%) G4 dendrimer play a important role in activating the binding ability of PAMAM dendrimer with BSA. The Nature of the Binding Interaction of PAMAMNH2-C12 (25%) G4 Dendrimer with BSA. In this report, eq 2 was used to obtain the binding constants (KA) and binding site (n) of PAMAM-NH2-C12 (25%) G4 dendrimer to BSA.38

In fact, it can also be seen from Figure 4B that PAMAMNH2-C12 (25%) G4 dendrimer absorbed photons from the excitation light (λex = 280 nm) and from the emitted fluorescence (λem = 345 nm). So, the inner filter effect in the fluorescence quenching of BSA should be considered. In addition, the absorption peak at 294 nm comes from the unprotonated tertiary amines of PAMAM-NH2-C12 (25%) G4 dendrimer.32 The Stern−Volmer equation were used to analyze the fluorescence quenching data:33 F0 = 1 + kqτ0[PAMAM] = 1 + K sv[PAMAM] Fcor

(1)

where F0 and Fcor are the corrected fluorescence intensities in the absence and presence of PAMAM-NH2-C12 (25%) G4 dendrimer corrected according to ref 34, respectively. kq, Ksv, [PAMAM], and τ0 are the quenching rate constant of BSA, the Stern−Volmer dynamic quenching constant, the concentration of PAMAM-NH2-C12 (25%) G4 dendrimer, and the average lifetime of BSA without PAMAM dendrimer, respectively. The Stern−Volmer plots of F0/Fcor versus [PAMAM] are shown in Figure 5. The results indicate that the Stern−Volmer

log

F0 − Fcor = n log KA Fcor ⎛ ⎞ 1 − n log⎜ ⎟ ⎝ [PAMAM] − (F0 − Fcor)[Pt ]/F0 ⎠ (2)

where [PAMAM] and [Pt] are the total PAMAM-NH2-C12 (25%) G4 dendrimer and the total BSA concentration, respectively. KA and n can be obtained from the slope and intercept by plotting of log(F0 − Fcor)/Fcor versus log (1/ ([PAMAM] − (F0 − Fcor)[Pt]/F0)). The results are listed in Table 2. It can be seen from Table 2 that BSA-PAMAM-NH2-C12 (25%) G4 dendrimer complex easy formed. The values of this binding constants were obviously bigger than those of other PAMAM dendrimers without hydrophobic-functionalized groups.39 The number of both binding sites were near 1, indicating that there were more than one binding sites with highly binding ability and selectivity on BSA to link PAMAM dendrimer. In addition, the values of binding constants at c(PAMAM) ≥ 4.5 μM were bigger than that of c(PAMAM) ≤ 3.0 μM. This result implied that the structure changes of BSA induced by PAMAM-NH2-C12 (25%) G4 dendrimer did not decrease the ability in binding with PAMAM molecule and the distances between the later PAMAM molecule and binding site on BSA became great. The values of KA in Table 2 were used to calculate the enthalpy change (ΔH°), the entropy (ΔS°) and free-energy change (ΔG°) from eqs 3−5. The results are shown in Table 3. Through the values of ΔH° and ΔS°, we can obtain the types of noncovalent interactions between PAMAM-NH2C12 (25%) G4 dendrimer and BSA. The hydrogen bonding, van der Waals forces, electrostatic, and hydrophobic interactions are the main four types of noncovalent interactions playing important roles in proteins binding with ligands.40 According to the views of Ross and Subramanian,41 when c(PAMAM) ≤ 3.0 μM, the values of ΔH° and ΔS° of BSA with PAMAM-NH2-C12 (25%) G4 dendrimer are both positive, which indicated that hydrophobic interaction might play a

Figure 5. Stern−Volmer plots for the quenching of BSA (3.0 μM) by PAMAM-NH2-C12 (25%) G4 dendrimer at different temperatures. pH 7.40, λex = 280 nm, and λem = 345 nm.

