Langmuir 2005, 21, 4083-4089
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Correlation between Mechanical Behavior of Protein Films at the Air/Water Interface and Intrinsic Stability of Protein Molecules Anneke H. Martin,†,‡,| Martien A. Cohen Stuart,‡ Martin A. Bos,†,§ and Ton van Vliet*,† Wageningen Centre for Food Sciences (WCFS), P.O. Box 557, 6700 AN, Wageningen, The Netherlands, Wageningen University, Laboratory of Physical Chemistry & Colloid Science, P.O. Box 8038, 6700 EK Wageningen, The Netherlands, and TNO Nutrition and Food Research Institute, P.O. Box 360, 3700 AJ Zeist, The Netherlands Received October 19, 2004. In Final Form: January 22, 2005 The relation between mechanical film properties of various adsorbed protein layers at the air/water interface and intrinsic stability of the corresponding proteins is discussed. Mechanical film properties were determined by surface deformation in shear and dilation. In shear, fracture stress, σf, and fracture strain, γf, were determined, as well as the relaxation behavior after macroscopic fracture. The dilatational measurements were performed in a Langmuir trough equipped with an infra-red reflection absorption spectroscopy (IRRAS) accessory. During compression and relaxation of the surface, the surface pressure, Π, and adsorbed amount, Γ (determined from the IRRAS spectra), were determined simultaneously. In addition, IRRAS spectra revealed information on conformational changes in terms of secondary structure. Possible correlations between macroscopic film properties and intrinsic stability of the proteins were determined and discussed in terms of molecular dimensions of single proteins and interfacial protein films. Molecular properties involved the area per protein molecule at Π ≈ 0 mN/m (A0), A0/M (M ) molecular weight) and the maximum slope of the Π-Γ curves (dΠ/dΓ). The differences observed in mechanical properties and relaxation behavior indicate that the behavior of a protein film subjected to large deformation may vary widely from predominantly viscous (yielding) to more elastic (fracture). This transition is also observed in gradual changes in A0/M. It appeared that in general protein layers with high A0/M have a high γf and behave more fluidlike, whereas solidlike behavior is characterized by low A0/M and low γf. Additionally, proteins with a low A0/M value have a low adaptability in changing their conformation upon adsorption at the air/water interface. Both results support the conclusion that the hardness (internal cohesion) of protein molecules determines predominantly the mechanical behavior of adsorbed protein layers.
Introduction In systems such as emulsions and foams, proteins are known to often form a cohesive viscoelastic film around oil droplets and air cells which appears to stabilize these colloids against flocculation, coalescence, or disproportionation. The process of network formation of proteins at the interface can be viewed as a three-step process, namely, the adsorption and initial anchoring of the protein at the interface, the accompanying conformational change and subsequent rearrangement of the adsorbed protein, and the formation of a protein network with specific mechanical properties.1,2 Adsorption of proteins at the air/water interface has been fairly well studied, and the molecular factors affecting the initial anchoring are partly understood.3-7 The conformational changes that take place upon adsorption at the air/water interface have been studied * Author to whom correspondence should be addressed. E-mail:
[email protected]. Phone: +31-317-475156. Fax: +31-317483669. † Wageningen Centre for Food Sciences (WCFS). ‡ Wageningen University. § TNO Nutrition and Food Research Institute. | Current affiliation: University of Guelph, Department of Food Science, Guelph Ontario, N1G 2W1 Canada. (1) Martin, A. H.; Grolle, K.; Bos, M. A.; Cohen Stuart, M. A.; van Vliet, T. J. Colloid Interface Sci. 2002, 254, 175. (2) Razumovsky, L.; Damodaran, S. Langmuir 1999, 15, 1392. (3) Damodaran, S.; Song, K. B. Biochim. Biophys. Acta 1988, 954, 253. (4) Dickinson, E.; Murray, B. S.; Stainsby, G.; Dickinson, E., Eds. Advances in food emulsions and foams; Elsevier: London, 1988; pp 123-162.
to a much lesser extent. Information on conformational changes has been frequently deduced from indirect evidence, namely by relating surface excess, surface pressure, and protein layer thickness to protein molecular dimensions.8-10 Recently, Meinders et al.,11 Renault et al.,12 and Martin et al.13 have reported on in situ measurements of structural changes in terms of secondary structure of proteins upon adsorption at the air/water interface using infra-red reflection absorption spectroscopy (IRRAS). Relatively small changes in secondary structure were observed, but no conclusions could be drawn about the extent to which tertiary structure changes. Conformational changes (both in secondary and tertiary structure) of proteins at the interface permit the formation of intermolecular bonds, and viscoelastic protein films are (5) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1979, 70, 403. (6) MacRitchie, F. In Proteins at Liquid Interfaces; Mo¨bius, D., Miller, R., Eds.; Elsevier: Amsterdam, 1998. (7) Pezennec, S.; Gauthier, F.; Alonso, C.; Graner, F.; Croguennec, T.; Brule´, G.; Renault, A. Food Hydrocolloids 2000, 14, 463. (8) Clark, D. C.; Smith, L. J.; Wilson, D. R. J. Colloid Interface Sci. 1988, 121, 136. (9) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1979, 70, 427. (10) Lu, J. R.; Su, T. J.; Thomas, R. K.; Penfold, J.; Webster, J. J. Chem. Soc., Faraday Trans. 1998, 94, 3279. (11) Meinders, M. B. J.; de Jongh, H. H. J. Biospectroscopy 2002, 67, 319. (12) Renault, A.; Pezennec, S.; Gauthier, F.; Vie´, V.; Desbat, B. Langmuir 2002, 18, 6887. (13) Martin, A. H.; Meinders, M. B. J.; Bos, M. A.; Cohen Stuart, M. A.; van Vliet, T. Langmuir 2003, 19, 2922.
