Activation energy of the disruption of gel networks in relation to elastically stored energy in fine-stranded ovalbumin gels
Munialo, C.D. ; Linden, E. van der; Jongh, H.H.J. de - \ 2016
Food Hydrocolloids 55 (2016). - ISSN 0268-005X - p. 163 - 171.
Recoverable energy - network structure - disruption - ovalbumin - activation energy - fine-stranded
The aim of this study was to relate the activation energy of the disruption of ovalbumin networks to elastically stored energy (i.e. recoverable energy, RE) obtained from mechanical deformation tests. To this end, heat-set ovalbumin gels were prepared at a fixed volume fraction and pH, but varying incubation temperatures. The activation energy required to disrupt the gels was derived from the Arrhenius equation. Increasing incubation temperature from 65 to 95 °C during gel formation resulted in a gradual increase in the activation energy up to a factor of ∼ 8. Gels obtained at or just below the protein denaturation temperature of around 75 °C had significantly lower recoverable energy (RE). These latter gels also had lower fracture stress and strain. At incubation temperatures above 70 °C RE was constant around 75 %, although a steady increase in activation energy was observed. This demonstrates that storing energy in a protein network is not directly related to the interactions that make up the network. A combination of electron microscopy, water holding, and stress relaxation experiments were performed to study the different energy dissipation modes. It was shown that different dissipation modes for various gels were comparable, and this explains why the RE was similar, with the exception of gels prepared at lower incubation temperatures where (micro) fracture events could have occurred that lowered the RE. These results suggest that RE is not a network characteristic related to microstructural or smaller length scale interactions, but the result of various material-related energy dissipation mechanisms.
Comparison of Heat-Induced Aggregation of Globular Proteins
Delahaije, R.J.B.M. ; Wierenga, P.A. ; Giuseppin, M.L.F. ; Gruppen, H. - \ 2015
Journal of Agricultural and Food Chemistry 63 (2015)21. - ISSN 0021-8561 - p. 5257 - 5265.
laser-light scattering - beta-lactoglobulin - ionic-strength - induced denaturation - reaction-kinetics - whey proteins - neutral ph - in-situ - ovalbumin - gels
Typically, heat-induced aggregation of proteins is studied using a single protein under various conditions (e.g., temperature). Because different studies use different conditions and methods, a mechanistic relationship between molecular properties and the aggregation behavior of proteins has not been identified. Therefore, this study investigates the kinetics of heat-induced aggregation and the size/density of formed aggregates for three different proteins (ovalbumin, ß-lactoglobulin, and patatin) under various conditions (pH, ionic strength, concentration, and temperature). The aggregation rate of ß-lactoglobulin was slower (>10 times) than that of ovalbumin and patatin. Moreover, the conditions (pH, ionic strength, and concentration) affected the aggregation kinetics of ß-lactoglobulin more strongly than for ovalbumin and patatin. In contrast to the kinetics, for all proteins the aggregate size/density increased with decreasing electrostatic repulsion. By comparing these proteins under these conditions, it became clear that the aggregation behavior cannot easily be correlated to the molecular properties (e.g., charge and exposed hydrophobicity).
Water holding of protein gels
Urbonaite, V. - \ 2015
Wageningen University. Promotor(en): Erik van der Linden, co-promotor(en): Laurice Pouvreau; H.H.J. de Jongh. - Wageningen : Wageningen University - ISBN 9789462574229 - 206
sojaeiwit - ovalbumine - wei-eiwit - waterbergend vermogen - gelering - structuur - morfologie - reologie - permeabiliteit - centrifugeren - soya protein - ovalbumin - whey protein - water holding capacity - gelation - structure - morphology - rheology - permeability - centrifugation
Food products are typically multicomponent systems, where often the spatial volume is set by a protein continuous network. The ability of protein-based food products to entrap water and to prevent its exudation upon mechanical deformation is important for the texture and thus sensory perception of food products. Understanding of structural origins that determine gel water holding is therefore essential, and would allow designing foods with controlled sensory perception. Water removal from the gel (quantity, kinetics and mechanism) is related to the coarseness and deformation of the network. An understanding of the interplay between the effect of coarseness and stiffness on WH in fine and coarse gels allows one to take a better control and tune juiciness and the release of tastants from food products.
