Thermodynamic and Microscopy Studies of Urea-Soy Protein

Chapter 5

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Thermodynamic and Microscopy Studies of Urea-Soy Protein Composites K. Venkateshan* and X. S. Sun Bio-Materials and Technology Lab, Department of Grain Science and Industry, Kansas State University, Manhattan, Kansas 66506, USA. *[email protected]

Biocomposites composed of organic molecules and confined in soy protein matrices have potential for various medical and drug-delivery applications. The objective of this study was to characterize the thermodynamic behavior and corresponding structural changes of urea-soy protein composite. Large melting temperature depression was observed for urea crystals interlayered with soy protein in comparison with bulk urea. In addition to a broader melting peak, the shape and size of the urea crystals interlayered with soy protein were significantly altered from those of bulk urea crystals. Formation of the interlayered morphology of urea crystals and soy protein involved dissolution and interpenetration of urea in the soy protein solution, which resulted in dissolution and denaturation of soy protein followed by moisture dehydration and subsequent precipitation and confinement of urea crystals in soy protein layers. Laser scanning microscopy was used to characterize the structural changes of the urea crystals and soy protein. The altered structure was composed of rod-shaped urea crystals of width 300 to 1500 nm confined between soy protein layers of width 500 to 1000 nm forming an interlayered morphology. The main conclusion drawn from the thermodynamic and microscopy studies is that the melting temperature depression and the corresponding crystal size of urea do not agree with the Gibbs-Thomson predictions. We attribute this to the configurational effects at the urea-soy protein interfaces © 2010 American Chemical Society In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

being more dominant than size effects as considered by Gibbs-Thomson theory.


Introduction In the past two decades, extensive studies have been performed to investigate the effect of confinement of low-molecular-weight organic materials and polymeric materials in inorganic and polymeric matrices on transition temperatures (1–3). Small sized molecules are embedded in the matrices by confined nucleation and growth or precipitation in inorganic matrix pores or polymer cross-linked network pores of well-known pore sizes (4–6). The studies have focused on both glass and melting transition temperatures. The melting temperature depression (ΔTm) studies (4, 7, 8) have focused on determining , where and are the melting temperatures of bulk and confined materials of size, r, respectively. In certain studies (4, 7, 8) in which size effects dominated, good agreement between the melting point depression and confined material size based on the Gibbs-Thomson equation (9) has been reported. Some studies (5, 6), however, showed that the Gibbs-Thomson equation does not always agree well when the bulk heat of melting in the Gibbs-Thomson equation is invalid because of interfacial inhomogeneities. Small sized molecules were also embedded in cross-linked polymer networks, such as highly cross-linked elastomer, and ΔTm has been reported (7, 8). In these studies, changes in ΔTm were explained by modifications in contributions to specific heats of the components due to the dominance of enthalpic and entropic effects. Similar to structural modifications of polymer networks by addition of small molecules, studies (10–12) describing protein structural changes with addition of organic molecules, acids, bases, and salts are extensive and well known. Among these modification studies, protein denaturation or structural changes with urea have been studied in depth by using calorimetry. Thermodynamic and simulation studies have focused on dissolution and protein denaturation mechanisms combined with the study of urea-protein interactions in solution to determine the enthalpic and entropic components of the protein modification process (10–15). These studies also described basic features of the hydrophobic effect and hydrogen bonding in relation to protein stability. Primarily, two mechanisms have been proposed to account for urea-induced perturbation of the hydrophobic regions of proteins in solutions. One mechanism is based on the indirect role of urea in perturbing the structure and dynamics of water, which, in turn, leads to the perturbation of structure and dynamics of hydrophobic regions (16). The other mechanism is based on the direct role of urea in interpenetrating the hydrophobic regions and forming open structures around hydrophobic regions (17). Despite the scope of these studies of urea-protein in solution, characterization of urea-soy protein thermal behavior and structure in the precipitated solid state has not been extensive. The modification and depression of melting temperature of urea confined between protein layers and corresponding morphological changes 60 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

have not been reported and characterized. Furthermore, the mechanism leading to the open structure at the urea-protein interface is not well understood. In this study, we investigate (1) changes in melting behavior of urea interlayered with protein by using differential scanning calorimetry (DSC) and (2) structural changes of urea-protein composite by using laser scanning microscopy (LSM).