plots have two regression curves intersecting at CPAMAM /CBSA = 1, indicating that the interaction mechanism became more complex and later PAMAM molecule coming close to the binding site in the neighborhood of Trp-213 or Trp-134 were disturbed by the former PAMAM molecule. Generally, the diameter of the PAMAM G4 dendrimer is about 4.5 nm and the dimensions of BSA are 140 × 40 × 40 Å.35,36 Considered the volume of BSA, it cannot provide enough big hydrophobic cavity to pack up PAMAM-NH2-C12 (25%) G4 dendrimer. Therefore, the binding site of PAMAM dendrimer to BSA might locate on the surface of BSA. Once the former PAMAM dendrimer occupies the position of advantage over other

Table 2. Binding Parameters of PAMAM-NH2-C12 (25%) G4 Dendrimer with BSA at Different Temperature c(PAMAM) ≤ 3.0 μM −1

c(PAMAM) ≥ 4.5 μM −1

T (K)

(KA)1 (M )

n1

R1

SD1

(KA)2 (M )

n2

R2

SD2

298 310

1.22 × 105 1.49 × 105

0.92 0.87

0.9588 0.9954

0.0498 0.0198

4.15 × 105 3.40 × 105

0.96 0.91

0.9991 0.9819

0.0043 0.0159

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Table 3. Relative Thermodynamic Parameters of the System of PAMAM-NH2-C12 (25%) G4 Dendrimer with BSA c(PAMAM) ≤ 3.0 μM

c(PAMAM) ≥ 4.5 μM

T (K)

ΔH°1 (kJ·mol−1)

ΔG°1 (kJ·mol−1)

ΔS°1 (J·mol−1·K−1)

ΔH°2 (kJ·mol−1)

ΔG°2 (kJ·mol−1)

ΔS°2 (J·mol−1·K−1)

298 310

12.80

−29.02 −30.07

140.33

−12.76

−32.05 −32.83

64.73

Figure 6. Predicted orientation of the binding conformation of PAMAM dendrimer with BSA (A) Electrostatic potential map of BSA with the different PAMAM dendrimer. Red color represents negatively charged surface, and blue color represents positively charged surface. (B) Cartoon representation of the lowest docking energy conformation of PAMAM dendrimer with BSA. (C) Representation of the amino acid residues binding with PAMAM dendrimer.

by molecular docking technique. From the docking calculation, 10 conformers with minimum binding energy were picked up from the cluster analysis. The cluster results are shown in Figure 6A and Supporting Information Table S2. It can be seen from the results that the energetically most favorable conformation of the docked pose are near to domain I and domain II. PAMAM-NH2-C12 (25%) dendrimer approached toward the linking gap between domain I and domain II of BSA. From the theory analysis, domain I (−7) and domain II (−9) are negatively charged, whereas domain III is neutral at physiological pH.15 In fact, it can be also seen from Figure 6A that the negatively charges of BSA mainly locate in domain I and domain II. The unlike charges attracting each other is one of the important binding forces. In addition, the steric, hydrogen bonding or hydrophobic forces might be involved in this binding process. From the docking simulation the observed free energy change of binding (ΔG) and the binding constants (KA) in the most favorable binding site are larger than the experimental results. The predicated binding model with the lowest docking energy was then used for binding orientation analysis (Figure 6B,C). It indicated that there were twenty-nine amino acid residues taking part in the binding interaction of BSA with PAMAM-NH2-C12 (25%) dendrimer. These amino acid residues are Asp-323, Arg-208, Glu-207, Lys242, Lys-239, Glu-243, Asp-248, Glu-251, Pao-96, Lys-93, Gln94, Glu-95, Arg-98, His-67, Thr-68, Ser-65, Glu-6, Leu-66, Lys64, Leu-69, Glu-63, Gly-61, Arg-10, Ile-7, Cys-62, Ser-5, Lys-4, His-9, and His-3. From above analysis, the interaction of PAMAM-NH2-C12 (25%) dendrimer with BSA does not be presumed to be only one kind of force, as there are

major role in the binding PAMAM-NH2-C12 (25%) G4 dendrimer to BSA.42 Since the positive value of ΔS° can also be thought that the electrostatic force might be involved in the binding process. The electrostatic force should not be excluded. When c(PAMAM) ≥ 4.5 μM, the values of ΔH° and ΔS° both changed. The values of ΔH° became negative, while ΔS° decreased obviously. This indicated that the electrostatic force and hydrogen bonds are executed in the binding interaction, but hydrophobic force is even involved in this interaction. As mentioned earlier, BSA contains two tryptophan residues of which Trp-213 is located within a more hydrophobic binding pocket compared with Trp-134 surrounded by acidic residues on the surface of BSA. PAMAM-NH2-C12 (25%) G4 dendrimer first bound to domain II by mainly hydrophobic force at low concentrations of PAMAM molecule. With PAMAM dendrimer distorting the structure of BSA, the later added PAMAM-NH2-C12 (25%) G4 dendrimer selected to bind another site by complex forces. ln