Quantitative description of the parameters affecting the adsorption behaviour of globular proteins
Delahaije, R.J.B.M. ; Gruppen, H. ; Giuseppin, M.L.F. ; Wierenga, P.A. - \ 2014
Colloids and Surfaces. B: Biointerfaces 123 (2014). - ISSN 0927-7765 - p. 199 - 206.
air-water-interface - bovine serum-albumin - beta-lactoglobulin - rheological properties - air/water interface - surface rheology - kinetics - ovalbumin - charge - denaturation
The adsorption behaviour of proteins depends significantly on their molecular properties and system conditions. To study this relation, the effect of relative exposed hydrophobicity, protein concentration and ionic strength on the adsorption rate and adsorbed amount is studied using ß-lactoglobulin, ovalbumin and lysozyme. The curves of surface elastic modulus versus surface pressure of all three proteins, under different conditions (i.e. concentration and ionic strength) superimposed. This showed that the interactions between the adsorbed proteins are similar and that the adsorbed proteins retain their native state. In addition, the adsorption rate (kadsorb) was shown to scale with the relative hydrophobicity and ionic strength. Moreover, the adsorbed amount was shown to be dependent on the protein charge and the ionic strength. Based on these results, a model is proposed to predict the maximum adsorbed amount (Gmax). The model approximates the adsorbed amount as a close-packed monolayer using a hard-sphere approximation with an effective protein radius which depends on the electrostatic repulsion. The theoretical adsorbed amount was in agreement with experimental Gmax (±10%).
Protein Concentration and Protein-Exposed Hydrophobicity as Dominant Parameters Determining the Flocculation of Protein-Stabilized Oil-in-Water Emulsions
Delahaije, R.J.B.M. ; Wierenga, P.A. ; Nieuwenhuijzen, N.H. van; Giuseppin, M.L.F. ; Gruppen, H. - \ 2013
Langmuir 29 (2013)37. - ISSN 0743-7463 - p. 11567 - 11574.
diffusing wave spectroscopy - beta-lactoglobulin - coalescence stability - disjoining pressure - drop size - adsorption - films - ph - interfaces - ovalbumin
DLVO theory is often considered to be applicable to the description of flocculation of protein-stabilized oil-in-water emulsions. To test this, emulsions made with different globular proteins (ß-lactoglobulin, ovalbumin, patatin, and two variants of ovalbumin) were compared under different conditions (pH and electrolyte concentration). As expected, flocculation was observed under conditions in which the zeta potential is decreased (around the isoelectric point and at high ionic strength). However, the extent of flocculation at higher ionic strength (>50 mM NaCl) decreased with increasing protein-exposed hydrophobicity. A higher exposed hydrophobicity resulted in a higher zeta potential of the emulsion droplets and consequently increased stability against flocculation. Furthermore, the addition of excess protein strongly increased the stability against salt-induced flocculation, which is not described by DLVO theory. In the protein-poor regime, emulsions showed flocculation at high ionic strength (>100 mM NaCl), whereas emulsions were stable against flocculation if excess protein was present. This research shows that the exposed hydrophobicity of the proteins and the presence of excess protein affect the flocculation behavior.