10.1021/la047417t CCC: $30.25 © 2005 American Chemical Society Published on Web 03/31/2005
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formed partly as a result of this. These films can be mechanically characterized by deformation in shear (shape changes) or in dilation (area changes) as has often been done in the literature.1,14-16 The results of these studies show differences in mechanical strength for various proteins, but a molecular explanation for the differences is not really available. The relation between protein molecular properties, such as secondary structure or hydrophobicity, and adsorption properties was earlier discussed by Razumovsky et al.,2 but they did not deal with viscoelastic properties of protein films. Pereira et al.17 studied three different conformational states of BSA to elucidate the role of protein conformation in dilational rheology and concluded that the states possessing a more rigid structural stability gave more elastic adsorbed layers with higher interfacial dilational moduli. Moreover, for hard, globular proteins, like lysozyme, the large values of the interfacial storage modulus (at the oil/water interface) appear to be dominated by intra-protein interactions, i.e., the stability of the native state of the protein.18 Thus, both intra-protein and inter-protein interactions contribute to the formation of an interfacial protein film, of which the properties can be assessed by surface rheology. Deformation in shear is usually associated with cohesive interactions between protein molecules, but it may also involve the internal cohesion and the surface density of the protein present at the surface. Area changes of the surface are related to adsorption and (partial) desorption processes but can also probe cohesive and repulsive interactions between protein molecules.14,19 Therefore, interfacial rheology appears to be a valuable tool to investigate conformational stability at the interface.19,20 Cohesive interactions between different protein molecules exist as a rule in ‘interfacial’ and bulk gels, i.e., they form physical, sometimes even chemical, intermolecular bonds. Examples of intermolecular bonding are hydrophobic interactions or disulfide bridging; the latter was found to be normally of less importance at air/water interfaces.21 Both Renault et al.12 and Martin et al.13 reported on the possibility of measuring one type of intermolecular aggregation at the air/water interface, i.e., the formation of antiparallel β-sheets by ovalbumin and (soy) glycinin, respectively. Intermolecular β-sheet formation due to hydrogen bonding between protein molecules was observed, and this is probably one of the reasons that ovalbumin and glycinin form a cohesive protein film. In these cases, changes in secondary structure may thus be related to the formation of a strong protein network at the interface. In this paper, we attempt to correlate the mechanical behavior of interfacial protein films to the structural stability or adaptability to change conformation of single protein molecules. The mechanical properties are derived from force measurements on films under shear and dilation (14) Bos, M. A.; van Vliet, T. Adv. Colloid Interface Sci. 2001, 91, 437. (15) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1980, 76, 227. (16) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1980, 76, 240. (17) Pereira, L. G. C.; Theodoly, O.; Blanch, H. W.; Radke, C. J. Langmuir 2003, 19, 2349. (18) Freer, E. M.; Yim, K. S.; Fuller, G. G.; Radke, C. J. J. Phys. Chem. B 2004, 108, 3835. (19) Martin, A. H., Thesis, Wageningen University, 2003. (20) Pereira, L. G. C.; Theodoly, O.; Blanch, H. W.; Radke, C. J. Langmuir 2003, 19, 2349. (21) Jones, D. B.; Middelberg, P. J. Langmuir 2002, 18, 5585. (22) van Vliet, T.; Martin, A.; Renkema, M.; Bos, M. In Plant Biopolymer Science, Food and Non Food Applications; Renard, D., Della Valle, G., Popineau, Y., Eds.; Royal Society of Chemistry: Cambridge, 2002.
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and from the relaxation behavior after exposure to shear/ dilation. Particularly, the latter served to assess the extent of elasticity of a protein film. IRRAS spectra were simultaneously recorded of the surfaces exposed to dilation. These spectra provide information on the adsorbed amount and on the level of secondary structure changes.13 The molecular characteristics are defined in terms of packing density, deformability, interactions, and molecular dimensions. Molecular dimensions may involve those of single proteins, e.g., the area occupied per film forming molecule (A0), or those of the interfacial protein network. In the latter case, the adsorbed protein layer is seen as a three-dimensional gel in which the dimension perpendicular to the interface is relatively small (
