Protein FTIR analysis

Despite the fact that crude APPL s are totally soluble in DMF, an important residue is obtained at the end of the solvent sequence (0 100, THF DMF) indicating that the protein-rich fractions require association with the polyphenolic part for their solubilization in DMF (Table VI). Because each APPL has a different amino acid composition, its solubility distribution is also different, but in both cases, the THF fraction is the most lignin-like, with only 1% nitrogen. This is confirmed by FTIR analysis (Fig. 7). As the fractionation proceeds with increasing solvent polarity, the lignin characteristic bands at 1515, 1460, 1265, 1095, 1035, and 810 cm-1 disappear, while the amide characteristic bands at 3290 and 3080 cm-1 appear. 

While these conclusions are directed towards the fibrinogen-albumin mixture study, this type of FTIR analysis of proteins has general applicability. These results indicate that FTIR can produce usable spectra of flowing aqueous protein solutions, and these spectra can in turn provide useful molecular-level information concerning protein-surface interactions. 

Fourier-transformed infrared (FTIR) is another excellent method to study protein folding. Unlike the well-known use of FTIR as a method for the identification of functional groups, in terms of protein structure this method allows the determination of secondary structure. The frequency of vibration of the amide I band of the peptide chain (1500-1600 cm M heavily depends on the structure of the protein. FTIR has the advantage of being more sensitive for the study of proteins that contain (3-sheet elements as compared to CD. Furthermore, since FTIR spectroscopy can be applied to solids also, it allows the structural analysis of aggregated protein deposits. The availability of the rapid step-scan method for FTIR is also very useful for the study of rapid folding reactions (see Vibrational Spectroscopy). 

Quantitative analysis of protein IR and VCD spectra in terms of the fractional components (FC) of their secondary structure has taken different approaches, as noted earlier. The FTIR approach of assigning frequencies to specific components can, in principle, identify amounts of unordered structure in a protein fold. The viability of this approach... 

Naidja A, Liu C, Huang PM (2002) Formation of protein-bimessite complex XRD, FTIR, and AFM analysis. J Colloid Interface Sci 251 46-56... 

Fig. 6. Deconvolved amide F region FTIR spectra of apo- and heme-hemopexin. The amide F FTIR spectra of apo- and heme-hemopexin in D2 O were recorded and curve-fitted to resolve the individual bands. The differences between the original and fitted curves are shown in the upper traces in the panels. The estimated helix (15%), beta (54%), turn (19%), and coil (12%) content of the apo-protein are not significantly changed upon heme binding 104). This analysis was required because of the positive 231-nm elhpticity band in hemopexin and is consistent with the derived crystal structure results.

Two Streptomyces strains, S. badius and S. viridosporus, were found to be able to grow on kraft lignin (In-dulin ATR) as sole carbon source. The resulting APPL (Acid Precipitable Polymeric Lignin) was characterized by FTIR and elemental analysis for C, H and N, and was found to contain proteins in addition to a relatively demethoxylated lignin component. The proteins were further characterized by amino acid analysis, while the lignin component was separated by solvent extraction and its molecular weight distribution determined by HPSEC. 

The wavelengths of IR absorption bands are characteristic of specific types of chemical bonds. In the past infrared had little application in protein analysis due to instrumentation and interpretation limitations. The development of Fourier transform infrared spectroscopy (FUR) makes it possible to characterize proteins using IR techniques (Surewicz et al. 1993). Several IR absorption regions are important for protein analysis. The amide I groups in proteins have a vibration absorption frequency of 1630-1670 cm. Secondary structures of proteins such as alpha(a)-helix and beta(P)-sheet have amide absorptions of 1645-1660 cm-1 and 1665-1680 cm, respectively. Random coil has absorptions in the range of 1660-1670 cm These characterization criteria come from studies of model polypeptides with known secondary structures. Thus, FTIR is useful in conformational analysis of peptides and proteins (Arrondo et al. 1993). 

Figure 2.22. Initial velocity of oxygen consumption as a function of the substrate (catechol) concentration in the presence of 0.074mg (7.11 x 1(T9M) tyrosinase (A), 2.0mg (2.8 x 1(T4M with a corresponding concentration of the mineral active sites, [M5]1.71xl(r6) of 8-Mn02 (B) and 10.0 mg (1.40 x 10 3M with a corresponding concentration of the mineral active sites, [M ]8.54 x 10-6) of S-Mn02 (C). Reprinted from Naidja, A., Liu, C., and Huang, P. M. (2002). Formation of protein-birnessite complex XRD, FTIR, and AFM analysis. J. Coll. Interface Sci. 251,46-56, with permission from Elsevier.

The possibilities of application of far-UV circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy in analysis of thermal stability of proteins and structural changes within protein molecules as well in explanation of cross reactivity between food allergens have been described in more detail in Section 3.4. Likewise nuclear magnetic resonance (NMR), especially 2D and multidimensional NMR as well as the method based on diffraction of monochromatic x-rays widely used in examination of tertiary structures of allergens have been described in Section 3.4 and by Neudecker et al. (2001) and Schirmer et al. (2005). 

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