Molecular Description of the Formation and Structure of Plasticized

Biomacromolecules 2005, 6, 3209-3219

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Molecular Description of the Formation and Structure of Plasticized Globular Protein Films Thierry Lefe` vre,*,† Muriel Subirade,‡ and Michel Pe´ zolet† Centre de recherches en Sciences et Inge´ nierie des Macromole´ cules (CERSIM), De´ partement de Chimie, Pavillon Alexandre-Vachon, and Canada Research Chair of Proteins, Bio-Systems and Functional Foods, Nutraceuticals and Functional Foods Institute (INAF), De´ partement de Sciences des Aliments et Nutrition, Pavillon Paul-Comtois, Universite´ Laval, Cite´ Universitaire, Que´ bec, Que´ bec, G1K 7P4 Canada Received August 1, 2005; Revised Manuscript Received September 30, 2005

To optimize the properties of plasticized globular proteins films, a clear comprehension of the structure and molecular events occurring during film formation is required. In this work, the structural organization of β-lactoglobulin (β-lg) films plasticized with diethyelene glycol are investigated for the first time during the entire film formation process by attenuated total reflectance and transmission infrared spectroscopy. The films are made by a common two-step procedure consisting of a first heat treatment (80 °C/30 min) followed by the casting of the film-forming solution for dehydration. Heating at 80 °C leads to the self-aggregation of the proteins with a conversion of regular secondary structures into antiparallel β-sheets. The kinetics of the conformational conversion shows that ∼10% of the amino acids are involved in β-sheets after the first step. Dehydration induces a further aggregation, with ∼46% of the amino acids involved in β-sheets in the final film. Water evaporation results in the association of the aggregates formed during the heating step. The presence of the plasticizer during water removal is essential as it allows specific conformational rearrangements into extended β-sheets and ordering of the polypeptide chains. This work underlines that the assembly of building blocks is common in β-lg networks and it emphasizes the widespread occurrence of β-structures in synthetic and natural protein networks. Introduction Plasticized self-supported films made of globular proteins are attractive biomaterials because of their potential applications as edible biodegradable coatings and wrappings.1,2 Edible films are used in the food industry to extend the shelf life of food products and to preserve their quality by inhibiting the migration of aroma, flavors, moisture, and oxygen. This type of matrix is also relevant for the pharmaceutical and medical areas since it may be intended for the encapsulation and release in vivo of bioactive substances at specific sites3 and for the growth of cell cultures.4 A clear understanding of the structure and formation of protein films is required to control their properties. Besides applications, globular protein films also arouse the interest for more fundamental aspects since they may be helpful in providing insights into the general rules and interactions that govern protein self-assembly and protein conformation. Edible films have been made with various food globular proteins including soy,5,6 wheat,7,8 pea,9 and whey proteins (WP).10 The latters received a particular attention since whey is a waste of the cheese industry that has to be valorized. In this respect, many efforts have been devoted to the optimization of the techno-functional properties of whey protein * Corresponding author. Phone: (418) 656-2131 #6233. Fax: (418) 6567916. E-mail: [email protected]. † De ´ partement de Chimie, Universite´ Laval. ‡ De ´ partement de Sciences des Aliments et Nutrition, Universite´ Laval.

isolates (WPI), a mixture of whey proteins. These properties, including gelifying, emulsifying, foaming and film-forming abilities are mainly based on the aggregation capacity of globular proteins that in turn allows the formation of different types of networks. The structure of the matrix is not only dictated by the type of applied treatment (heat, pressure, etc.) but also strongly depends on environmental factors such as pH, ionic strength, type of ions, and protein concentration. Since it is the most abundant protein in whey, β-lactoglobulin (β-lg) dominates the techno-functional properties of WPI. As an example, films prepared with WPI and β-lg have similar mechanical and barrier characteristics.11,12 β-Lg is a small globular protein of 164 amino acids that bears two disulfide bonds (Cys66-Cys160 and Cys106-Cys119) and one free thiol group (Cys121). They are frequently involved in the formation of disulfide bridges, via thiol oxidation and/or S-H/S-S exchange reaction, a phenomenon that plays a major role in many properties of β-lgbased networks.13-15 This protein is composed of nine β-strands that form a calyx, a three-turn R-helix and other minor secondary structures.16 Globular protein-based films are often made in two steps.5,6,10 A protein aqueous solution is first subject to a heat or alkaline treatment followed by the deposition of the film-forming solution on a substrate for dehydration in a controlled relative humidity (RH) atmosphere (typically RH ) 50%). The comprehension of the entire film formation process is only partial. It is generally admitted that proteins

10.1021/bm050540u CCC: $30.25 © 2005 American Chemical Society Published on Web 10/22/2005

