Complexes of Dendrimers with Bovine Serum Albumin

Biomacromolecules 2010, 11, 465–472

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Complexes of Dendrimers with Bovine Serum Albumin J. S. Mandeville and H. A. Tajmir-Riahi* De´partement de Chimie-Biologie, Universite´ du Que´bec a` Trois-Rivie`res, C. P. 500, Trois-Rivie`res (Que´bec), G9A 5H7, Canada Received October 21, 2009; Revised Manuscript Received December 21, 2009

We report the complexation of bovine serum albumin (BSA) with several dendrimers of different compositions mPEG-PAMAM (G3), mPEG-PAMAM (G4), and PAMAM (G4) at physiological conditions using constant protein concentration and various dendrimer contents. FTIR, CD, and fluorescence spectroscopic methods were used to analyze polymer binding mode, the binding constant, and the effects of dendrimer complexation on BSA stability and conformation. Structural analysis showed that dendrimers bind BSA via hydrophilic and hydrophobic interactions with a number of bound polymers (n): 1.30 for mPEG-PAMAM-G3, 1.30 for mPEG-PAMAM-G4, and 1.0 for PAMAM-G4. The polymer-BSA binding constants were KmPEG-G3 ) 5.0 ((0.8) × 103 M-1, KmPEG-G4 ) 1.0 ((0.3) × 104 M-1, and KPAMAM-G4 ) 1.1 ((0.4) × 104 M-1. Dendrimer binding altered BSA conformation with a major reduction of R-helix and an increase in random coil and turn structures, indicating a partial protein unfolding.

Introduction Dendrimers are unique synthetic macromolecules of nanometer dimensions with a highly branched structure and globular shape.1-3 Among dendrimers, polyamidoamines (PAMAM; Scheme 1A) are often used as potential gene and drug delivery systems.4-7 However, PAMAM dendrimers are toxic in cells and animals due to their polycationic character.8 It has been demonstrated that modification of the amino groups on the periphery of the dendrimer with poly(ethylene glycol) chains (Scheme 1B) reduces the toxicity and increases the biocompatibility of the resulting polymer.9 This is because poly(ethylene glycol) is nontoxic, nonimmunogenic, and water-soluble and its conjugation with other substrates produces conjugates that combine the properties of both the substrate and the polymer. However, conjugate formation can alter the binding affinity of PAMAM to DNA, drug, and protein. Recent studies showed dendrimer complexation with human serum albumin alters protein conformation and causes a partial protein unfolding.10 Even though the interaction of dendrimers with bovine serum albumin (BSA) has been reported, the effect of polymer complexation on protein stability and secondary structure is not yet known.11,12 Serum albumins are the major soluble protein constituents of the circulatory system and have many physiological functions.13 The most important property of this group of proteins is that they serve as transporters for a variety of compounds. BSA (Scheme 2) has been one of the most extensively studied of this group of proteins, particularly because of its structural homology with human serum albumin (HSA). The BSA molecule is made up of three homologous domains (I, II, III) that are divided into nine loops (L1-L9) by 17 disulfide bonds. The loops in each domain are made up of a sequence of large-small-large loops forming a triplet. Each domain in turn is the product of two subdomains (IA, IB, etc.). X-crystallographic data14,15 show that the albumin structure is predominantly R-helical with the remaining polypeptide, occurring in turns and in extended or flexible regions between subdomains * To whom correspondence should be addressed. Tel.: 819-376-5011 (ext. 3310). Fax: 819-376-5084. E-mail: [email protected].

with no β-sheets. BSA has two tryptophan residues that possess intrinsic fluorescence.16,17 Trp-134 in the first domain and Trp212 in the second domain. Trp-212 is located within a hydrophobic binding pocket of the protein, and Trp-134 is located on the surface of the molecule. While there are marked similarities between BSA and HSA in their compositions, HSA has only one tryptophan residue Trp-214, while BSA contains two tryptophan, Trp-212 and Trp-134, as fluorophores capable of fluorescence quenching. Fluorescence quenching is considered as a technique for measuring binding affinities. Fluorescence quenching is the decrease of the quantum yield of fluorescence from a fluorophore induced by a variety of molecular interactions with quencher molecule.18 Therefore, it was of interest to use quenching of the intrinsic tryptophan fluorescence of BSA as a tool to study the interaction of dendrimers with BSA in an attempt to characterize the nature of polymer-protein complexation. We present spectroscopic analysis of BSA complexes with dendrimers PAMAM ([NH2(CH2)2NH2] (G4) polyamidoamine), m-PEG (poly(ethylene glycol))-PAMAM (G3), and m-PEGPAMAM (G4) in aqueous solution at physiological conditions, using constant protein concentration and various dendrimer compositions. Structural information regarding dendrimer binding mode and the effect of polymer-BSA complexation on the protein stability and secondary structure is reported here.