Protein–Peptide Interaction: Study of Heat-Induced Aggregation and Gelation of ß-Lactoglobulin in the Presence of Two Peptides from Its Own Hydrolysate
Kosters, H.A. ; Wierenga, P.A. ; Vries, R.J. de; Gruppen, H. - \ 2013
Journal of Agricultural and Food Chemistry 61 (2013)18. - ISSN 0021-8561 - p. 4218 - 4225.
rheological properties - alpha-lactalbumin - gels - isolate - acid - ovalbumin - fractions - soy
Two peptides, [f135–158] and [f135–162]-SH, were used to study the binding of the peptides to native ß-lactolobulin, as well as the subsequent effects on aggregation and gelation of ß-lactoglobulin. The binding of the peptide [f135–158] to ß-lactoglobulin at room temperature was confirmed by SELDI-TOF-MS. It was further illustrated by increased turbidity of mixed solutions of peptide and protein (at pH 7), indicating association of proteins and peptides in larger complexes. At pH below the isoelectric point of the protein, the presence of peptides did not lead to an increased turbidity, showing the absence of complexation. The protein–peptide complexes formed at pH 7 were found to dissociate directly upon heating. After prolonged heating, extensive aggregation was observed, whereas no aggregation was seen for the pure protein or pure peptide solutions. The presence of the free sulfhydryl group in [f135–162]-SH resulted in a 10 times increase in the amount of aggregation of ß-lactoglobulin upon heating, illustrating the additional effect of the free sulfhydryl group. Subsequent studies on the gel strength of heat-induced gels also showed a clear difference between these two peptides. The replacement of additional ß-lactoglobulin by [f135–158] resulted in a decrease in gel strength, whereas replacement by peptide [f135–162]-SH increased gel strength.
Designing microcapsules based on protein fibrils and protein - polysaccharide complexes
Hua, K.N.P. - \ 2012
Wageningen University. Promotor(en): Erik van der Linden, co-promotor(en): Leonard Sagis. - S.l. : s.n. - ISBN 9789461733801 - 136
lysozym - ovalbumine - pectinen - aggregatie - inkapseling in microcapsules - lysozyme - ovalbumin - pectins - aggregation - microencapsulation
Keywords: encapsulation, microcapsule, protein, fibril, protein-polysaccharide complex, controlled release, interfacial rheology, lysozyme, ovalbumin
This thesis describes the design of encapsulation systems using mesostructures from proteins and polysaccharides. The approach was to first investigate the physical properties of the encapsulating materials (protein fibrils and protein – polysaccharide complexes). Subsequently, microcapsules with tunable release rate and mechanical strength were developed.
Firstly, the effect of steady shear and turbulent flow on the formation of protein fibrils from lysozyme was studied. We determined the conversion and size distribution of fibrils obtained by heating lysozyme solutions at pH 2. The formation of fibrils was quantified using flow-induced birefringence. The size distribution was fitted using decay of birefringence measurements and Transmission Electron Microscopy. The morphology of Lys fibrils and kinetics of their formation varied considerably depending on the flow applied. With increasing shear or stirring rate, more rod-like and shorter fibrils were obtained, and the conversion into fibrils was increased.
Secondly, we have investigated the surface rheological properties of oil – water interfaces stabilized by fibrils from lysozyme (long and semi-flexible, and short and rigid ones), fibrils from ovalbumin (short and semi-flexible), lysozyme – pectin complexes, or ovalbumin – pectin complexes. We have compared these properties with those of interfaces stabilized by the native proteins. The surface dilatational and surface shear moduli were determined using an automated drop tensiometer, and a stress controlled rheometer with biconical disk geometry. Results show that interfaces stabilized by protein – pectin complexes have higher surface shear and dilatational moduli than interfaces stabilized by the native proteins only. At most of the experimental conditions, interfaces stabilized by protein fibrils have the highest surface rheological moduli. The difference between long semi-flexible lysozyme fibrils or short rigid lysozyme fibrils is not pronounced in interfacial dilation rheology but significant in interfacial shear rheology. The complex surface shear moduli of interfaces stabilized by long semi-flexible fibrils are about ten times higher than those of interfaces stabilized by short rigid fibrils, over a range of bulk concentrations. Interfaces stabilized by short and more flexible ovalbumin fibrils have a significantly higher surface shear modulus than those stabilized by the somewhat longer and more rigid short lysozyme fibrils.
