Protein surface denaturation

These estimates oversimplify both the enthalpic effects of interaction and the entropic effects of size differences. First, interactions between adsorbed molecules as described by Frumkin type models do not allow for the formation of dimers or larger aggregates in the surface layer which can occur in practice. Equations of state for monolayers showing such two-dimensional aggregation have been proposed for various models [32-42]. Second, proteins differ from surfactants in more than just the size. For proteins, surface denaturation can take place, leading to their unfolding at the surface, at least at low surface pressures. The partial molar surface area for proteins, in contrast to surfactants, is large and variable. The interrelation between the... 

Protein tertiary structure is also influenced by the environment In water a globu lar protein usually adopts a shape that places its hydrophobic groups toward the interior with Its polar groups on the surface where they are solvated by water molecules About 65% of the mass of most cells is water and the proteins present m cells are said to be m their native state—the tertiary structure m which they express their biological activ ity When the tertiary structure of a protein is disrupted by adding substances that cause the protein chain to unfold the protein becomes denatured and loses most if not all of Its activity Evidence that supports the view that the tertiary structure is dictated by the primary structure includes experiments m which proteins are denatured and allowed to stand whereupon they are observed to spontaneously readopt their native state confer matron with full recovery of biological activity... 

Mechanical forces, such as shearing, shaking, and pressure, may also denature proteins [44,45], Shaking proteins may lead to inactivation owing to an increase in the area of the gas/liquid interface. At the interface, the protein unfolds and maximizes exposure of hydrophobic residues to the air. Surface denaturation may also occur at the protein/container interface and has been observed following adsorption of proteins to filter materials [46]. 

Although ionic surfactants are often associated with denaturation of proteins [104], the nonionic surfactant polysorbate 80 has been included in several marketed formulations and serves to inhibit protein aggregation. The mechanism may be the greater tendency of the surfactant molecules to align themselves at the liquid/ air interface, excluding the protein from the interface and inhibiting surface denaturation. 

The manufacture and processing of the protein microarray should be conducted in such a manner that the arrayed proteins remain in their native and active state. For most proteins, this usually means the hydrated state in order to avoid surface denaturation. For antibody arrays which are perhaps more forgiving than other proteins, it has been our experience that while these could be stored cold and dry, it is most important to rehydrate them prior to use. This process is in sharp contrast to the preparation of nucleic acid arrays in which strand melting or denaturahon is necessary to achieve optimal binding to the solid support. While the hybridization process is well understood and can be controlled under thermodynamic principles, the folding and renaturation of proteins on planar (microarray) surfaces is under study. 

Membranes such as NC supported on glass may be more applicable for protein microarrays than glass substrates. Supported charged nylon membranes for microarrays are currently entering the marketplace as well. The essential ingredient for protein is water. Protein hydration reduces the likelihood for surface denaturation. Hydrophilic membranes allow proteins to... 

The authors suggest that while antibodies are structurally similar and relatively stable proteins that can be easily labeled at lysine residues in the Fc region, antigens are much more variable both in structure and relative stability. An alternative explanation is that antibodies likely recognize surface-denatured antigens even better than native antigens. Thus, performance of antigen arrays may be more related to an increase in the number... 

This phenomenon is known as surface denaturation. Adsorption area and, consequently, the extent of denaturation are decreased with an increasing specific adsorption of the anion. The dependence of the molecular weight versus adsorption area shows that the proteins of large molecular weight (above 15 kD) behave differently from the smaller proteins [94], as they possibly do not denaturate and spread on mercury to the same extent as... 

Graham, D.E., Phillips, M.C. (1979a). Proteins at liquid interfaces. I. Kinetics of adsorption and surface denaturation. Journal of Colloid and Interface Science, 70, 403 114. 

Another aspect of polysorbates is that they are inherently susceptible to oxidative degradation. Often, as raw materials, they contain sufficient quantities of peroxides to cause oxidation of protein residue side chains, especially methionine (59). The potential for oxidative damage arising from the addition of stabilizer emphasizes the point that the lowest effective concentrations of excipients should be used in formulations. For surfactants, the effective concentration for a given protein will depend on the mechanism of stabilization. It has been postulated that if the mechanism of surfactant stabilization is related to preventing surface-denaturation, the effective concentration will be around the detergent s critical micellar concentration. Conversely, if the mechanism of stabilization is associated with specific protein-detergent interactions, the effective surfactant concentration will be related to the protein concentration and the stoichiometry of the interaction (39). 

Cumper and Alexander ( ) and Cumper (T) explained that during foam formation, a monolayer of surface denatured protein surrounded by liquid is rapidly adsorbed at the interface of the colloidal mixture, trapping air and forming bubbles (Table I,... 

Proteins of egg white denature more rapidly than those of whey protein concentrate (13, 34). However, isolated p-lactoglobulin from the whey concentrate was more susceptible to surface denaturation than egg white ovalbumin. These data suggest that whey contains substances that protect the proteins from surface denaturation and may account for the lower stability of whey protein concentrate foams than those of egg white protein. A balance between the disaggregation effect of select pH values and the tendency toward greater aggregation of proteins at higher heating temperatures were correlated closely with maximum foam stability (13, 15). 

The packing material first described for direct injection of biological samples was prepared by simply saturating the accessible adsorption sites of a Cis reversed-phase silica with human plasma proteins (105). After saturation, the human plasma proteins were denatured at the external surface, and their native conformation was destroyed. With this treatment, the proteins formed a hydrophilic layer with weak ion-exchange properties, which provided protection from contact with the sample proteins, whereas the alkyl ligands inside the pores remained unchanged and thus served for analyte retention. The retention behavior of the saturated phase did not alter with this treatment, but the efficiency was reduced dramatically. Such protein-coated columns have shown a lifetime of several months (106). 

In the experiments with galactose oxidase and dopamine -monooxygenase very low enzyme concentrations have often been used. The addition of an inert protein might help to approximate the natural conditions by providing a protection against surface denaturation and against radicals, and by regulating the trace metal concentrations. 

Removal of calcium ions makes unfolding of a-lactalbumin irreversible. The denaturation temperature of a-lactalbumin decreased 20 °C when calcium ions were removed by a chelator (Bemal and Jelen 1984). Hillier et al (1979) found that an increase in the calcium concentration up to 0.4 mg/ml slowed the heat denaturation of a-lactalbumin, but additional calcium had little effect. There is a slow conformational change at pH 4 as calcium is released from carboxyl groups on the protein surface (Kronman et al 1964). Failure to measure the heat of denaturation for a-lactalbumin at pH 3 shows the protein chain is already unfolded at low pH (de Wit and Klarenbeek 1984). 

DNA is precipitated, the solution must be deproteinized. This is brought about by treatment with chloroform-isoamyl alcohol followed by centrifugation. Upon centrifugation, three layers are produced an upper aqueous phase, a lower organic layer, and a compact band of denatured protein at the interface between the aqueous and organic phases. Chloroform causes surface denaturation of proteins. Isoamyl alcohol reduces foaming and stabilizes the interface between the aqueous phase and the organic phases where the protein collects. 

If a small amount of protein solution is suitably spread at the surface of an aqueous substrate, most of the protein will be surface-denatured, giving an insoluble monomolecular film before it has a chance to dissolve. The techniques already described for studying spread monolayers of insoluble material can, therefore, be used in... 

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