Materials and Method Downloaded by PENNSYLVANIA STATE UNIV on August 21, 2013 | http://pubs.acs.org Publication Date (Web): August 11, 2010 | doi: 10.1021/bk-2010-1043.ch005

Materials The water-based soy proteins used in this work were extracted from defatted soy flour with a protein dispersability index (PDI) of 90 according to the acidic precipitation method described by Sun et al. (18, 19) and labeled as soy protein isolate (SPI). The defatted soy flour was purchased from Cargill, Inc. The weight fraction of soy protein in solution is 32%. The molecular weight of the protein is approximately 100 to 400 kDa. The soy protein is predominantly composed of two globulins, 7S and 11S. It has been reported that the extracted soy protein in solution is partially denatured (18, 19). The semidenatured soy protein will undergo further denaturation with addition and dissolution of urea. Urea in granular form was purchased from Fisher Scientific. The molecular weight of urea is 60 Da. Synthesis of Urea-Soy Protein Composites The urea-soy protein composite was synthesized by the following procedure. An accurately weighed amount of SPI was added to a clean glass beaker, followed by the addition of weighed urea to the same beaker. The weight fraction ratio of urea and SPI was 33:67. Since SPI contains 68 wt% water, the concentration of urea in SPI was 7 M. The urea-SPI mixture was mixed gently for 10 minutes and then dried in an oven at 363 K for one hour. Further, the mixture was removed from the oven and stored at room temperature for 24 hours until drying was complete. During the thermal conditioning and drying process, urea and SPI precipitated and evolved from a clear transparent solution to a solid composite. The moisture content in the dried composite sample was 4.5 % by weight and was determined by using thermal gravimetric analysis (TGA) measurements (scan not shown) and heater oven weight loss measurements. Furthermore, the peak and final temperature of water vaporization obtained from TGA scan was 425 K and 438 K respectively.

Experimental Methods Differential Scanning Calorimetry (DSC) Technique Melting behavior was studied by using a differential scanning calorimeter (DSC 7; PerkinElmer, Norwalk, CT) calibrated with indium and zinc. About 3 mg of the sample was accurately weighed and transferred into the DSC pans, and the pans were sealed. The DSC scans were obtained between temperature range 61 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

of 313 K to 415 K and for a heating rate of 10 K/minute. The DSC scan were obtained for bulk SPI, bulk urea, and urea-SPI composite. Onset, peak, and final melting transition temperatures of urea were determined, and the corresponding enthalpy of melting was calculated.


Laser Scanning Microscopy (LSM) Technique Images were obtained by using an Axioplan 2 MOT research microscope (Carl Zeiss, Inc., Thornwood, NY, USA) equipped with a Zeiss Axiocam HR digital camera, a fully motorized stage with mark-and-find software, plan neofluor objectives (1.25_/0.035, 10_/0.3, 20_/0.5, 40_/0.75, 40_/1.3 oil), plan apochromat objectives (63_/1.4 oil, 100_/1.4 oil), an achroplan objective (4_/0.1), differential contrast interference (DIC), phase contrast (ph), dark field, bright field, and Axiovision 3.1 software with interactive measurements and D deconvolution modules. A small amount of the solid composite sample was placed onto a 3-inch × 1-inch glass slide (Fisher Scientific) without spreading force. The sample was allowed to set at room conditions for 2 min. Differential contrast interference images were taken at various magnifications and locations in the sample. Images were obtained for bulk SPI, bulk urea, and urea-SPI composite.

Results Melting Studies Figures 1A and 1B show the plots of heat flow, dH/dt, where H is enthalpy and t is time against temperature, T, of bulk urea and urea-soy protein composite. The melting peak temperature of bulk urea is was 407.7 K with ΔT = 11 K, the difference between the onset and end temperatures of the melting endotherm and enthalpy of melting of bulk urea, was 15.37 kJ/mol, which are in agreement with the literature values (20, 21). The DSC scan of urea-soy protein composite (Figure 1B) showed significant depression of urea melting peak temperature, observed at 373.9 K, a larger ΔT of 52 K, and a lower enthalpy of melting,

(enthalpy of melting of confined urea crystal of size r in

composite) value of 9.44 kJ/mol in comparison with

. In calculating

, it is important to note that the accurate weight fraction of urea in the urea-soy protein composite was considered and that is reported as enthalpy per mol of urea. In the scan for urea-soy protein composite, peaks corresponding to denaturing of 7S and 11S were absent, evidence that the soy protein globulins had undergone complete denaturation.