Finally, encapsulation systems are developed using layer-by-layer adsorption of food-grade polyelectrolytes on an emulsion droplet template. The first encapsulation system was built with alternating layers of ovalbumin fibrils and high methoxyl pectin. By varying the number of layers, the release of active ingredients can be controlled: increasing the number of layers of the shell from four to eight, decreases the release rate by a factor six.
The other encapsulation systems were built with alternating layers of protein – pectin complexes and protein fibrils. Two types of proteins (ovalbumin and lysozyme) and three types of fibrils were used: short and semi-flexible from ovalbumin, short and rod-like, and long and semi-flexible from lysozyme. At low number of layers (less than five), microcapsules from ovalbumin complexes and fibrils were stronger than microcapsules prepared from lysozyme complexes and fibrils. Increasing the number of layers, the mechanical stability of microcapsules from lysozyme complexes and fibrils increased significantly, and capsules were stronger than those prepared from ovalbumin complexes and fibrils with the same number of layers. The contour length of the Lys fibrils did not have a significant effect on mechanical stability of the lysozyme complexes and fibrils capsules. These results show that mechanical properties of this type of capsule can be tuned by varying the flexibility of the protein fibrils.
Effect of Saccharide Structure and Size on the Degree of Substitution and Product Dispersity of a-Lactalbumin Glycated via the Maillard Reaction
Haar, R. ter; Schols, H.A. ; Gruppen, H. - \ 2011
Journal of Agricultural and Food Chemistry 59 (2011)17. - ISSN 0021-8561 - p. 9378 - 9385.
ionization mass-spectrometry - beta-lactoglobulin - electrospray-ionization - state glycation - improvement - stability - ovalbumin - proteins - sugars
The course of the Maillard reaction between a-lactalbumin and various mono- and oligosaccharides in the solid state was studied using UPLC–ESI-TOF-MS. Individual reaction products were monitored for their degree of substitution per protein molecule (DSP). The Maillard reaction rate depended on the saccharide type and decreased when the saccharide size increased. Conjugation with charged saccharides was hindered when a specific average DSP was reached, probably resulting from electrostatic repulsion. The DSP varied between 0 and 15, and the standard deviation of the average DSP, which is a measure for product dispersity, increased to 1.9. Similar experiments were performed with a dipeptide. Relative reaction rates in these experiments were 1 for glucose, 0.28 for maltose, and 0.16 for maltotriose. Comparison of the results obtained using a-lactalbumin and the dipeptide made clear that the Maillard reaction rate is determined by a number of factors, including saccharide reactivity and lysine accessibility
Improvement of foaming properties of cuttlefish skin gelatin by modification with N-hydroxysuccinimide esters of fatty acid
Aewsiri, T. ; Benjakul, S. ; Visessanguan, W. ; Wierenga, P.A. ; Gruppen, H. - \ 2011
Food Hydrocolloids 25 (2011)5. - ISSN 0268-005X - p. 1277 - 1284.
air-water-interface - proteins - proteolysis - adsorption - surfactant - ovalbumin - charge
Conformation and foaming properties of cuttlefish skin gelatin modified by N-hydroxysuccinimide esters of different saturated fatty acids including capric acid (C10:0), lauric acid (C12:0) and myristic acid (C14:0) at different molar ratios (0.25, 0.50, 1.00 and 2.00) were investigated. Covalent attachment of fatty acids into gelatin was observed as evidenced by the decrease in amino groups. Fourier transform infrared spectroscopic study indicated the presence of alkyl group of modified gelatin. The higher increase in surface activity with coincidental increase in surface hydrophobicity was observed in gelatin modified with fatty acid ester having a longer chain, especially at the higher molar ratio. The increase in foam expansion was related with the improved surface activity mediated by the modification by N-hydroxysuccinimide esters of fatty acid.