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Biomacromolecules, Vol. 6, No. 6, 2005

are denatured during the first step resulting in the exposition to water of the initially buried thiol groups and hydrophobic side-chains. Upon dehydration, these “reactive” unfolded proteins can interact with each other, thus allowing the formation of a network. The role of protein polymerization by covalent bonds on the film characteristics has been underlined since disulfide bonds have been found to develop during the first and second steps.13 However, physical interactions including hydrophobic effects and hydrogen bonds are clearly also involved,17,18 but the contribution of each type of interaction remains unclear. Since films made with globular proteins are very brittle, a plasticizer has to be added before dehydration to provide flexibility to the final film. Plasticizers are usually lowmolecular weight molecules such as sorbitol, glycerol, or other polyols. As the plasticizer content increases, the tensile strength of the films decreases, and the breaking strain and oxygen permeability (OP) increase.7,10 This stems from the ability of the plasticizer to disrupt intermolecular association and to increase intermolecular spacing between the biopolymers which results in an increase of the chain mobility and free volume of the polypeptide chains.19 The ability of a plasticizer to form hydrogen bonds with the proteins as well as its ability to associate with water20 seems also to influence its efficiency. Since water also acts as a plasticizing agent, the RH is an important factor that influences the mechanical properties and permeability of the films. Most of the studies on edible films have focused on some macroscopic characteristics: stress and strain at break and permeability to oxygen, moisture, and lipids. Due to their hydrophilic nature, globular protein films have a poor water vapor permeability and a good OP.7,10 The influence of the type and concentration of plasticizer, pH, and heating conditions has been investigated. For example, more severe heat treatments result in improved film properties.10,21 Films become stiffer, stronger, and more stretchable for higher heating temperatures and longer heating times.21 Other (understudied) factors may also influence the macroscopic properties of protein films among which are the cross-linking density and the crystallinity.22,23 A higher cross-linking density decreases water vapor permeability, whereas cystallinity is usually assumed to be impermeable to oxygen.23 Higher crystallinity and cross-linking density results in an increase of the glass transition temperature, Tg, which is another important parameter since permeability is enhanced above Tg due to the increased mobility of the chains in the rubbery state. Whereas macroscopic properties of films made with globular proteins have been investigated extensively, only a few data are available about their structure. Images of β-lg films obtained by atomic force microscopy24 have shown a porous structure with large particles of ∼1 µm × 1 µm to ∼15 µm × 15 µm attributed to protein aggregation. Features of the size of the protein molecule have also been identified. The microstructure of WPI films as studied by transmission electronic microscopy is described as a protein matrix surrounded by a water and plasticizer phase.25 The microstructure depends on the protein concentration, plasticizer, and pH.25 At high WPI concentrations, a very aggregated

Lefe` vre et al.

structure with large pores is formed, whereas a fine structure is formed at low concentrations. As the pH increases from 7 to 9, a denser structure is formed. Changes in the mechanical properties and OP and WVP have been related to differences in film microstructures.25 Films made of soybean glycinin26 and wheat gliadin8 have been examined at a molecular level using Fourier transform infrared (FTIR) spectroscopy. These studies revealed the presence of intermolecular β-sheets after the heating and dehydration steps which underlines the importance of hydrogen bonding in the protein network. β-Sheets have then been proposed to act as junction zones in the cohesion of the film, and a relation between protein conformation and mechanical properties has been proposed.26 A recent study by Raman spectroscopy has confirmed the predominance of β-sheet structures in β-lg films.27 The β-sheets seem to be perpendicular to the filamentous aggregate long axis, in a similar way than in amyloids. Such reports underline the role that the β-sheet motif may play in the structure and properties of globular protein films but more data are needed to evaluate the importance of this type of structure. In this study, the contribution of β-sheet in plasticized films made with β-lg from heat-denatured solutions and the film structural organization have been analyzed using attenuated total reflection (ATR) and transmission FTIR spectroscopy. Since only a few data are available about the molecular events occurring during film formation, especially during the dehydration step, we sought to describe the protein conformational changes occurring during the entire film formation process. The influence (or the absence) of the heat treatment and the role played by the plasticizer on the film structure have also been considered. Relations between globular protein films in one hand and heat-induced and cold-set gels on the other hand are underlined. Experimental Section Materials. Bovine milk β-lg, a crystallized and thrice lyophilized powder containing genetic variants A and B, was purchased from Sigma Chemical Co. (Ref L-0130, lot 124H7045). The purity is ∼90% as certified by the manufacturer. Deuterated water (D2O) was obtained from CDN Isotopes, Inc. (Pointe-Claire, Canada). The plasticizer used was diethylene glycol (DEG) and was obtained from Aldrich, Ltd. All products were used without further purification. Sample Preparation. Self-supported films were prepared according to the procedure of McHugh et al.10 β-lg was first dissolved in distilled H2O or in D2O at 10% w/v. The use of D2O is an efficient way to study the conformation of protein by FTIR spectroscopy during the whole film formation process since the protein amide I band is free from D2O absorption bands, whereas in H2O, it overlaps with the O-H bending mode. As a standard procedure, the first step of the film preparation consists of heating the protein solution for 30 min in a water bath set at 80 °C followed by a rapid cooling to room temperature in a bath of water. For some investigations, samples have been heated at different temperatures (between 68 and 80 °C) during various times. The

