Protein adsorption model testing

The adsorption of proteins at interfaces is a key step in the stabilization of numerous food and non-food foams and emulsions. Our goal is to improve our understanding of the relationships between the sequence of proteins and their surface properties. A theoretical approach has been developed to model the structure and properties of protein adsorption layers using the analogy between proteins and multiblock copolymers. This model seems to be particularly well suited to /5-casein. However, the exponent relating surface pressure to surface concentration is indicative of a polymer structure intermediate between that of a two-dimensional excluded volume chain and a partially collapsed chain. For the protein structure, this would correspond to attractive interactions between some amino acids (hydrogen bonds, for instance). To test this possibility, guanidine hydrochloride was added to the buffer. A transition in the structure and properties of the layer is noticed for a 1.5 molar concentration of the denaturant. Beyond the transition, the properties of the layer are those of a two-dimensional excluded volume chain, a situation expected when there are no attractive interac-... 

So far, we have summarized strategies to exploit the chemical versatility of polymer brushes to either immobilize biomolecules by covalent attachment or for significantly decreasing protein adsorption. However, the extended interface created by the brush in a good solvent also provides a swellable, soft layer that can promote the nonspecific immobilization of enzymes and provide an environment that supports their activity. We have tested the functionality of enzymes physisorbed from solution [11]. Because this type of binding is weak, the conformation and activity of the proteins is expected to remain largely intact. To assess the influence of polymer brush chemistry, wettability, and swellability on the physisorption of proteins, model enzymes were chosen. Alkaline phosphatase (ALP) and horseradish peroxidase (HRP) were selected because they both catalyze the transformation of a colorless substrate to a colored product, and the enzymatic activity can therefore be easily monitored with colorimetry. The substrate of choice for ALP is /lara-nitrophenyl phosphate (pNPP), which is hydrolyzed to yield yellow /lara-nitrophenol (pNP) (Figure 4.14). 

Although protein adsorption from aqueous solutions onto various types of surfaces has been studied for several decades, no models accurately describe the process in detail [4]. Protein adsorption is typically analyzed by measuring a series of surface coverage data (F, usually in mg m ) against either the equilibrium protein concentration (Ceq, usually in mgmL ) or the time elapsed (f). F can be evaluated by measuring the amount of protein remaining in solution in contact with a particulate surface in a series of test tubes or in a stirred ceU. Alternatively, the... 

Separations in hydrophobic interaction chromatography have been modeled as a function of the ionic strength of the buffer and of the hydrophobicity of the column, and tested using the elution of lysozyme and ovalbumin from octyl-, butyl- and phenyl-Sepharose phases.2 The theoretical framework used preferential interaction analysis, a theory competitive to solvophobic theory. Solvophobic theory views protein-surface interaction as a two-step process. In this model, the protein appears in a cavity in the water formed above the adsorption site and then adsorbs to the phase, with the free energy change... 

An example of the immobilization of antibodies on channel surfaces was presented by Eteshola and Leckband [395]. A microfluidic sensor chip was developed to quantify a model analyte (sheep IgM) with sensitivities down to 17 nM. This was achieved by first immobilizing a layer of bovine serum albumine (BSA) onto the channel wall, followed by specific adsorption of protein A to which the primary antibody for IgM was coupled covalently. This antibody could capture IgM, which was detected with the secondary antibody, labeled with horseradish peroxidase (Scheme 4.91). This enzyme catalyzes the conversion of the fluorogenic substrate 3-(p-hydroxyphenyl)propioni c acid into a fluorophore, which was quantified off-chip with a spectrofluorometer. The measured fluorescence signal was proportional to the analyte concentration in the test sample. 

The phenomenon of viral adsorption to various surfaces was extensively studied from an environmental standpoint as reviewed by Daniels (14) and Gerba (15) for prevention of various waterborne viral transmissions. The problem of virus removal from complex protein solutions is very different from that of sewage and drinking water treatment processes because most protein molecules compete for the active sites of the adsorbents. Hence, both the adsorption rate and capacity diminish in the presence of protein molecules (16). It is the intention of this paper to demonstrate and to compare the antiviral activity of a surface-bonded QAC in aqueous solutions against 2 model viruses with and without the presence of proteins. The efficacy of the accepted antiviral thermo-inactivation was compared with the viral inactivation method by the surface-bonded QAC treatment. Beta-lactamase was used as a thermolabile model protein (17), and bacteriophage T2 and herpes simplex virus type 1 (HSV-1, an enveloped animal virus) were used as model hydrophilic and hydrophobic viruses to test these chemical inactivation methods. 

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