Reconsidering the importance of interfacial properties in foam stability
Wierenga, P.A. ; Norel, L. van; Basheva, E.S. - \ 2009
Colloids and Surfaces. A: Physicochemical and Engineering Aspects 344 (2009)1-3. - ISSN 0927-7757 - p. 72 - 78.
bovine beta-lactoglobulin - maillard reaction - whey proteins - soap films - egg-white - ovalbumin - behavior - manufacture - emulsions - glycation
In food industry, protein isolates are often used to help in the formation and stabilisation of food foams. Subsequently there is great interest in (1) understanding the effect of processing parameters on the functional properties of the isolate, and (2) methods and techniques that can help to predict the foam properties. This article describes the foaming properties of proteins that were modified in the Maillard reaction. From these relatively simple experiments results were obtained that indicate that for certain protein solutions the foam properties can vary significantly, while the interfacial properties are constant. Commercial protein isolates originate from only a few sources, mainly egg white and whey, and sometimes plant proteins (e.g. soy). Despite these limited sources a large variety of isolates with a wide range of properties is produced. One source of variation is the isolation procedure, but at least equally important are the conditions used before, during and after drying the protein solution to form the dry powder. From the literature it was found that one of the major changes to the protein during processing of the isolates is the covalent coupling of sugars via the Maillard reaction. To study the effects of these reactions, a model system was produced that consists of proteins that were glycated to different degrees using Maillard reaction. For each sample, interfacial properties (e.g. surface pressure, dilatational modulus) were determined, and foam experiments were performed. The results show that at constant concentration of both the protein (0.5 g/L) and sugar (0.7 g/L), the foam-ability and stability could be significantly improved (e.g. non-modified lysozyme does not foam, the highest modification is easily foamed and the foam has a half-life time of 200 s). Interestingly, the improved foam properties could not be related to any change in interfacial properties. While foam stability improved with increasing modification, the measured interfacial properties were not significantly affected. These observations seem to go against the general view that changes in foam behaviour should be reflected in changes in the interfacial properties. Additional experiments on thin liquid films were performed, where the disjoining isotherm was measured. These isotherms did not show significant differences in the interactions between the adsorbed layers. This indicates that the electrostatic and steric interactions between the adsorbed layers do not depend on the degree of modification. Only the thin film stability against rupture was found to increase with increasing modification. The thin film experiments lead to the hypothesis that aggregates (or oligomeric proteins) formed during modification might become trapped in the film. The presence of these oligomeric proteins could result in an increase of the apparent viscosity in these films, or in gelling or jamming of the liquid phase between the two interfaces. In other words, the observed behaviour is the result of the confined geometry of the thin films. The results confirm other observations that Maillard reactions improve foaming properties. Moreover, strong indications were found that to predict foam stability we need more than the traditional parameters (i.e. (dynamic) surface pressure, interfacial reology, and disjoining pressure).
The Critical Aggregation Concentration of ß-Lactoglobulin-Based Fibril Formation
Kroes-Nijboer, A. ; Venema, P. ; Bouman, J. ; Linden, E. van der - \ 2009
Food Biophysics 4 (2009)2. - ISSN 1557-1858 - p. 59 - 63.
heat-induced denaturation - amyloid fibrils - globular-proteins - whey-protein - ionic-strength - low ph - gels - gelation - ovalbumin - lysozyme
The critical aggregation concentration (CAC) for fibril formation of ß-lactoglobulin (ß-lg) at pH 2 was determined at 343, 353, 358, 363, and 383 K using a Thioflavin T assay and was approximately 0.16 wt%. The accuracy of the CAC was increased by measuring the conversion into fibrils at different stirring speeds. The corresponding binding energy per mol, as determined from the CAC, was 13 RT (~40 kJ mol¿1) for the measured temperature range. The fact that the CAC was independent of temperature within the experimental error indicates that the fibril formation of ß-lg at pH 2 and the measured temperature range is an entropy-driven process.
