The research field of protein adsorption on surfaces appears to be as popular as ever. In the past year, several hundred published papers tackled problems ranging from fundamental aspects of protein surface interactions to applied problems of surface blood compatibility and protein surface immobilization. Although some parts of the protein adsorption process, such as kinetics and equilibrium interactions, can be accurately predicted, other aspects, such as the extent and the rate of protein conformational change, are still somewhat uncertain. The whole field is ripe for a comprehensive theory on protein adsorption.
The purpose of the present study is to find a quantitative relationship between the adsorption behavior and the secondary structure of proteins. The adsorption of two monoclonal IgGs which differ in their isoelectric point and their corresponding F(ab′)2 fragments is followed in time. The proteins are adsorbed on hydrophilic silica and on hydrophobic methylated surfaces at different values of pH and ionic strength. The adsorption behavior is studied by measuring FTIR spectra of the adsorbed proteins. The adsorbed amount is related to the integrated area of the amide II region of the spectrum. The secondary structure of the adsorbed proteins is evaluated by analyzing the second derivatives of the amide I region. Quantitative information on the secondary structure is obtained by applying a fitting procedure which assumes the absorption bands for the different structural components to be Lorentzian shaped. The results show that for IgG the adsorbed amounts decrease with an increasing net charge density on the protein. This decrease is correlated to a reduction in the β-sheet content which suggests that IgG molecules adsorb in a less compact conformation. The adsorption-induced reduction in the β-sheet content is larger at hydrophobic methylated surfaces than at hydrophilic silica surfaces. The dependence of the amount of structural elements on the pH diminishes at higher ionic strength. F(ab′) 2 fragments contain a higher fraction of β-sheet content than whole IgG molecules, and these fractions are less strongly influenced by the adsorption conditions. Therefore, it can be concluded that the F(ab′)2 fragments have a higher structural stability toward adsorption than whole IgG molecules.
Interactions of recombinant human growth hormone and lysozyme with solid surfaces are studied using total internal reflection fluorescence (TIRF) and monitoring the protein's intrinsic tryptophan fluorescence. The intensity, spectra, quenching, and polarization of the fluorescence emitted by the adsorbed proteins are monitored and related to adsorption kinetics, protein conformation, and fluorophore rotational mobility. To study the influence of electrostatic and hydrophobic interactions on the adsorption process, three sorbent surfaces are used which differ in charge and hydrophobicity. The chemical surface groups are silanol, methyl, and quaternary amine. Results indicate that adsorption of hGH is dominated by hydrophobic interactions. Lysozyme adsoption is strongly affected by the ionic strength. This effect is probably caused by an ionic strength dependent conformational state in solution which, in turn, influences the affinity for adsorption. Both proteins are more strongly bound to hydrophobic surfaces and this strong interaction is accompanied by a less compact conformation. Furthermore, it was seen that regardless of the characteristics of the sorbent surface, the rotational mobility of both proteins' tryptophans is largely reduced upon adsorption.
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