Experimental Section Materials. BSA fraction V was purchased from Sigma Chemical Company and used as supplied. PAMAM-G4 [NH2(CH2)2NH2] (G4) polyamidoamine dendrimer (MW 14214 g/mol) was purchased from Aldrich Chemical Co. and used as supplied. mPEG-PAMAM-G3 (methoxypoly(ethylene glycol) [NH2(CH2)2NH2] (G3) polyamidoamine; MW 5697 g/mol) and mPEG-PAMAM-G4 (methoxypoly(ethylene glycol) [NH2(CH2)2NH2] (G4) polyamidoamine; MW 8423 g/mol; mPEG block has a molecular weight of 5000 g/mol) with dendrimer-conjugate stoichiometry n ) 112.9 were synthesized according to published methods.19,20 Other chemicals were of reagent grade and used without further purification. Preparation of Stock Solutions. BSA was dissolved in aqueous solution (40 mg/mL or 0.5 mM) containing 10 mM Tris-HCl buffer

10.1021/bm9011979  2010 American Chemical Society Published on Web 01/19/2010

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Mandeville and Tajmir-Riahi

Scheme 1. (a) Chemical Structure of PAMAM-G4 Dendrimer; (b) Chemical Structure mPEG-PAMAM-G3 Dendrimer

(pH 7.4). The protein concentration was determined spectrophotometrically using the extinction coefficient of 36 500 M-1 cm-1 at 280 nm.21

Dendrimer 1 mM was prepared in distilled water and diluted to various concentrations in 10 mM Tris-HCl.

Dendrimer-BSA Interaction Scheme 2. Structure of Bovine Serum Albumin, with Tryptophan Residues Shown in Green Color

Fourier Transform Infrared (FTIR) Spectroscopic Measurements. Infrared spectra were recorded on a FTIR spectrometer (Impact 420 model) equipped with deuterated triglycine sulfate (DTGS) detector and KBr beam splitter, using AgBr windows. Solution of dendrimer was added dropwise to the BSA solution with constant stirring to ensure the formation of homogeneous solution and to have dendrimer concentrations of 0.025, 0.05, and 0.1 mM, with a final protein concentration of 0.25 mM (20 mg/mL). Spectra were collected after a 2 h incubation of BSA with polymer solution at room temperature using hydrated films. Interferograms were accumulated over the spectral range 4000-600 cm-1, with a nominal resolution of 2 cm-1 and 100 scans. The difference spectra [(protein solution + dendrimer solution) - (protein solution)] were generated using the polypeptide antisymmetric and symmetric C-H stretching bands22 at 2952, 2932, 2912, and 2868 cm-1 as internal standard. These bands, which are due to protein C-H stretching vibrations, do not undergo any spectral changes (shifting or intensity variation) upon polymer complexation, and therefore, they are commonly used as internal standard. When producing difference spectra, these bands were adjusted to the baseline level to normalize difference spectra. Details regarding infrared spectral treatment are given in our recent publication.23 Analysis of Protein Conformation. Analysis of the secondary structure of BSA and its dendrimer complexes was carried out on the basis of the procedure previously reported.24 The protein secondary structure is determined from the shape of the amide I band, located around 1660-1650 cm-1. The FT-IR spectra were smoothed and their baselines were corrected automatically using Grams AI software. Thus, the root-mean square (rms) noise of every spectrum was calculated. By means of the second derivative in the spectral region 1700-1600 cm-1, six major peaks for BSA and the complexes were resolved. The above spectral region was deconvoluted by the curve-fitting method with the Levenberg-Marquadt algorithm, the peaks corresponding to R-helix (1658-1656 cm-1), β-sheet (1638-1614 cm-1), turn (1670-1665 cm-1), random coil (1648-1640 cm-1), and β-antiparallel (1692-1680 cm-1) were adjusted, and the area was measured with the Gaussian function. The area of all the component bands assigned to a given conformation were then summed up and divided by the total area.25 The curve-fitting analysis was performed using the GRAMS/AI version 7.01 software of the Galactic Industries Corporation. Circular Dichroism (CD). CD spectra of BSA and its dendrimer complexes were recorded with a Jasco J-720 spectropolarimeter. For measurements in the far-UV region (178-260 nm), a quartz cell with a path length of 0.01 cm was used in a nitrogen atmosphere. BSA concentration was kept constant (12.5 µM) while varying each polymer concentration (0.0125, 0.025, 0.05 and 0.1 mM). An accumulation of