Plasticized Globular Protein Films

chosen protein concentration was a compromise between two goals: obtaining a dense and stiff film using high concentration while avoiding gelation of the solution during the heating step by using too high concentrations which would had hampered further casting. At 10% w/v of β-lg, the concentration is just below the critical gelation concentration28,29 and the solutions remain liquid after heating allowing casting for dehydration. In addition, the ionic strength has to be sufficiently low and the pH far from the isoelectric point (pI ) 5.2) to avoid gelation and coagulation that would have occurred under such conditions during heating even at low protein concentrations due to reduced electrostatic repulsions. In this study, the ionic strength was minimized by using unbuffered solutions. The resulting pH was 7 in H2O and 7.4 in D2O. DEG was mixed with the heat-denatured protein solution which is then referred to as the film-forming solution. DEG content ranged from 1:0.4 to 1:1.2 β-lg:DEG weight ratio, but in most experiments the ratio was 1:1. Filmforming solutions were cast on glass Petri dishes and allowed for water evaporation at a 50% RH. Dehydration of the D2O film-forming solution was investigated in situ in the spectrometers purged with dried air (RH ∼ 0%). Typically, complete drying of a 50 µL sample takes around 90 min. Other films were dried from D2O film-forming solutions in a desiccator containing a D2O vapor environment (RH ∼ 50%). The RH of the sample compartment was controlled with an aqueous solution saturated with NaCl. It was verified at each step of the film preparation that the D2O samples were not contaminated by the water vapor from the atmosphere. Because the molar absorptivity of the amide I band is high,30 very thin films have to be used to record nonsaturated spectra in the transmission mode. Thus, 15 µL of the filmforming solution was deposited on a 22 × 30 mm Parafilm surface, resulting in self-supported films having a thickness of 5-6 µm. Parafilm was used since this deformable substrate appeared very suitable to delicately pulled off such fragile films. FT-IR Spectroscopy. The spectra were recorded using either a Magna 850 or a Magna 560 ThermoElectron spectrometer (Madison, WI) equipped with a KBr beam splitter and a liquid-nitrogen-cooled mercury-cadmiumtelluride detector. Each spectrum is the result of the average of 128 scans at 2 cm-1 resolution apodized with a HappGenzel function. ATR infrared spectroscopy has allowed the study in situ of the dehydration process within the spectrometer purged with dry air on ZnSe or germanium crystals. A drop of the solution was deposited on the crystal and spectra were recorded as a function of time. The spectra of the films dehydrated in situ on the ATR crystals were close to those dehydrated in a desiccator at a controlled RH atmosphere (RH ∼ 50%) and subsequently deposited on the ATR crystals. Only small variations in relative intensities at 1655 and 1629 cm-1 (see below) were observed. Therefore, films made in situ on ATR crystals are highly representative of conventional films (i.e., dehydrated in desiccators at a controlled RH). Transmission infrared spectroscopy was used to study free-standing films and heat-induced denaturation of aqueous solutions. For the latter, liquid samples were

Biomacromolecules, Vol. 6, No. 6, 2005 3211

Figure 1. (a) Transmission infrared spectra in the amide I′ region of a solution of β-lg in D2O (10% w/v) heated at 80 °C as a function of time from the onset of aggregation up to 100 min. Spectra were normalized to give an area of 1. Arrows indicate the direction of intensity changes as time increases. (b) Same spectra as in (a) after subtraction of the first spectrum. The absorbance of the band at 1616 cm-1 at end of the heating step (after 30 min) is indicated by an arrow. (c) Area of the components at 1685 and 1616 cm-1, A(1685+1616), as a function of time. The level of aggregation at the end of the heating step is indicated by an arrow.

placed between two CaF2 Biocell windows from Biotools, Inc. (Elmhurst, IL) manufactured with a calibrated path length of 40 µm. The windows were placed in a homemade heating cell using a Peltier element as a heating/cooling device. To study the first step of the film formation process, film-forming solutions were heated progressively from 25 to 80 °C at a rate of ∼2 °C‚min-1 and equilibrated at 80 °C. A spectrum was recorded every 1.5 min during the whole heating treatment. The free-standing films were mounted between the two jaws of a stretcher that is especially designed to be installed within the spectrometer. No stress was applied on the sample. If necessary, water vapor was subtracted. For the D2O solutions, the spectra of pure D2O was also subtracted so that a flat baseline was obtained between 2000 and 1700 cm-1. A linear baseline was also subtracted in the 17501500-cm-1 region. For the transmission spectra of heated solutions, the area was normalized to a value of 1 to avoid possible non desirable and irrelevant intensity variations. For the analysis of secondary structure contents, spectra were curve-fitted in the 1720-1500 cm-1 region by using Gaussian-Lorentzian functions. The minimun number of components that gave reasonable fits was used. The initial values of the peak positions were determined from self-deconvolution and from the literature. The band parameters (position, intensity, bandwidth and Lorentzian/Gaussian ratio) were free to evolve during fitting. The uncertainty on the band area corresponds to standard deviation calculated on identical samples. All data treatments were performed with GRAMS/ AI 7.0 (ThermoGalactic, Salem, NH). Results First Step (Heating Step). The first step consists of a heat treatment of a 10% w/v β-lg in D2O at 80 °C for 30 min. Figure 1a shows the transmission infrared spectra of β-lg in D2O at 80 °C as a function of heating time in the