Effect of Protein Charge on the Generation of Aggregation-Prone Conformers
Broersen, K. ; Weijers, M. ; Groot, J. de; Hamer, R.J. ; Jongh, H.H.J. de - \ 2007
Biomacromolecules 8 (2007)5. - ISSN 1525-7797 - p. 1648 - 1656.
amyloid fibril formation - beta-lactoglobulin - electrostatic interactions - thermal-stability - egg-albumin - ovalbumin - ph - surface - succinylation - denaturation
This study describes how charge modification affects aggregation of ovalbumin, thereby distinguishing the role of conformational and electrostatic stability in the process. Ovalbumin variants were engineered using chemical methylation or succinylation to obtain a range of protein net charge from -1 to -26. Charge modification significantly affected the denaturation temperature. From urea-induced equilibrium denaturation studies, it followed that unfolding proceeded via an intermediate state. However, the heat-induced denaturation process could still be described as a two-state irreversible unfolding transition, suggesting that the occurrence of an intermediate has no influence on the kinetics of unfolding. By monitoring the aggregation kinetics, the net charge was found not to be discriminative in the process. It is concluded that the dominant factor determining ovalbumin aggregation propensity is the rate of denaturation and not electrostatic repulsive forces.
Structure and rheological properties of acid-induced egg white protein gels
Weijers, M. ; Velde, F. van de; Stijnman, A. ; Pijpekamp, A. van de; Visschers, R.W. - \ 2006
Food Hydrocolloids 20 (2006)2-3. - ISSN 0268-005X - p. 146 - 159.
heat-induced aggregation - induced gelation - beta-lactoglobulin - functional-properties - light-scattering - denatured whey - disulfide bond - ionic-strength - ovalbumin - lysozyme
This study compares the rheological properties of acid-induced gels prepared of industrial spray-dried egg white proteins (EWP) with the acid-induced gels prepared of ovalbumin (OA) and whey protein isolate (WPI). Also we aimed to form transparent gels of EWP by means of the cold-gelation process. We showed that it was not possible to prepare cold-set gels because ovotransferrin (OT), present in EWP, was found to interfere with fibril formation. Therefore, we developed a new purification method in which first Or was selectively denatured by a heating step, subsequently precipitated by acidification and removed by centrifugation. Finally, the supernatant was desalted by ultra filtration. This resulted in a preheated EWP preparation, which mainly contains OA (> 80%). By removing OT using this new preheat procedure transparent gels were obtained after acid-induced gelation. Fracture properties of various EWP preparations were determined and compared with those of acid-induced gels of OA and WPI. Gels formed from different EWP preparations were weak (fracture stress 1-15 kPa, fracture strain 0.3-0.7), and the networks consisted of thin strands with hardly any additional disulphide bonds formed during the gelation step. In conclusion, the microstructure of the aggregates formed in the first step of the cold-gelation process and the amount of additional disulphide bonds formed during the second step appeared to be the determining factors contributing to the hardness and deformability of acid-induced gels of egg white proteins.
Assessing the Extent of Protein Intermolecular Interactions at Air-Water Interfaces Using Spectroscopic Techniques
Jongh, H.H.J. de; Wierenga, P.A. - \ 2006
Biopolymers 82 (2006)4. - ISSN 0006-3525 - p. 384 - 389.
surface shear rheology - air/water interface - ovalbumin - adsorption - layers - dynamics
There is an ongoing debate about whether a protein surface film at an air-water interface can be regarded as a gelled layer. There is literature reporting that such films show macroscopic fracture behavior and a rheology comparable to three-dimensional protein bulk-networks. Ifthis is the case, a complete description of the formation of adsorbed layers should include a transition from single, freely moving proteins to a gelled layer. This report presents studies using spectroscopic techniques, such as infrared, fluorescence and neutron spectroscopy, or ellipsometry, to derive molecular insight in situ to substantiate the intermolecular networking in surface films of chicken egg ovalbumin. It is concluded that protein films, generated by equilibrium adsorption from the bulk, behave as a densely packed colloidal repulsive particle system, where the proteins still have a significant rotational mobility, have a predominantly retained globular fold, and show distinct (lateral) diffusion. Applied stresses on the surface film (by compressions of the interface) may result in protein denaturation and aggregation. This process renders a surface film from a colloidal particle into that of a gelled system.
