CHANGES IN THE SECONDARY STRUCTURE OF ADSORBED IGG AND F(AB')(2) STUDIED BY FTIR SPECTROSCOPY

The purpose of the present study is to find a quantitative relationshi p between the adsorption behavior and the secondary structure of prote ins. The adsorption of two monoclonal IgGs which differ in their isoel ectric point and their corresponding F(ab')(2) fragments is followed i n time. The proteins are adsorbed on hydrophilic silica and on hydroph obic methylated surfaces at different values of pH and ionic strength. The adsorption behavior is studied by measuring FTIR spectra of the a dsorbed proteins. The adsorbed amount is related to the integrated are a of the amide II region of the spectrum. The secondary structure of t he adsorbed proteins is evaluated by analyzing the second derivatives of the amide I region. Quantitative information on the secondary struc ture is obtained by applying a fitting procedure which assumes the abs orption bands for the different structural components to be Lorentzian shaped. The results show that for IgG the adsorbed amounts decrease w ith an increasing net charge density on the protein. This decrease is correlated to a reduction in the beta-sheet content which suggests tha t IgG molecules adsorb in a less compact conformation. The adsorption- induced reduction in the beta-sheet content is larger at hydrophobic m ethylated surfaces than at hydrophilic silica surfaces. The dependence of the amount of structural elements on the pH diminishes at higher i onic strength. F(ab')(2) fragments contain a higher fraction of beta-s heet content than whole IgG molecules, and these fractions are less st rongly influenced by the adsorption conditions. Therefore, it can be c oncluded that the F(ab')(2) fragments have a higher structural stabili ty toward adsorption than whole IgG molecules.