62 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


Figure 1. DSC scans of bulk urea (A) and urea-soy protein composite (B) wherein urea is interlayered with soy protein obtained by heating at a rate of 10 K/minute. Laser Scanning Microscope Figure 2 shows the LSM image of bulk SPI. The bright regions in the image are protein clusters, and it is evident from the image that the clusters are interconnected and the average size of the clusters is one micron. Because the clusters were nonconventional in shape, the size of the clusters corresponds to the largest dimension of the cluster. Figure 3 shows the LSM image of bulk urea. The urea particles exhibited rectangular and tetragonal faceted shapes. Based on the rectangular geometry, the size of urea crystals were determined to range in size from 50 to 300 microns. The LSM images of urea-soy protein composite (Figure 4) show evidence of size reduction and structure modification of both protein clusters and urea particles. The urea crystals (dark rods) are randomly oriented and interlayered with the protein matrix (bright layers). The average size, or width, of the layers of urea and protein are 500 nm and 750 nm, respectively. These images depict the coexistence of urea and soy protein in an interlayered morphology. The structural changes 63 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

observed with the interpenetration of urea in the protein matrix are a result of fine dispersion of the urea in protein matrix in both the particulate and emulsion states.

Discussion


Melting Temperature Depression of Confined Urea in Protein Matrix In previous studies, the melting temperature depression has been attributed to the dominance of either (1) size effects as described by Gibbs-Thomson theory (19) or (2) configurational effects arising from higher interatomic spacing at surface and interfacial regions. In the following section, we consider these two aspects that are attributed to melting temperature depression.

1. Size Effects and Validity of Gibbs-Thomson Predictions According to the Gibbs-Thomson equation, the equilibrium melting temperature,

, of a crystal of radius r with a free surface is written as (22):

also written alternatively as:

By rearranging Eq. 1a, we obtain:

where is the melting temperature of the bulk crystal, γs,l is the interfacial tension between the solid and its melt, Vs is the molar volume of the solid, and is the molar enthalpy of melting of crystal of radius r at

, which is

taken to be equal to that of the enthalpy of melting of the bulk crystal, , in Eq. 1a. In the course of investigating the validity of Gibbs-Thomson theory with our experimental findings, assumptions that were used to derive the Gibbs-Thomson equation and its relevance to our work are briefly summarized as follows: i)

Inhomogeneities composed of coexisting crystalline and amorphous regions and contributions from heterogeneous nucleation are ignored (2). 64 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


ii) Surface interactions between the confined crystals and surrounding heterogeneous and amorphous phase are considered to be minimal, and the theory considers the attainment of equilibrium to be exclusively between the confined crystals and their liquid phase (22). iii) The theory is derived for crystals that are in equilibrium with their liquid and vapor phase and not for crystals that are tightly confined within a cavity, where pressure may change with changes in both temperature and volume on melting (22). iv) The presence of crystals in the solvated state and the resulting incomplete crystallization are considered to be negligible (12). These assumptions lead to the consideration that the enthalpy of melting of the confined urea crystals of size r, of the bulk urea crystal,

, is equal to the enthalpy of melting

, in deriving the Gibbs-Thomson equation. In

addressing and with our results, values of and were calculated and compared. The melting peaks in Figures 1B and 1A were integrated, and the calculated values of enthalpies of melting of urea, and

were 9.44 and 15.37 kJ/mol, respectively. The different

and values indicate the possibilities of (1) coexisting crystalline and amorphous regions, (2) surface interactions where urea is consumed because of chemical bonding with SPI, and (3) incomplete crystallization. Hence, we consider using ΔHm,r in Eq. 1b to determine r for the observed melting point depression value of 33.8 K. In studying the validity of Gibbs-Thomson theory, we consider Figures 1A and 1B, which show that the difference in melting temperature between the confined ) is 33.8 urea crystals in soy protein and the bulk urea crystal ( K. By substituting the known values for urea of γs,l = 45.3 mJ/m2 (23), Vs = 45.45 cm3/mol (molecular wt = 60 Da and density = 1.32 g/cc),

= 9.44 kJ/mol

= 33.8 K in Eq. 1b, the size of confined urea crystals in (from Figure 1B), and soy protein was calculated to be 5.26 nm. In contrast, the size of interlayered urea crystals determined from Figure 4, ranged from 300 nm to 1500 nm. Evidently, the decrease in size of urea crystals is much less than that predicted by the GibbsThomson equation, thus exhibiting a clear disagreement with the Gibbs-Thomson prediction.