X-ray and light scattering study of the structure of large protein aggregates at neutral pH
Pouzot, M. ; Nicolai, T. ; Visschers, R.W. ; Weijers, M. - \ 2005
Food Hydrocolloids 19 (2005)2. - ISSN 0268-005X - p. 231 - 238.
heat-induced denaturation - beta-lactoglobulin - globular-proteins - ovalbumin - gels - gelation - kinetics
The structure of large ovalbumin and ß-lactoglobulin aggregates formed after heat-denaturation at neutral pH was studied using a combination of light and small-angle X-ray scattering. The effect of the electrostatic interactions was investigated by varying the ionic strength. The results were compared with images obtained using cryo-TEM. The structure of ovalbumin aggregates is compatible with that of semi-flexible strings of monomers that are more flexible and increasingly branched with increasing ionic strength. The persistence length increases with decreasing ionic strength. ß-lactoglobulin aggregates consist of clusters of primary aggregates that are formed in the first step of the aggregation process. At low ionic strength the association of primary aggregates is mostly head to tail, while with increasing ionic strength denser clustering of the primary aggregates is observed.
Quantitative description of the relation between protein net charge and protein adsorption to air-water interfaces
Wierenga, P.A. ; Meinders, M.B.J. ; Egmond, M.R. ; Voragen, A.G.J. ; Jongh, H.H.J. de - \ 2005
The Journal of Physical Chemistry Part B: Condensed Matter, Materials, Surfaces, Interfaces & Biophysical 109 (2005)35. - ISSN 1520-6106 - p. 16946 - 16952.
der-waals contributions - beta-casein adsorption - surface concentration - electrostatic forces - air/water interface - circular-dichroism - globular-proteins - ovalbumin - kinetics - ph
In this study a set of chemically engineered variants of ovalbumin was produced to study the effects of electrostatic charge on the adsorption kinetics and resulting surface pressure at the air-water interface. The modification itself was based on the coupling of succinic anhydride to lysine residues on the protein surface. After purification of the modified proteins, five homogeneous batches were obtained with increasing degrees of modification and -potentials ranging from -19 to -26 mV (-17 mV for native ovalbumin). These batches showed no changes in secondary, tertiary, or quaternary structure compared to the native protein. However, the rate of adsorption as measured with ellipsometry was found to decrease with increasing net charge, even at the initial stages of adsorption. This indicates an energy barrier to adsorption. With the use of a model based on the random sequential adsorption model, the energy barrier for adsorption was calculated and found to increase from 4.7kT to 6.1kT when the protein net charge was increased from -12 to -26. A second effect was that the increased electrostatic repulsion resulted in a larger apparent size of the adsorbed proteins, which went from 19 to 31 nm2 (native and highest modification, respectively), corresponding to similar interaction energies at saturation. The interaction energy was found to determine not only the saturation surface load but also the surface pressure as a function of the surface load. This work shows that, in order to describe the functionality of proteins at interfaces, they can be described as hard colloidal particles. Further, it is shown that the build-up of protein surface layers can be described by the coulombic interactions, exposed protein hydrophobicity, and size.
Effects of succinylation on the structure and thermostability of lysozyme
Veen, M. van der; Norde, W. ; Cohen Stuart, M.A. - \ 2005
Journal of Agricultural and Food Chemistry 53 (2005)14. - ISSN 0021-8561 - p. 5702 - 5707.