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amide I′ region. The broad band between 1700 and 1600 cm-1 is due to the amide I′ mode and that at 1575 cm-1 is due to the antisymmetric COO- stretching vibration (νas(COO-)) of Glu and Asp residues.31,32 The transient heating period up to 80 °C corresponding to the protein unfolding is not shown since many details have been reported about the effect of increasing temperature on β-lg using FTIR spectroscopy.33-35 These studies have shown that the amide I′ band is dramatically altered upon heating due to the loss of native structures such as R-helix/unordered structures (1645 cm-1), turns (1660 cm-1), and intramolecular β-sheets (1634 cm-1). The first spectrum in Figure 1a precisely corresponds to the unfolded protein at the onset of aggregation (the very first spectrum for which the band at 1616 cm-1 starts to appear, see below). As shown by the peak maximum at 1632 cm-1, the denatured state still contains nativelike structures such as intramolecular β-sheets. Thus, the unfolded state of β-lg is not a completely unordered polypeptide chain, a fact that is now well established for proteins in general.36 Upon heating, two bands near 1685 and 1616 cm-1 (Figure 1a) increase in intensity due to the formation of intermolecular antiparallel β-sheets. These bands arise from a splitting of the amide I′ band due to intermolecular transition dipole coupling.37-39 Extended antiparallel β-sheets are commonly found in aggregated proteins,40,41 especially in heat-denatured proteins.33,42,43 The heat-induced aggregation/ gelation of β-lg is a well-known phenomenon which has been extensively investigated using many techniques.44-46 These studies and the current IR spectra lead to the view that proteins are not only unfolded after the first step of the film preparation, but aggregation does occur and the aggregates are partly composed of β-sheets. Moreover, the appearance of the bands at 1685 and 1616 cm-1 is concomitant to a decrease in intensity of a broad feature at 1680-1627 cm-1. This observation represents a progressive conversion of residual regular structures and unordered segments into intermolecular β-sheets. The three isosbestic points at 1680, 1627, and 1593 cm-1 indicate that aggregation is a two-state process and that nativelike structures are directly converted into intermolecular β-sheets without any accumulated intermediate species. Figure 1b shows the spectra of Figure 1a after subtraction of the spectrum at the onset of aggregation (first trace in Figure 1a). These difference spectra clearly show the isosbestic points as well as the development and the position of the two aggregation bands at 1685 and 1616 cm-1. The position of the latter one can provide insights into the molecular structure of the aggregates.35,47 It is known that two types of heat-induced gel with different structures can be formed depending on the aqueous environment.28,48 When electrostatic repulsions are high (low ionic strength or pH far from the pI), the so-called fine stranded gels are formed. The aggregates result from an ordered agregation and lead to the formation of linear aggregates. These gels are transparent, homogeneous at a micron scale, and have a high water-holding capacity. When electrostatic repulsions are minimized (high ionic strength/pH near the pI), particulate gels occur. The aggregates result from a nonspecific ag-

Lefe` vre et al.

Figure 2. Spectra in the amide I′ region of the denatured β-lg solution (10% β-lg w/v) after the heating step (30 min at 80 °C) in the absence (full line) and presence (broken line) of the plasticizer (DEG), and spectrum of the dehydrated film dissolved in D2O (dotted line). Spectra are recorded at room temperature.