2. Configurational Contribution to the Melting Process In our studies, because the calculated size of the nanocrystal (5.26 nm) from Gibbs-Thomson equation for ΔT = 33.8 K is much lower than the experimentally observed crystal size (ranging from 300 nm to 1500 nm), it is very probable that 65 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


Figure 2. High-magnification LSM image of bulk SPI. configurational effects at the solid-liquid and urea-soy protein interfaces play a more significant role than size effects in causing a high melting temperature depression of 33.8 K.

Surface Energy and Configurational Entropy The size-dependent melting temperature, described as (23):

, of metallic nanocrystals is

where r is the radius of the crystal, Svib is the vibrational component of the melting entropy of bulk crystals at the bulk melting temperature, R is the ideal gas constant, and ro is a critical radius at which all atoms of a particle are located on its surface. For most crystals, the overall melting entropy, Sm, is dominated by its vibrational component, Sm = Svib (24, 25). For semiconductors and embedded 66 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


Figure 3. LSM image of bulk urea crystals. The magnification scale is shown in red. crystals (26), the electronic and configurational components of the melting entropy are not negligible, and Sm ≠ Svib. When r>>ro, Eq. 2 can be mathematically expressed as:

By comparing Eqns. 3 and 1a, we obtain:

From Eq. 4, it is evident that γs,l is dependent on Sm, which, in turn, is affected by vibrational and configurational components. In scenarios in which the configurational component is significant, the assumption of Sm = Svib would be erroneous and thus underestimate the γs,l value. Instead, Sm = Svib + Sconfig must be incorporated, where Sconfig is the configurational entropy. The significant configurational contribution to the melting processes arises from higher interatomic spacing and disorder and anharmonic forces at surface and interfacial 67 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


Figure 4. High-magnification LSM image of urea-soy protein composite. The urea crystals (dark rods) are randomly oriented and interlayered with the protein matrix (bright layers). regions that result in lowering of vibrational frequencies (22). On the basis of the water amount (4.5 % by weight) and the effects of dissolution or solvation of urea in water, we consider the role of water in influencing the melting point depression of urea in the composite sample to be small. Furthermore, the evidence of lack of secondary chemical interactions between urea and soy protein at the interface leads us to believe that the configurational effects at the interface are the primary cause of melting temperature depression. The molecular origin of the configurational component of entropy in nanocrystals and hydrated proteins and its consequence in exhibiting higher heat capacity, Cp, has been studied by using both standard and temperature-modulated calorimetry (22, 27, 28).

Conclusion The observed melting temperature depression and corresponding reduction in urea crystal size do not obey the Gibbs-Thomson theory. We attribute this to configurational effects at the urea-soy protein interface being more dominant than size effects as considered by Gibbs-Thomson theory. The higher configurational entropy, Sconfig, of urea nanocrystals interlayered with soy protein can be attributed to (1) a large fraction of atoms in the interfacial region and a more open structure that lead to weaker interatomic coupling that decreases the vibrational frequencies and increases the vibrational and configurational entropy, (2) thermally induced variation of the vibrational and configurational entropy of materials due to lattice vibrations and variation of equilibrium defect concentration, especially in the 68 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

interface region, and (3) strong anharmonic forces that lower the vibrational frequencies because of increased interatomic spacing. In general, the disordered interfacial atoms of interlayered urea crystals have a higher configurational entropy contribution and heat capacity (Cp) than the bulk crystal.


Acknowledgments The authors thank the U.S. Department of Agriculture and Kansas Agricultural Experiment Station for grant sponsorship, contribution no. 10-195-B from the Kansas Agricultural Experiment Station. The authors also appreciate Dr. Dan Boyle’s assistance with the LSM images.

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70 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

Thermodynamic and Microscopy Studies of Urea-Soy Protein

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