air-water-interface - circular-dichroism - cold denaturation - adsorption - proteins - ovalbumin - stability - silica
The influence of succinylation on lysozyme is studied using circular dichroism, fluorescence spectroscopy, and differential scanning calorimetry. The spectroscopic data reveal that at room temperature the structures of succinylated lysozyme and native lysozyme are similar. However, the calorimetric results show that the thermal stability of succinylated lysozyme is lower than that of native lysozyme. For succinylated lysozyme, the denaturation temperature (Td) varies in the range of 325-333 K (52-60 degrees C) and the associated denaturation enthalpy (DeltadenH) varies between 225 and 410 kJ/mol. For lysozyme, Td is 342-349 K (69-76 degrees C) and DeltadenH is 440-500 kJ/mol. From these data, the change in the heat capacity (DeltadenCp) upon thermal denaturation is derived. For lysozyme, DeltadenCp is 7.5 kJ/mol/K, and for succinylated lysozyme, it is 16.7 kJ/mol/K. The value of DeltadenCp for lysozyme is comparable to previously reported values. The high value of DeltadenCp for succinylated lysozyme is explained in terms of an extended degree of unfolding of the secondary structure and exposure of the apolar parts of the succinyl groups. Furthermore, the Gibbs energy of denaturation, as a function of temperature, derived from the thermodynamic analysis of the calorimetric data, indicates a cold-denaturated state of succinylated lysozyme below 20 degrees C. However, because a denatured state at low temperatures could not be detected by CD or fluorescence measurements, the native state may be considered to be metastable at those conditions.
Correlation between mechanical behavior of protein films at the air/water interface and intrinsic stability of protein molecules
Martin, A.H. ; Cohen Stuart, M.A. ; Bos, M.A. ; Vliet, T. van - \ 2005
Langmuir 21 (2005)9. - ISSN 0743-7463 - p. 4083 - 4089.
air-water-interface - surface rheological properties - liquid interfaces - conformational aspects - beta-lactoglobulin - shear rheology - adsorption - layers - ovalbumin - reflection
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, ¿f, and fracture strain, ¿f, 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, ¿, and adsorbed amount, ¿ (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 ¿ ¿ 0 mN/m (A0), A0/M (M = molecular weight) and the maximum slope of the ¿-¿ curves (d¿/d¿). 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 A 0/M have a high ¿f and behave more fluidlike, whereas solidlike behavior is characterized by low A0/M and low ¿f. 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
Disulphide bond formation in food protein aggregation and gelation
Visschers, R.W. ; Jongh, H.H.J. de - \ 2005
Biotechnology Advances 23 (2005)1. - ISSN 0734-9750 - p. 75 - 80.
beta-lactoglobulin - egg-white - denaturation - ovalbumin - heat - gels
In this short review we discuss the role of cysteine residues and cystine bridges for the functional aggregation of food proteins. We evaluate how formation and cleavage of disulphide bonds proceeds at a molecular level, and how inter- and intramolecular disulfide bonds can be detected and modified. The differences between heat-, high-pressure-, and denaturant-induced unfolding and aggregation are discussed. The effect of disulphide bonding between aggregates of proteins and protein mixtures on the functional macroscopic properties of space filling networks in protein gels is briefly presented
The effect of shear flow on the percolation concentration of fibrillar protein assemblies
Veerman, C. ; Sagis, L.M.C. ; Venema, P. ; Linden, E. van der - \ 2005
Journal of Rheology 49 (2005)2. - ISSN 0148-6055 - p. 355 - 368.
rod-like macromolecules - rodlike chains subject - rheooptical response - transition-temperatures - collagen protein - light-scattering - gels - ovalbumin - viscoelasticity - birefringence
The objective of this study was to investigate the effect of shear flow on the percolation concentration (cp) for solutions of fibrillar protein assemblies. Theoretical calculations were performed to obtain cp versus Peclet number. They were based on a random contact model for rodlike particles, making use of a shear dependent excluded volume per fibril. We found cp to increase with increasing Peclet number. Results of flow birefringence measurements were used to obtain the rotational diffusion coefficient at cp, which enables one to transform the theoretically obtained cp(Pe) into a prediction for cp versus shear rate. This prediction was used to fit viscosity measurements as a function of shear rate, near the percolation threshold. A satisfactory fit was found indicating that the percolation threshold, cp, as function of shear rate can be predicted by combining theory and optical measurements.