# Peak Fitting Applied to Fourier Transform Infrared and Raman Spectroscopic Analysis of Proteins
## 4 Discussion
Therefore, \(\beta\)-sheet and random coil are the main secondary structural elements of gluten at low moisture contents. Upon hydration (up to 50% _w/w_), [PERSON] and [PERSON] [18] reported an increase in \(\beta\)-turn (from 14 to 65%) at the expense of decrease in \(\beta\)-sheet and random coil structure. They suggested that the \(\beta\)-turn is the preferred secondary structure of gluten in its hydrated state. These results are not in line with our results as \(\beta\)-sheets are found to be the dominating secondary structures in the here studied hydrated gluten samples. However, the systems studied by [PERSON] and [PERSON] [18] were completely different compared with the here studied gluten-only samples. The buildup of this high \(\beta\)-sheet content in the \begin{table}
\begin{tabular}{c c c} \hline \hline
**Amino acid** & **Absorbance Band Assignment** & **IR Frequency Position (cm\({}^{-1}\))** \\ \hline \multirow{2}{*}{Asparagine} & C=O (stretching) & 1678 \(\pm\) 3 \\  & NH bending & 1622 \(\pm\) 2 \\ \hline \multirow{2}{*}{Glutamine} & C=O stretching & 1670 \(\pm\) 4 \\  & N-H bending & 1610 \(\pm\) 4 \\ \multirow{2}{*}{Arginine} & C=N asymmetric & 1673 \(\pm\) 3 \\  & C=N symmetric stretching & 1633 \(\pm\) 3 \\ \hline \multirow{2}{*}{Lysine} & NH\({}^{+}\) asymmetric bending & 1629 \(\pm\) 1 \\ \multirow{2}{*}{Tyrosine} & Ring vibration & 1602 \(\pm\) 2 \\ \hline \multicolumn{3}{c}{\({}^{a}\) Data adopted from refs [6; 51].} \\ \end{tabular}
\end{table} 
Table 3: Absorbance bands of amino acid side chains in the 1600–1700 cm\({}^{-1}\)\({}^{a}\). hydrated gluten is a result of mechanical input energy during the mixing process [11], which also possibly resulted in the formation of intermolecular \(\beta\)-sheets observed at 1616 and 1623 cm\({}^{-1}\) (Figure 6). Zein proteins are highly hydrophobic. Commercial zein is protein-body-free and mainly contains \(\alpha\)-zein [60]. Four domains can be distinguished in the \(\alpha\)-zein structure: an N-terminal signal sequence, an N-terminal \(\beta\)-turn domain, a domain consisting of nine repeating sequences (flanked by glutamine residues), and a C-terminal domain [61]. The latest model to describe \(\alpha\)-zein structure has been proposed by [PERSON] et al. [62]. The proposed complete structure is built of nine \(\alpha\)-helix structures, present in a central domain, in addition to an N-terminal segment. According to [PERSON] and [PERSON] [63], the secondary structure of \(\alpha\)-zein dissolved in aqueous ethanol includes 40-60% \(\alpha\)-helix conformations and a small amount of \(\beta\)-sheet structures. When the protein's temperature is raised above its glass transition temperature (T\({}_{\beta}>35\)\({}^{\circ}\)C), a reversible change from an \(\alpha\)-helix-dominated conformation to more \(\beta\)-sheet structures has been observed imparting viscoelastic properties to zein [57]. \(\alpha\)-helix and \(\beta\)-sheet are generally considered \"ordered\" secondary structures, while \(\beta\)-turn and random coil are considered \"unordered\" protein secondary structures [64]. The transition from the glassy state to the rubbery state (upon reaching T\({}_{\beta}\)) is accompanied by an increase in ordered protein structures [64]. This can explain the fact that \(\beta\)-sheet and \(\alpha\)-helix structures are the dominant structures in the here studied hydrated zein samples mixed at above 40 \({}^{\circ}\)C. On the other hand, the unordered \(\beta\)-turn and random coil secondary structures are often linked to protein surface hydrophobicity as these structures facilitate the exposure of hydrophobic residues [65]. It has been suggested that the random coil structure formation is promoted by weakening of hydrogen bonds. Conversely, development of \(\alpha\)-helix structures is promoted by protein hydration and the subsequent hydrogen bond formation [64; 65]. This might explain the undetectable low levels of random coil structures in the hydrated zein sample. [PERSON] et al.