gregation process with coarse particles (500-1000 nm). The resulting gels are opaque, heterogeneous at a micron scale, and have a low water-holding capacity. In IR spectroscopy, the low-wavenumber aggregation component corresponding to fine-stranded agregates is located below ∼1617 cm-1 at 85 °C, whereas for particulate gels, this band is located above ∼1620 cm-1.35,47 Therefore, the band at 1616 cm-1 in Figure 1 shows that fine-stranded aggregates are formed during the heating step of the film peparation. In addition, these aggregates are irreversibly formed (data not shown), most probably due to the formation of disulfide bridges.14,49 From the spectra in Figure 1b, the area of the two aggregation components, A(1685+1616), can be measured as a function of time (Figure 1c) thus providing a way to analyze the kinetics of β-sheet formation. The kinetics is exponential at the beginning of the process and then is linear up to 100 min (Figure 1c). The formation of β-sheets even continues at the same rate for at least 5 h (data not shown). Consequently, longer heating times, which are known to induce higher aggregation levels (more and larger aggregates), also generate more β-sheets. Similarly, higher heating temperatures (see below) or higher protein concentration50 result in higher contents of intermolecular β-sheets and in more aggregated structures. These observations highlight the relationship between aggregation level and the amount of β-sheets, which may have an impact on the properties of the final films. In the present study, the heating step (30 min of heating) ends approximately at the begining of the linear region (see Figure 1c). Based on the area of the aggregation bands with respect to the area of the whole amide I′ band, it is found that about 10% of the amino acids are involved in intermolecular β-sheets after 30 min of heating. The remaining amino acids may correspond to proteins that belong to aggregates but for which not all of the amino acids are converted into intermolecular β-sheets, and/or they may correspond to isolated and unfolded (nonaggregated) proteins. Figure 2 (solid and dashed lines) shows the comparison of the denatured protein solution in the absence and in the presence of the plasticizer (DEG added after the heat treatment). In agreement with another FTIR study,8 no difference is observed in the amide I′ region indicating that the conformation of the proteins, whether they are isolated or involved in aggregates, is not affected by the plasticizer. In particular, the intermolecular β-sheets formed during the first step are



Figure 3. ATR spectra in the amide I′ region of a film-forming β-lg solution (10% w/v in D2O, after heating 30 min at 80 °C, β-lg:DEG weight ratio of 1:1) as a function of time during dehydration. The arrow indicates the direction of intensity changes as time increases. In the inset, the broken and the full lines correspond to the first (film-forming solution) and last (final film) spectrum of the figure, respectively. They are normalized relative to the peak intensity maximum.

stable in the sense that they are unaltered by DEG. The plasticizer does not perturb the structure of the aggregates in solution and the interactions between DEG and proteins are equivalent to those occurring between water and proteins. Finally, DEG, which is water miscible, seems to form with water a continuous liquid phase in which the aggregates are dispersed. Second Step (Dehydration Step). The next step of the film preparation consists of the dehydration of the filmforming solution. The dehydration step has been studied in situ within the spectrometer purged with dry air (0% RH) on an ATR crystal. Figure 3 shows the ATR spectra of the film-forming solution in the amide I′ region as a function of time during water evaporation (the spectra are recorded during ∼3 h). The intensities of the amide I′ and νas(COO-) bands dramatically increase as a result of the rise of the protein content at the crystal surface upon dehydration. The small band at 1515 cm-1 due to the ν(CC) + δ(CH) ring vibration of Tyr residues32,51 increases for the same reason. The DEG content also increases upon dehydration as revealed by the rise in intensity of DEG bands in the low-frequency region of the spectrum (data not shown). The first spectrum in Figure 3 corresponds to the film-forming solution and is close to that obtained in transmission (Figure 2). Upon water loss, the peak maximum of the amide I′ band shifts from 1632 to 1622 cm-1 due to the appearance of a strong band at 1622 cm-1. The development of this band and the simultaneous increase in intensity at 1684 cm-1 shows that a large amount of additional intermolecular antiparallel β-sheets are formed during water evaporation. The broad component at ∼1642 cm-1 reflects the presence of residual regular structures. At this position, mainly R-helices and unordered structures are likely to contribute in D2O.39,40 The analysis of the normalized spectra (not shown) reveals no isosbestic point suggesting that protein film dehydration is a complex multistep process. Water removal may involve conformational changes, aggregation, and the effect of decreasing interactions between water and proteins on the vibrational modes. For comparison purposes, the inset in Figure 3 shows the first and last spectra (film-forming solution and film, respectively), the absorbance being normalized with respect to the peak maximum. As discussed above, the amide I′ band

Figure 4. Curve-fitting of a ATR spectrum corrsponding to a β-lg film plasticized with DEG. Table 1. Band Parameters Obtained after Curve-Fitting of the Spectrum of a Plasticized β-lg Film in the 1750-1500 cm-1 Region band position (cm-1)

assignment

areaa (%)

1683 ( 1 1662 ( 1 1640 ( 1 1620 ( 1 1602 ( 1 1585 ( 1 1567 ( 1 1514 ( 1

intermolecular antiparallel β-sheet turns R-helix/unordered structures intermolecular antiparallel β-sheet side-chain vibration (Arg) νas(COO-) of Asp νas(COO-) of Glu ν(CC) + δ(CH) ring of Tyr

5(1 13 ( 2 25 ( 2 30 ( 3 1 ( 0.5 8(1 17.5 ( 2 0.4 ( 0.2

a Areas are percentages of the total area in the 1720-1500-cm-1 region. The uncertainty on the parameters correspond to standard deviations calculated on identical samples.

