Susanne Wrang Bruun

Correcting attenuated total reflection-Fourier transform infrared spectra for water vapor and carbon dioxide.

Fourier transform infrared (FT-IR) spectroscopy is a valuable technique for characterization of biological samples, providing a detailed fingerprint of the major chemical constituents. However, water vapor and CO2 in the beam path often cause interferences in the spectra, which can hamper the data analysis and interpretation of results. In this paper we present a new method for removal of the spectral contributions due to atmospheric water and CO2 from attenuated total reflection (ATR)-FT-IR spectra. In the IR spectrum, four separate wavenumber regions were defined, each containing an absorption band from either water vapor or CO2. From two calibration data sets, gas model spectra were estimated in each of the four spectral regions, and these model spectra were applied for correction of gas absorptions in two independent test sets (spectra of aqueous solutions and a yeast biofilm (C. albicans) growing on an ATR crystal, respectively). The amounts of the atmospheric gases as expressed by the model spectra were estimated by regression, using second-derivative transformed spectra, and the estimated gas spectra could subsequently be subtracted from the sample spectra. For spectra of the growing yeast biofilm, the gas correction revealed otherwise hidden variations of relevance for modeling the growth dynamics. As the presented method improved the interpretation of the principle component analysis (PCA) models, it has proven to be a valuable tool for filtering atmospheric variation in ATR-FT-IR spectra.

Antigenic Specificity of Serum Antibodies in Mice Fed Soy Protein

Background: Soybean protein is used in a number of food products but unfortunately is also a common cause of food allergy. Upon ingestion of soy protein, healthy mice like other animals and humans generate a soy-specific antibody response in the absence of signs of illness. Not much is known about the relationship between the immunogenic proteins involved in this nondeleterious antibody response and the pathological response associated with food allergy.The objective of the present study was to characterize the antigenic specificity of the soy protein-specific antibody response generated in healthy mice ingesting soy protein. Methods: Blood from mice fed a soy-containing diet was analyzed using ELISA and immunoblot for antibody reactivity towards various soy protein fractions and pure soy proteins/subunits. Mice bred on a soy-free diet were used as controls. Results: The detectable antigenic specificity of the serum antibodies of soy-consuming mice comprised glycinin and β-conglycinin. Immunoblots with soy protein extract demonstrated antibody reactivity towards both the basic and the acidic chains of glycinin and the β-conglycinin subunits with an individual response pattern among mice. Moreover, antibody reactivity was found towards the native quaternary structure of glycinin. Conclusions: Mice ingesting soy protein generate an antibody response with reactivity towards glycinin and β-conglycinin. Antibody reactivity found towards the native quaternary structure of glycinin indicates an oral immunogenicity of the highly processing-resistant oligomerized glycinin.

A Chemometric Analysis of Ligand-Induced Changes in Intrinsic Fluorescence of Folate Binding Protein Indicates a Link between Altered Conformational Structure and Physico-Chemical Characteristics

Ligand binding alters the conformational structure and physico-chemical characteristics of bovine folate binding protein (FBP). For the purpose of achieving further information we analyzed ligand (folate and methotrexate) -induced changes in the fluorescence landscape of FBP. Fluorescence excitation and emission two-dimensional (2D) spectra were recorded over a wide range of wavelengths on a Perkin-Elmer LS 55 spectrofluorometer at varying pH in different buffers, and the resulting three-dimensional data were subjected to a chemometric analysis, parallel factor analysis (PARAFAC). The most important finding was the occurrence of two maximum intensity emission wavelengths of tryptophan, 350 nm (component one) and 330 nm (component two). In contrast to the first component, the score of the short wavelength component increased with increasing ligation of FBP. Since the emission wavelengths of indole groups in tryptophan shorten with increasing distance from the solvent surface of proteins, an increasing number of the 11 tryptophan residues seem to reorientate from the solvent surface to the interior of FBP with increasing ligation. The sharp decrease in hydrophobicity at pI=7–8 following binding of folate accords fairly well with the disappearance of strongly hydrophobic tryptophan residues from the solvent-exposed surface of FBP. The PARAFAC has thus proven useful to establish a hitherto unexplained link between parallel changes in conformational structure and physico-chemical characteristics of FBP induced by folate binding. Parameters for ligand binding derived from PARAFAC analysis of the fluorescence data were qualitatively and quantitatively similar to those obtained from binding of radiofolate to FBP. Herein, methotrexate exhibited a higher affinity for FBP than in competition with radiofolate. This could suggest a rapid and firm complexation of folate to FBP, blocking access of competing ligands.

Application of Near-Infrared and Fourier Transform Infrared Spectroscopy in the Characterization of Ligand-Induced Conformation Changes in Folate Binding Protein Purified from Bovine Milk: Influence of Buffer Type and pH

Fourier transform infrared (FT-IR) and near-infrared (NIR) spectroscopy have been applied to detect structural alterations in folate binding protein (FBP) induced by ligation in different buffer types. The amide I region pointed to a β-sheet to α-helix transition upon ligation in acetate and phosphate buffers, and the formation of intermolecular β-sheet was indicated at pH 5.0, in agreement with a dimerization of FBP taking place at this pH. The ligand-induced changes in the 2100–2300 nm NIR region were significant for FBP in acetate and phosphate buffers of pH 5.0, and the variations were interpreted as secondary structure changes, based on previous assignments of secondary structures to the combination bands in the NIR region. In the case of acetate buffer, variations in the amide combination bands agreed with the amide I analysis, but for the other buffer types some discrepancies were found and explained by side-chain contributions to the NIR, which could reflect the tertiary and quaternary structure differences. NIR spectra of FBP at pH 7.4 and 5.0 revealed contradictory effects on the side chains, reflecting different polymerization events at the two pH values, whereas the amide I region indicated similar changes at the two pH values. Therefore, we suggest that FT-IR and NIR spectroscopy may complement each other, such that the two techniques in combination may give information on all three types of protein conformational changes. While the secondary structure changes are revealed by FT-IR, the tertiary and quaternary structure changes are reflected in the NIR spectra, although the general influence of the latter changes on the NIR spectra remains to be confirmed.