Changes in the Secondary Structure of Adsorbed IgG and F(ab‘)₂ Studied by FTIR Spectroscopy

Abstract

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.

Immune complement activation is attenuated by surface nanotopography

“…These changes have previously been shown to increase with an increase in hydrophobicity of the surface for a number of proteins including IgG 32,33 (see also the Supplementary data for detailed information on QCM-D measurements of IgG adsorption). An increase in IC activation by precoated IgG on hydrophobic surfaces has earlier been reported by Wettero et al 34 and has been proposed to originate from surface-induced conformational changes of the IgG molecule.…”

FTIR Spectroscopic Study of Biogenic Mn-Oxide Formation byPseudomonas putidaGB-1

“…The amide I ATR peak of GB-1 cells does not shift upon Mn-oxidation (Table 5). Second derivative analysis of GB-1 and GB-1/MnO x ATR spectra in Figure 6 both reveal a protein mixture comprising β-sheet (1629 and 1637 cm −1 ), random coil (1645 cm −1 ), and α-helical (1652 cm −1 ) conformations (Buijs et al 1996). The amide I peak maximum for hydrated samples (ATR) occurs at 1637 cm −1 , corresponding to β-sheets (Table 5).…”

“…A shift in the amide I region to higher wavenumber in DRIFT and transmission spectra (Table 5) is consistent with previous studies (Hübner and Blume 1988) indicating that this change reflects the impact of sample dehydration. The presence of a small doublet peak in transmission spectra at 1658 (α-helical) and 1648 cm −1 (random coil/α-helical) (Buijs et al 1996;Jung 2000) suggest a change in protein conformation due to Mn-oxidation/-binding ( Figure 3, Table 5). The peak shift from 1652 cm −1 (α-helical) represents an increase in randomly coiled proteins after Mn-oxidation.…”

Vibrational Studies of Different Human Body Disorders Using FTIR Spectroscopy

“…The amide I band is a peculiar band which provides the most insight into secondary structure under biologically relevant conditions. Buijs, J. et al [35] have studied change in secondary structure of adsorbed IgG and F(ab') 2 . They have shown that the adsorbed amounts decrease with an increasing net charge density on the protein for IgG.…”