Adsorbed protein structure

Atkinson, P.J., Dickinson, E., Horne, D.S., Leermakers, F.A.M., Richardson, R.M. (1996). Theoretical and experimental investigations of adsorbed protein structure at a fluid interface. Berichte der Bunsen-Gesellschaftfur Physikalische Chemie, 100, 994-998. 

Haynes C A and Norde W 1995 Structural stabilities of adsorbed proteins J. Colloid Interface Sci. 169 313-28... 

It is generally assumed the fluorescence and Fourier transform mid-infrared (FT-IR) spectroscopies do not suffer from the above-mentioned inconveniences and may be applied to turbid samples. Front-face (fluorescence) and attenuated total reflection (FT-IR) techniques may provide information on the structure of adsorbed proteins. 

SAMs was correlated to the affinities of the Fn-specific monoclonal antibodies. Although antibody-based measurements could not distinguish between conformational (structural) and orientational changes in the adsorbed proteins, they provided information about the biological activity of adsorbed proteins. 

The 3D structure of a native protein (in aqueous solution) is only marginally thermodynamically stable and it is sensitive to changes in its environment. It is, therefore, not surprising that adsorption is often accompanied by rearrangements in the protein s 3D structure. It is commonly observed experimentally that the thickness of an adsorbed protein layer is comparable to the dimensions of the protein molecule in solution. It indicates that the adsorbed protein molecules remain compactly structured. 

This would prevent intimate contact between the adsorbed protein molecules and the PS surface. As a result, the structural integrity of the protein is expected to be less perturbed. 

Another important factor proved to be the influence of trace quantities of moisture in the compacts, from both the environment and also within the protein itself. Dry proteins adsorb water with avidity because of the exposed hydrophilic regions within the protein structure, changing the properties of the hydrogen bonding within the structure and, therefore, the flexibility of the overall structure. Excessive drying, on the other hand, can result in the collapse of an otherwise water-soluble protein to the point where it is effectively denatured and will no longer dissolve. 

Dickinson, E. (1999b). Adsorbed protein layers at fluid interfaces interactions, structure and surface rheology. Colloids and Surfaces B Biointerfaces, 15, 161-176. 

Globular proteins form close-packed monolayers at fluid interfaces. Hence a large contribution to the adsorbed layer viscoelasticity arises from short-range repulsive interactions between hard-sphere particles. In addition to, or instead of, this glass-like5 structure from hard spheres densely packed in two dimensions, many adsorbed proteins can exhibit attractive interactions leading to a more gel-like5 network structure. Hence the mechanical properties of an adsorbed layer depend on many... 

Figure 8.15 Cartoon showing how proteins, polysaccharides and surfactants (emulsifiers) might be distributed at the triglyceride-water interface. Inter-facial complexation in vivo between adsorbed protein and charged polysaccharide in the gastrointestinal tract could affect digestion of protein and fat by forming structures that inhibit the accessibility and activity of enzymes (proteases and lipases). Reproduced from Dickinson (2008) with permission.

Specific formulation strategies need to be employed for macromolecule compounds. An excellent review of protein stability in aqueous solutions has been published by Chi et al. (92). In addition to solution stability of proteins and peptides, aerosolization may result in significant surface interfacial destabilization of these compounds if no additional stabilization excipients are added. This is due to the fact that protein molecules are also surface active and adsorb at interfaces. The surface tension forces at interfaces perturb protein structure and often result in aggregation (92). Surfactants inhibit interface-induced aggregation by limiting the extent of protein adsorption (92). 

Most food products and food preparations are colloids. They are typically multicomponent and multiphase systems consisting of colloidal species of different kinds, shapes, and sizes and different phases. Ice cream, for example, is a combination of emulsions, foams, particles, and gels since it consists of a frozen aqueous phase containing fat droplets, ice crystals, and very small air pockets (microvoids). Salad dressing, special sauce, and the like are complicated emulsions and may contain small surfactant clusters known as micelles (Chapter 8). The dimensions of the particles in these entities usually cover a rather broad spectrum, ranging from nanometers (typical micellar units) to micrometers (emulsion droplets) or millimeters (foams). Food products may also contain macromolecules (such as proteins) and gels formed from other food particles aggregated by adsorbed protein molecules. The texture (how a food feels to touch or in the mouth) depends on the structure of the food. 