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three scans with a scan speed of 50 nm per min was performed and data were collected for each nm from 260 to 180 nm. Sample temperature was maintained at 25 °C using a Neslab RTE-111 circulating water bath connected to the water-jacketed quartz cuvettes. Spectra were corrected for buffer signal, and conversion to the Mol CD (∆ε) was performed with the Jasco Standard Analysis software. The protein secondary structure was calculated using CDSSTR, which calculates the different assignments of secondary structures by comparison with CD spectra, measured from different proteins for which high quality X-ray diffraction data are available.26,27 The program CDSSTR is provided in CDPro software package, which is available at http://lamar.colostate.edu/∼sreeram/CDPro. Fluorescence Spectroscopy. Fluorometric experiments were carried out on a Varian Cary Eclipse. Stock solutions of dendrimer 1 mM in buffer (pH ) 7.4) were prepared at room temperature (24 ( 1 °C). Various solutions of polymers (10-400 µM) were prepared from the above stock solutions by successive dilutions also at 24 ( 1 °C. A solution of BSA (25 µM) in 10 mM Tris-HCl (pH. 7.4) was also prepared at 24 ( 1 °C. The above solutions were kept in the dark and used soon after. Samples containing 0.4 mL of the above BSA solution and 0.4 mL of various polymer solutions were mixed to obtain final polymer concentration of 5 to 200 µM with constant BSA content 12.5 µM. The fluorescence spectra were recorded at λexc ) 280 nm and λem from 287 to 500 nm. The intensity at 337 nm (tryptophane) was used to calculate the binding constant (K) according to literature reports.28-31 Model of Bovine Serum Albumin. The structure of BSA was predicted by automated homology modeling using SWISS-MODEL Workspace32,33 from the amino acid sequence NP-851335. The structure of free HSA (PDB id: 1AO6, chain A) obtained by X-ray crystallography34 was used as a template. These two proteins share 78.1% of sequence identity, which is sufficient to obtain reliable sequence alignment.35 Images of the structures were generated using Pymol (DeLano Scientific, Palo Alto, CA); RMSD between model and template proteins was 0.20 Å for positions of backbone atoms, as calculated with DeepView/Swiss-PdbViewer 4.0.1 (Scheme 2). The quality of the predicted BSA structure was found to be similar to the structure of free HSA, used here as a template using structure and model assessment tools of SWISS-MODEL workspace.

Results and Discussion FTIR Spectra of Dendrimer-BSA Complexes. The dendrimer-BSA interaction was characterized by infrared spectroscopy and its derivative methods. Because there was no major spectral shifting for the protein amide I band at 1656 cm-1 (mainly CdO stretch) and amide II band at 1545 cm-1 (C-N stretching coupled with N-H bending modes)22-24 upon polymer interaction, the difference spectra [(protein solution + dendrimer solution) - (protein solution)] were obtained to monitor the intensity variations of these vibrations, and the results are shown in Figure 1. Similarly, the infrared selfdeconvolution with second derivative resolution enhancement and curve-fitting procedures24 were used to determine the protein secondary structures in the presence of dendrimers (Figure 2 and Table 1). CD spectroscopy was also used to analyze the protein conformation in the polymer-BSA complexes, and the results are shown in Figure 3 and Table 2. At low dendrimer concentration (0.025 mM), an increase in intensity was observed for the protein amide I at 1656 and amide II at 1545 cm-1, in the difference spectra of the mPEGPAMAM-G3-, mPEG-PAMAM-G4-, and PAMAM-G4-BSA complexes (Figure 1, diff. 0.025 mM). Positive features are located in the difference spectra for amide I and II bands at 1654 and 1540 cm-1 (mPEG-PAMAM-G3), 1654 and 1540 cm-1 (mPEG-PAMAM-G4), and 1654 and 1540 cm-1 (PAMAMG4, Figure 1, diff., 0.025 mM). These positive features are