of the film-forming solution is broad so that no component is clearly distinguishable. This spectrum corresponds to denatured and partially aggregated proteins. The large bandwidth indicates that the peptide bonds of the polypeptide chain experience many different dihedral angles corresponding to various secondary structural elements. On the contrary, the amide I′ band of the film exhibits well-defined components which is characteristic of an ordered conformation. Many of the polypeptide segments are restricted to a narrow range of different conformations, the main secondary structure being the antiparallel β-sheet. These ordered β-sheets are likely to provide a partial crystalline character to the film matrix. The spectrum of the film has the same features as the spectra obtained for other β-lg networks characterized by a large amount of β-sheets (gels, emulsions, etc.).35,41,47 To estimate the β-sheet content in the film, a curve-fitting of the spectrum of the fully dehydrated solution (last spectrum in Figure 3a) has been carried out using Lorentzian-Gaussian functions (Figure 4). Eight components were used in the 1760-1500 cm-1 region. Four are associated with the amide I′ band, the others corresponding to side-chain vibrations. The positions, area, and assignments of the components are given in Table 1. The components at 1662 and 1640 cm-1 are representative of residual regular secondary structures in the film. Two components were necessary to adequately fit the νas(COO-) band at 1575 cm-1. The two bands are located at 1585 and 1567 cm-1, which is very good agreement with the values of the νas(COO-) frequencies of Asp and Glu residues in D2O, respectively.32 The relative intensity of these bands is consistent with the larger number of Glu (16 Glu against 11 Asp for β-lg A, 10 Asp for β-lg B). The area of the aggregation bands at 1683 and 1620 cm-1 indicates that

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Figure 5. The effect of heating temperature on the spectra in the amide I′ region of (a) the film-forming solutions and (b) films. Heating temperatures are indicated in (a) while arrows in (b) indicate the direction of spectral changes as the heating temperature increases. The dotted lines represent the film-forming solution in (a) and the film in (b) without prior heat treatment. Spectral intensitites are normalized to the maximum of the amide I′ band. The inset (c) presents the position of the aggregation band near 1622 cm-1 as a function of heating temperature. Spectra are recorded at room temperature and normalized with respect to the band intensity at 1622 cm-1.

48 ( 3% of the amide I′ band is due to intermolecular β-sheets. A similar curve-fitting carried out for samples made in H2O (not shown) gives a contribution of β-sheets of 45 ( 3%. Therefore, it can be assessed that ∼46% of the amino acids are engaged in β-sheets, whereas ∼54% form other structures such as R-helix, random coil segments and turns. To study the redispersibility of the films, D2O-based films were put in D2O. Films slowly dissolved, a process that takes about 96 h. The time for complete dissolution increases as the dehydration step was longer. Surprisingly, the spectrum of a dissolved film (dotted line in Figure 2) is almost identical to that of the solution before dehydration. This indicates that the film network formation is reversible under these conditions. More importantly, the network appears to be formed by the reversible assembly of the aggregates (particles) formed during the heating step. These particles can then be considered as building blocks that associate via physical interactions to constitute the network. It also appears that these building blocks are irreversibly formed due to S-S bonds formation as already noted, and that their cohesion and structure are not altered upon water removal. Effect of the Heat Treatment (Heating Step). Figure 5 shows the effect of the heating temperature during the first step of the film preparation on the spectra of the film-forming solutions (Figure 5a) and final films (Figure 5b). Since films can also be formed with unheated (native) β-lg,52 the spectra corresponding to unheated protein solutions are also shown. Interestingly, such films are very brittle and friable. Thus, the comparison with “conventional” films (dehydrated from heat-denatured solutions) may provide information about the relations that exist between mechanical

Lefe` vre et al.

Figure 6. Transmission spectra recorded as a function of time in (a) the 1800-850 cm-1 and (b) 1720-1485 cm-1 region of a selfsupported β-lg film made from H2O solution and plasticized with DEG. Arrows indicate the direction of the spectral changes as time increases. In (b), spectra are normalized with respect to the intensity at 1630 cm-1. (c) Plot of the position of the aggregation band at 1629 cm-1 (white squares) and intensity ratio of the bands at 1629 and 1655 cm-1 (black triangles), A(1629)/A(1655), as a function of the area of νC-O DEG bands, A(DEG), at 1130, 1078, and 1061 cm-1. The area A(DEG) is normalized so that it is equal to unity at the beginning of the experiment.

properties and molecular conformation. The spectrum of native β-lg in solution in the amide I′ region (Figure 6a) has been described elsewhere.33,34,47,50 As judged from the development of the shoulder at 1616 cm-1 in Figure 5a, heating at increasing temperatures clearly leads to a higher content of intermolecular β-sheets after the first step, as expected. This result is consistent with a higher aggregation level with increasing temperatures. None of these spectra is modified by addition of DEG (not shown). On the other hand, the spectra of the films (Figure 5b) exhibit small differences, with the predominance of the aggregation bands at ∼1685 and ∼1622 cm-1 which indicates that different heating temperatures lead to similar protein conformations. In particular, films made of unheated proteins exhibit the same structural features showing that native proteins also aggregate as water evaporates. Therefore, aggregation of the proteins during water evaporation is not sufficient to form stiff and stretchable films, and the formation of aggregates prior dehydration is required. The shape, structure, composition and size of the aggregates are likely to affect the film macroscopic properties. In particular, the higher the aggregation level after the heating step, the stiffer the films.21 Figure 5b shows that the relative absorbance at 1622/1642 cm-1, A(1622)/A(1642), slightly decreases with heating temperature indicating that the β-sheet content is slightly higher for films obtained from solutions that initially contain a smaller amount of heat-induced β-sheets. Films evaporated from native protein solutions