The phenomena of association colloids in which the limiting structure of a lamellar micelle may be pictured as composed of a bimolecular leaflet are well known. The isolated existence of such a limiting structure as black lipid membranes (BLM) of about two molecules in thickness has been established. The bifacial tension (yh) on several BLM has been measured. Typical values lie slightly above zero to about 6 dynes per cm. The growth of the concept of the bimolecular leaflet membrane model with adsorbed protein monolayers is traceable to the initial experiments at the cell-solution interface. The results of interfacial tension measurements which were essential to the development of the paucimolecular membrane model are discussed in the light of the present bifacial tension data on BLM. 

The adsorption process is often entropically driven with the gain in entropy arising from dehydration of the adsorbent surface and structural rearrangements inside the protein molecule (the state of hydration and field overlap changes inside the adsorbed protein included). 

We have already established that the protein adsorption process may result in significant conformational changes. In addition to adsorbed amounts and rates, the orientation and conformation of the adsorbed protein are critical (Fig. 16). Conformation refers to the secondary (a-helix, P-sheet), tertiary, and quaternary structures. 

Clearly specific antibodies, and particularly monoclonal antibodies, may be very useful in probing the properties of adsorbed proteins. Specific antibodies have been used to probe the structure of antigens in solution 88). Consider the adsorption of a simple protein with a small number of reasonably well-defined epitopes (surface sites with antibody binding activity), as in Fig. 19. Clearly epitopes E and A are not accessible for binding, while B, C, and D would be sterically accessible. One could also envision a conformational change upon adsorption which produces an epitope... 

Tween 20 was considerably more effective at reducing the stability of foams of a-la than was the case with /3-lg. There was a significant decrease in a-la foam stability in the presence of Tween, at R values as low as 0.05. Minimal foam stability was observed at R = 0.15. There was no observed change in film drainage behavior or onset of surface diffusion in the adsorbed protein layer up to this R value. The only observed change was a progressive decrease in film thickness. Therefore, it is likely that disruption of adsorbed multilayers is responsible for a reduction in the structural integrity of the adsorbed protein layer and that this increases the probability of film rupture. 

Interestingly, protein adsorption is also a field of biological interfacial chemistry which parallels that of synthetic materials at the solid - liquid interface. A number of spectroscopic advances have been made which allow FT-IR to be used in kinetic monitoring of protein adsorption on metals and "biocompatible" polymers. In addition to providing in - situ measurements of total adsorbed protein, FT-IR can also yield information about perturbation of protein secondary structure in adsorbed layers. 

These kinetics studies required development of reproducible criteria of subtraction of foe H-O-H bending band of water, which completely overlaps foe Amide I (1650 cm 1) and Amide II (1550 cm"1) bands (98). In addition, correction of foe kinetic spectra of adsorbed protein layers for foe presence of "bulk" unadsorbed protein was described (99). Examination of kinetic spectra from an experiment involving a mixture of fibrinogen and albumin showed that a stable protein layer was formed on foe IRE surface, based on foe intensity of the Amide II band. Subsequent replacement of adsorbed albumin by fibrinogen followed, as monitored by foe intensity ratio of bands near 1300 cm"1 (albumin) and 1250 cm"1 (fibrinogen) (93). In addition to foe total amount of protein present at an interface, foe possible perturbation of foe secondary structure of foe protein upon adsorption is of interest. Deconvolution of foe broad Amide I,II, and m bands can provide information about foe relative amounts of a helices and f) sheet contents of aqueous protein solutions. Perturbation of foe secondary structures of several well characterized proteins were correlated with foe changes in foe deconvoluted spectra. Combining information from foe Amide I and m (1250 cm"1) bands is necessary for evaluation of protein secondary structure in solution (100). 

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