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Figure 1. FTIR spectra in the region of 1800-600 cm-1 of hydrated films (pH 7.4) for free BSA (0.25 mM), (A) free mPEG-PAMAM-G3 (0.5 mM), (B) free mPEG-AMAM-G4 (0.5 mM), (C) free mPEGAMAM-G4 (0.5 mm), and their BSA complexes with difference spectra (diff.; bottom two curves) obtained at different polymer concentrations (indicated on the figure).

related to an increase in the intensity of the amide I and amide II bands upon polymer complexation The increase in intensity of the amide I and amide II bands is due to polymer binding to protein CdO, C-N, and N-H groups. Additional evidence to support the polymer interaction with C-N and N-H groups comes from the shifting of the protein amide A band at 3290 cm-1 (N-H stretching mode) in the free BSA to 3288 (mPEGPAMAM-G3), 3288 (mPEG-PAMAM-G4), and 3287 cm-1 (PAMAM-G4), upon dendrimer complexation (spectra not shown). As polymer concentration increased to 0.1 mM, a decrease in intensity of the amide I was observed, with features at 1654 cm-1 (mPEG-PAMAM-G3), 1654 cm-1 (mPEG-PAMAM-G4), and 1654 cm-1 (PAMAM-G4) in the difference spectra of dendrimer-BSA complexes (Figures 1, diff, 0.1 mM). The observed decrease in intensity of the amide I band at 1656 cm-1 in the spectra of the polymer-protein complexes suggests a major reduction of protein R-helical structure at high dendrimer concentrations. Similar infrared spectral changes were observed for the protein amide I band in several ligand-protein complexes where the major protein conformational changes occurred.36 A quantitative analysis of the protein secondary structure for the free BSA and its polymer adducts in hydrated films has been carried out, and the results are shown in Figure 2 and Table 1. The free protein has 63% R-helix (1658 cm-1), β-sheet 14% (1632 and 1621 cm-1), turn structure 10% (1680 cm-1), β-antiparallel 7% (1692 cm-1), and random coil 6% (1644 cm-1) (Figure 2A and Table 1). The results are consistent with the spectroscopic studies of BSA previously reported.37,38 Upon dendrimer interaction, a major decrease of R-helix from 63%


Figure 2. Second derivative resolution enhancement and curve-fitted amide I region (1700-1600 cm-1) for free BSA and its polymer adducts with 0.1 mM dendrimer and 0.25 mM protein concentrations at pH 7.4.

(free BSA) to 55% (mPEG-PAMAM-G3-BSA), 56% (mPEGPAMAM-G4-BSA), and 48% (PAMAM-G4) with an increase in random coil from 6% (free BSA) to 10% (mPEG-PAMAMG3-BSA), 7% (mPEG-PAMAM-G4-BSA), and 12% (PAMAMG4-BSA) (Figure 2 and Table 1). A similar increase was also observed for the turn structure from 10% (free BSA) to 13% (mPEG-PAMAM-G3-BSA), 14% (mPEG-PAMAM-G4-BSA), and 13% (PAMAM-G4-BSA; Table 1). These results are consistent with the decrease in the intensity of the protein amide I band discussed above. The major decrease in R-helix structure and increase in random coil suggest a partial protein unfolding at high polymer concentration. Hydrophobic Interactions. The dendrimer antisymmetric and symmetric CH2 stretching vibrations39,40 in the region of 3000-2800 cm-1 of IR spectra were used to examine the presence of hydrophobic contact in the polymer-BSA complexes. The CH2 bands of the free mPEG-PAMAM-G3 located at 2952, 2942, 2919, and 2890 cm-1 shifted to 2958, 2933, 2913, and 2871 cm-1 (mPEG-PAMAM-G3-BSA); free mPEGPAMAM-G4 with CH2 bands at 2944, 2919, 2883, and 2857 cm-1 shifted to 2958, 2933, and 2871 cm-1 (mPEG-PAMAMG4-BSA) and free PAMAM-G4 with CH2 bands at 2938, 2981, and 2838 cm-1 shifted to 2956, 2933, and 2871 cm-1 (PAMAMG4-BSA) in the dendrimer-protein complexes (Figure 3). The shifting of the polymer antisymmetric and symmetric CH2 stretching vibrations in the region 3000-2800 cm-1 of the infrared spectra suggest the presence of hydrophobic interactions via polymer aliphatic chain and hydrophobic pockets in BSA. However, a major spectral shifting of dendrimer O-H, N-H, and C-O stretching and bending modes39,40 in the region of