(KA )2 ΔH ° ⎛ 1 1⎞ = ⎜ − ⎟ (KA )1 R ⎝ T1 T2 ⎠

ΔG° = −RT ln KA

(3) (4)

ΔH ° − ΔG° (5) T The Putative Binding Sites of PAMAM-NH2-C12 (25%) Dendrimer on BSA. As a transport protein, BSA has numerous sites for ligand binding. The putative binding sites of PAMAM-NH2-C12 (25%) dendrimer on BSA were studied ΔS° =

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Province (Grant No. BK2011422, BK2012671), the Natural Science Foundation of Education Department of Jiangsu Province (Grant No. 11KJB150019, 12KJA180009), and Jiangsu Provincial Key Laboratory of Coastal Wetland Bioresources and Environmental Protection (JLCBE11008), the Jiangsu Fundament of “Qilan Project” and “333 Project”, and the sponsorship of Jiangsu Overseas Research & Training Proggram for University Prominent Young & Middle-aged Teachers and Presidents.

hydrophobic amino residues (Leu-66, 69, Ile-7, Pro-96), negatively charged amino residues (Asp-323, Glu-6, 63, 95, 207, 243, 251), and other polar residues taking part in the binding interaction. So hydrophobic and electrostatic forces were involved in the binding interaction of PAMAM-NH2-C12 (25%) dendrimer with BSA. In addition, four hydrogen bonds (Thr-68: −NH−, 3.242 Å; Glu-63: −NH2, 2.542 Å; Glu-6: −OH, 2.982 Å; Ser-65: −OH, 3.068 Å) were also predicted from the most favorable binding site. In conclusion, hydrophobic, electrostatic and hydrogen bonding forces are all involved in this binding interaction. The computational results were in accordance with the experimental results.

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CONCLUSION In this paper, we have reported how one hydrophobicfunctionalized PAMAM dendrimer affects the structure and stability of BSA. The features are as follows. First, contrary to the case of PAMAM G4 dendrimers without hydrophobicfunctionalized groups, the hydrophobic forces have an important role in the binding interaction of dendrimer with BSA. The binding process of hydrophobic-functionalized dendrimer with BSA remarkably disrupted the stable helical structures of BSA. Second, the hydrophobic-functionalized dendrimer acted as a structure destabilizer for BSA and made BSA lose more reversibility of the structural change in the thermal denaturation. Third, the complex between the hydrophobic-functionalized PAMAM dendrimer and BSA easily formed with high values of binding constants. Many noncovalent interactions including steric, hydrophobic, electrostatic, and hydrogen bonds are involved in the binding process. In addition, the putative binding sites of PAMAM-NH2-C12 (25%) dendrimer on BSA were near domain I and domain II. This work demonstrates the importance of hydrophobicfunctionalized groups to dendrimer binding with protein. We believe that the present study will provide important insight into the nature of polymer−protein complexation and understanding of biological properties, cytotocicity of other functionalized PAMAM dendrimers.

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ASSOCIATED CONTENT

S Supporting Information *

Stern−Volmer quenching constants of PAMAM-NH2-C12 (25%) G4 dendrimer with BSA at different temperature; Docking summary of BSA with PAMAM dendrimer by the AutoDock program; RLS spectra of BSA, BSA-PAMAM-NH2C12 (25%) G4 dendrimer (b), and PAMAM-NH2-C12 (25%) G4 dendrimer. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*(J.C.) E-mail: [email protected]. Tel: +86 515 88233005. Fax: +86 515 88233005. *(Y.-Q.W.) E-mail: [email protected]. Tel: +86 515 88233188. Fax: +86 515 88233188. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge financial support of the Fund for the National Natural Science Foundation of China (Project No. 21201147), the Natural Science Foundation of Jiangsu 5543

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