exhibit the highest content of β-sheets which is likely to provide a more crystalline character to the films. A shift of the 1622-cm-1 component with the heating temperature during the first step can also be observed in Figure 5b. The position is plotted as a function of heating temperature in Figure 5c. The higher the temperature, the lower the frequency, suggesting an increase in the strength of the intermolecular hydrogen bonds and/or a change in the amount of strands in the sheet. A similar trend has been observed for films made from solutions heated during longer heating times (data not presented). Therefore, although different heat treatments do not lead to dramatic spectral differences in the films, some variations in the strength of the intermolecular hydrogen bonds may partly explain differences in the film mechanical properties. For example, the fact that longer heating times lead to stiffer films21 is certainly related to a higher aggregation level after the heating step, but it may also be partly related to an increase in the strength of the intermolecular hydrogen bonds. As a matter of fact, the macroscopic consolidation of heat-induced β-lg gels during cooling is characterized by a progressive increase of ∼7000 Pa of the storage modulus G′. This consolidation is directly related a strengthening of the intermolecular β-sheet hydrogen bonds characterized by a shift of ∼2.5 cm-1 of the aggregation band (unpublished data). Effect of Plasticizer Content. The molar absorptivity of the amide I band is very high so that the thickness of selfsupported films have to be smaller than 6 µm to be investigated by transmission FTIR spectroscopy. The spectra between 1800 and 850 cm-1 of such a film are presented in Figure 6a. Since the films evolve upon aging, the spectra are presented as a function of time during 30 h. Bands at 1130, 1078 and 1061 cm-1 attributed to the C-O stretching vibrations (νC-O) of DEG decrease in intensity indicating a progressive loss of DEG. The other bands due to DEG (not shown) also decrease in intensity confirming that the plasticizer evaporates in films. Evaporation of various plasticizers including DEG has also been noted in wheat gliadin films.53 Moreover, the amide I and amide II bands increase in intensity showing that the density of proteins increases within the film area probed by the laser beam. This is confirmed by the observation that the film, which is pinned at its two extremities in these experiments, ultimately craks before all of the plasticizer molecules are evaporated. The necessity for the protein molecules to fill the voids created by the evaporation of DEG induces the development of stresses within the film that leads to its breakage. This observation evidences the crucial role of the plasticizer on the free volume and mobility of the polypeptide chains. To determine whether conformational changes of the polypeptide chains are associated to the loss of plasticizer, the amide I/II region was enlarged in Figure 6b. In H2O, the amide I band has similar features than in D2O with a predominance of the aggregation bands at 1629 and 1695 cm-1. Upon DEG loss, the 1629-cm-1 component shifts toward higher wavenumber values, whereas the position of the 1655-cm-1 component is not altered. In Figure 6c the position of the 1629-cm-1 band is plotted as a function of


the sum of the area of DEG bands at 1130, 1078, and 1061 cm-1, A(DEG). This plot is in turn related to the position of the aggregation band as a fonction of DEG content. The value increases from 1628 to 1630 cm-1 suggesting that the plasticizer molecules interact with the peptide bonds via hydrogen bonds between the O-H groups of DEG and the amide CdO groups. When the plasticizer evaporates (whether it is a polyol or water), CdO- - -OH hydrogen bonds are released. The CdO amide bond is then strengthened which leads to an increase in the wavenumber of the amide I′ mode. The fact that the position of the 1655-cm-1 band is unchanged shows that DEG is not hydrogen bonded to the R-helices. The rate of the shift at 1629 cm-1 is small at the beginning of the DEG loss and accelerates as DEG evaporates to reach a maximum value at the end of the experiment, i.e., when the film craks. Thus, the hydrogen bonds in β-sheets are more affected by evaporation as the plasticizer content decreases, showing that the first DEG molecules that evaporate are not protein-bonded and that the last ones are linked to the CdO amide groups. This finding is consistent with Lieberman and Gilbert,19 who found that water sorption initially ocurs at specific sites on the protein molecules, followed by clustering of water. Figure 6, panels b and c, shows that, concomitantly to the bandshift, the relative absorbance at 1629/1655 cm-1, A(1629)/A(1655), increases. This is supported by the observation that the relative absorbance A(1622)/A(1642) of films made in D2O increases as the film-forming solution contains a decreasing amount of DEG (data not shown). Such results indicate that the content of R-helix/unordered structures is smaller (the content of β-sheets is higher) for smaller DEG concentrations. The plasticizer seems then to promote the formation of these structures. Since films are more flexible as the plasticizer content increases, the content of R-helix/unordered structures with respect to the content of intermolecular β-sheets may partly explain some mechanical properties. Very brittle films are obtained upon aging. From the present data, this characteristic may be associated with an evaporation of plasticizer and a replacement of unordered/ R-helix structures by β-sheets. Therefore, the plasticizer not only provides elasticity to the films by reducing proteinprotein interaction but also determines the amount of polypeptide segments that adopt other secondary structures than β-sheets. The influence of the plasticizer can also be evaluated by studying films dehydrated from plasticizer-free solutions. Such films crak because of a lack of flexibility and it may be asked whether such rigid films are different at a molecular level than plasticized films. In Figure 7 are compared films made from film-forming solutions in the absence and in the presence of DEG. The former films lack so much in flexibility that the contact is lost with the ATR crystal before all water is evaporated. As a consequence, the absorbance of the dried film is very low as revealed by the noise on the spectrum, but even so the protein conformation can be compared with confidence. In the absence of DEG, the amide I′ band components are broader and less defined than in the presence of DEG. The spectrum does not have the characteristic shape