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Table 1. Secondary Structure Analysis (Infrared Spectra) from the Free BSA and its Dendrimer Complexes in Hydrated Film at pH 7.4a amide I components (cm-1) 1695-1674 1686-1664 1657-1648 1648-1643 1642-1623 a

β-anti ((1%) turn ((2%) R-helix ((3%) random coil ((1%) β-sheet ((2%)

free BSA (%), 0.1 mM

mPEG-PAMAM G3-BSA (%), 0.1 mM

mPEG-PAMAM G4-BSA (%), 0.1 mM

PAMAM-G4-BSA (%), 0.1 mM

7 10 63 6 14

9 13 55 10 13

9 14 56 7 14

12 13 48 12 15

References 54 and 55.

212 in the second domain. Trp-212 is located within a hydrophobic binding pocket of the protein and Trp-134 is located on the surface of the molecule (Scheme 2). Tryptophan emission dominates BSA fluorescence spectra in the UV region. When other molecules interact with BSA, tryptophan fluorescence may change, depending on the impact of such an interaction on the protein conformation.18 On the assumption that there are (n) substantive binding sites for quencher (Q) on protein (B), the quenching reaction can be shown as follows:

nQ + B S QnB

(1)

The binding constant (KA) can be calculated as

KA ) [QnB]/[Q]n[B]

(2)

where [Q] and [B] are the quencher and protein concentration, respectively, [QnB] is the concentration of nonfluorescent fluorophore-quencher complex, and [B0] gives the total protein concentration

Figure 3. Spectral changes of dendrimer CH2 symmetric and antisymmetric stretching vibrations upon BSA complexation (the contribution from free protein vibrations has been subtracted in this region).

1700-600 cm-1 are evidenced for polymer-protein hydrophilic interactions (Figure 1). CD Spectra and Protein Conformation. The CD spectroscopic results shown in Figure 4 and Table 2 exhibit marked similarities with those of the infrared data. Secondary structure calculations based on CD data suggest that free BSA has a high R-helix content, 62%, β-sheet 13% turn, 14%, and random coil, 11% (Figure 3 and Table 2), which is consistent with the literature report.37 Upon dendrimer complexation, a major reduction of R-helix was observed from 62% free BSA to 50% in mPEG-PAMAM-G3, 54% mPEG-PAMAM-G4, and 49% PAMAM-G4 (Figure 4 and Table 2). The major decrease in R-helix was accompanied by an increase in the β-sheet and random coil structures (Table 2). The major reduction of the R-helix with an increase in the β-sheet and random is consistent with the infrared results that showed reduction of R-helix and an increase of random coil and turn structure due to a partial protein unfolding (Tables 1 and 2). Fluorescence Spectra and Stability of Dendrimer-BSA Complexes. BSA has two tryptophan residues that possess intrinsic fluorescence:17 Trp-134 in the first domain and Trp-

[QnB] ) [B0] - [B]

(3)

KA ) [B0] - [B]/[Q]n[B]

(4)

The fluorescence intensity is proportional to the protein concentration as described:

[B]/[B0] ∝ F/F0

(5)

Results from fluorescence measurements can be used to estimate the binding constant of the polymer-protein complex. From eq 4,

log[(F0 - F)/F] ) log KA + nlog[Q]

(6)