3216


Figure 7. Comparison of the ATR spectra of β-lg films dehydrated made from film-forming solutions in the absence (dotted line) and in the presence (full line) of DEG.

that is commonly found for β-lg matrices, including heatinduced gels, emulsions, and films. It shows that the polypeptide chain conformation in such films is not as ordered as in the presence of the plasticizer. The presence of the plasticizer appears then to have a strong impact on the conformational rearrangement and aggregation of proteins during water evaporation. Therefore, one of the roles of the plasticizer seems to lie in providing a favorable environment for specific molecular reorganizations of the polypeptide chains which is prevented in its absence. Discussion Formation of Protein Matrices Consists of the Assembly of Soluble Aggregates. The present study leads to the conclusion that the heating step induces unfolding and aggregation of the proteins. The plasticizer does not affect the protein conformation, and the aggegates are irreversibly formed. The heat-induced aggregates can be considered as building blocks involved in the formation of the film network. Because the procedure for film formation involves a heating step, there is an obvious, often neglected, relation between films and heat-induced gels/aggregates. The relation between both systems is in fact even closer since heatinduced aggregation at pH 7 of β-lg is now accepted to be a two-step process,49,54-57 with the formation of primary particles followed by their assembly into larger aggregates. The elementary units remain intact upon dilution.58 These hierarchical molecular events ressemble those proposed for plasticized films, athough in the latter case they are forced by the procedure required to make films. For WPI, the relation between heat-induced gels and films has recently been pointed out.12,25 In particular, the optimal concentration for the films was found at the critical gelation concentration. The similarity between protein films and gels is more evident if one refers to the so-called cold-set gels. Like films, such protein gels are formed in two steps. The first step consists of a heat treatment that is very similar to the one applied for films and that is carried out in similar ranges of concentration, ionic strength, and pH. In the second step, two methods are employed. A salt can be added to the denatured solution (Ca2+,59 Na+,60 and Fe2+ 61) or the pH gradually lowered toward the pI of the protein.62-64 Such treatments leads in both cases to a decrease of the Coulombic repulsions, which in turn allows the formation of intermolecular interactions and formation of a network. van der Waals interactions control the aggregation process at very

Lefe` vre et al.

low electrostatic repulsions, whereas the hydrophobic effect predominates at higher electrostatic repulsions.61 Research in this field leads to the view that cold gelation consists of the aggregation of the soluble particles formed during the first step. The particles formed during heating are then considered as elementary units that constitute the network.61 It can then be acknowledged that a film is a cold-set gel for which the network is formed by dehydration. Therefore, recent studies on heat-induced gels, cold-set gels, and films suggest that the formation of globular protein matrices via the assembly of primary particles may be a common phenomenon. Characteristics of the Building Blocks. It may then be proposed, as emphasized for cold-set gels,60 that macroscopic properties of the films may depend on both the characteristics (shape, structure, and size) and type of assembly of the primary particles. It is shown in this study that heat-induced aggregation of β-lg is accompanied by the formation of intermolecular antiparallel β-sheets, a well-known phenomenon for globular protein aggregation in general33,42,43 and for β-lg.33-35 The heat-induced particles are found by FTIR spectroscopy to correspond to fine-stranded aggregates. After heating, around 10% of the peptide bonds are involved in such structures. Therefore, aggregates are made of linked proteins with intermolecular β-sheets, although other types of interactions are involved including intermolecular disulfide bonds.14,49,65 Many data relative to heat-induced β-lg aggregates can be found in the literature. However, because the aggregation process and aggregation rate are strongly affected by many parameters (temperature, heating time, pH, ionic strength, type of ion, protein concentration, and protein source), it is difficult to find information that exactly corresponds to given conditions. However, two studies have been performed in similar conditions. A recent study60 has shown that at pH 7 and low ionic strength the structure of β-lg aggregates consist of short linear rods of sizes

Molecular Description of the Formation and Structure of Plasticized

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