The accessible fluorophore fraction (f) can be calculated by modified Stern-Volmer equation:

F0 /(F0 - F) ) 1/fK[Q] + 1/f

(7)

where F0 is the initial fluorescence intensity and F is the fluorescence intensities in the presence of quenching agent (or interacting molecule), K is the Stern-Volmer quenching constant, [Q] is the molar concentration of quencher, and f is the fraction of accessible fluorophore to a polar quencher, which indicates the fractional fluorescence contribution of the total emission for an interaction with a hydrophobic quencher.18 The plot of F0/(F0 - F) versus 1/[Q] yields f-1

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Table 2. Secondary Structure of BSA Complexes (CD spectra) with Dendrimers at pH 7.4a dendrimer concentration

R-helix % ((3%)

β-sheet % ((2%)

turn % ((2%)

random % ((2%)

free BSA (12.5 µM) mPEG-PAMAM-G3-BSA (0.1 mM) mPEG-PAMAM-G4-BSA (0.1 mM) PAMAM G4-BSA (0.1 mM)

62 50 54 49

13 20 15 24

14 16 16 15

11 14 15 12

a

Calculated by CDSSTR software.

Figure 4. Circular dichroism of free BSA and its dendrimer complexes in aqueous solution with protein concentration of 12.5 µM and polymer concentrations of 0.0125, 0.025, and 0.1 mM in 10 mM Tris-HCl buffer, pH 7.4, at 25 °C.

as the intercept on the y axis and (fK)-1 as the slope. Thus, the ratio of the ordinate and the slope gives K. The decrease of fluorescence intensity of BSA is monitored at 337 nm for BSA-polymer systems (Figure 5 shows representative results for each system). The plot of F0/(F0 - F) versus 1/[dendrimer] (Figure 5A-C shows representative plots). Assuming that the observed changes in fluorescence come from the interaction between polymer and BSA, the quenching constant can be taken as the binding constant of the complex formation. The K values given here are averages of four replicate and six replicate runs for BSA/polymer systems, each run involving several different concentrations of dendrimer (Figure 5). The binding constants obtained were KmPEG3 -1 and KmPEG-G4 ) 1.0 × 104 M-1 and G3 ) 5.0 × 10 M

KPAMAM ) 1.1 × 104 M-1 (Figure 5A′, B′, and C′). The binding constants calculated for the polymer-BSA suggest low affinity dendrimer-BSA binding, compared to the other strong ligand-protein complexes.41,42 However, lower binding constants (104 M-1 to 105 M-1) were also reported for several other ligand-protein complexes using fluorescence spectroscopic methods.43-45 The binding constant for PAMAM-G4/BSA is expected to be the highest, as there are 64 amino groups on the periphery of the dendrimer compared with 16 on mPEG-PAMAM-G4 and 8 on mPEG-PAMAMG3 dendrimers. Additionally, the intramolecular hydrogen bonding between mPEG and the PAMAM dendron should result in a weaker interaction in mPEG-PAMAM/BSA complexes.

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Figure 5. Fluorescence emission spectra of dendrimer-BSA systems in 10 mM Tris-HCl buffer pH 7.4 at 25 °C for (A) mPEG-PAMAMG3-BSA: (a) free BSA (12.5 µM), (b-h) with mPEG-PAMAM-G3 at 20, 40, 50, 60, 70, 80, and 90 µM; (B) mPEG-PAMAM-G4-BSA: (a) free BSA (12.5), (b-h) with mPEG-PAMAM-G4 at 20, 30, 40, 50, 60, 70, 80, 90, and 100 µM; (C) PAMAM-G4-BSA: (a) free BSA (12.5 µM); (b-g) with PAMAM-G4 at 2.5, 10, 20, 30, 60, and 160 µM. The plot of F0/(F0 - F) as a function of 1/dendrimers concentration. The binding constant K being the ratio of the intercept and the slope for (A′) mPEG-PAMAM-G3-BSA, (B′) mPEG-PAMAM-G4-BSA, and (C′) PAMAM-G4-BSA complexes.

The f values shown in Figure 5 suggest that polymers interact with fluorophore via hydrophobic interactions. As a result, we predict that polymer binds mainly with the two fluorophores Trp-212 buried inside and Trp-134 located on the surface of BSA. This argument is based on the fact that the emissions λmax of Trp-212 and Try-134 are at 340 nm (Figure 5A-C), which is the emission region of hidden tryptophan molecules, while fluorescence emission of exposed tryptophan molecule is at higher wavelength (350 nm) due to solvent relaxation.44-46 The tightening of the protein structure through intramolecular interactions, such as hydrogen bonds, seems to bury Trp-212 in a more hydrophobic environment. The change in fluorescence intensity of Trp212 and Trp-134 in the presence of dendrimers may arise as a direct quenching or as a result of protein conformational changes induced by polymer-BSA complexation. The results indicate that polymer interaction does not change the emission λmax at 340 nm. No spectral shift was observed for the emission spectra upon polymer-BSA complexation, indicating that tryptophan molecules are not exposed to any change in polarity. The emission λmax of quenched tryptophan remains at 337 nm, suggesting that dendrimers interact with BSA via the hydrophobic region located inside and on the surface. This argument is consistent with the infrared analysis of dendrimer CH2 antisymmetric and symmetric stretching

Figure 6. Plot of log (F0 - F)/F as a function of log[dendrimer] for the calculation of the number of bound polymer (n) in dendrimer-BSA complexes.

vibrations that showed hydrophobic contact in polymer-BSA complexes (Figure 3). The number of dendrimers bound (n) is calculated from log[(F0 - F)/F] ) log KS + n log[dendrimer] for the static quenching.46-53 The linear plot of log [(F0 - F]/F] as a function of log[dendrimer], shown in Figure 6. The n values from the slope of the straight line are 1.30 (mPEG-PAMAM-G3), 1.30 (mPEG-PAMAM-G4), and 1.0 (PAMAM-G4; Figure 6A-C). It seems more than one molecule of the mPEG-PAMAM-G3 and mPEG-PAMAM-G4 bind BSA, while only one molecule of the bulkier PAMAM-G4 can tightly bind BSA in these polymer-protein complexes. Comparison Between Dendrimer-BSA and Dendrimer-HSA Complexes. HSA and BSA are homologue proteins showing 78.1% of sequence identity. BSA contains two tryptophan residues (Trp-212 and Trp-134), while HSA has one tryptophan (Trp- 214). The BSA three-dimensional structures are very similar to HSA, as suggested by the 3D modeling presented here (Scheme 2). Based on our spectroscopic data, dendrimer binding to BSA occurs via hydrophilic and hydrophobic interactions and causes a partial protein unfolding. Similarly, the dendrimer complexation with HSA induced

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protein unfolding.10 However, stronger polymer-protein interaction was observed for HSA-dendrimer10 with a binding constant of KmPEG-G3 ) 1.3 × 103 M-1, KmPEG-G4 ) 2.2 × 104 M-1, and KPAMAM ) 2.6 × 104 M-1 than those of BSA reported here KmPEG-G3 ) 5.0 × 103 M-1, KmPEG-G4 ) 1.0 × 104 M-1, and KPAMAM ) 1.1 × 104 M-1. The reason can be attributed to the polymer interaction with the HSA hydrophobic Trp-214 pocket buried inside the protein, while polymer-BSA complexation occurs partly with the Trp-134 in the surface. The order of binding is PAMAM-G4 > mPEG-PAMAM-G4 > mPEG-PAMAM-G3 for both proteins. It seems the addition of amino groups on the periphery of the dendrimer increases the stability of polymer-protein complexation for both HSA and BSA. It is clear that dendrimers are big macromolecules with positively charged surfaces at neutral pH. Similarly, BSA and HSA are large biopolymers with positively charged surfaces. The interaction is simply weak and mainly between dendrimer polar groups (NH2) and protein hydrophilic groups (CdO, CN, and NH) as well as hydrophobic contacts with Trp-212 of BSA and Trp-214 of HSA located inside the protein. Acknowledgment. This work is supported by a grant from Natural Sciences and Engineering Research Council of Canada (NSERC). We thank Professor R. Sedagaht-Herati of Missouri State University (U.S.A.) for the gift of mPEG-G3 and mPEGG4.

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BM9011979

Complexes of Dendrimers with Bovine Serum Albumin

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