## 3 Results and Discussion
In Figure 3A the normalized UV absorption spectra for Syn solutions prepared with no filtration and with centrifugal filtration using membrane centrifugal filters with different cut-off of 200 nm, 100 kDa, 50 kDa and 30 kDa, are represented. The UV absorption spectra of Syn solutions submitted to centrifugation were collected at 5 min after centrifugation. It is clear from Figure 3A that a spectral perturbation arises at ca. 290-340 nm, which resembles the spectral perturbation observed for the NAYA compound (Figure 2A). Another aspect is the fact that increased light scattering was noted in the Syn solution when no filtration was performed, in comparison to what was observed when the protein solutions were submitted to the mentioned centrifugation procedure (Figure 3A). The UV absorption spectra in Figure 3A is intriguing and it means that UV absorption spectroscopy of Syn solutions, as also observed for NAYA solutions, can be used to identify and to determine the nature of Syn protein tyrosl groups interactions. Nevertheless, it cannot distinguish _a priori_ in the Syn molecular structure which protein tyrosl groups participate in such contacts. The normalized UV absorption spectra in Figure 3A are from Syn solutions with a similar and high optical density at 275 nm, of ca. 0.7. In Figure 3B, the normalized far UV absorption spectra for the same solutions presented in Figure 3A are shown. Still referring to Figure 3B, two overlapping bands with maximums at ca. 205 and ca. 223 nm, corresponding to the peptide bond absorption and tyrosl group absorption bands, respectively, can be seen [10]. In fact, in Figure 3B it can be observed that both the tyrosl group and the peptide bond showed roughly similar absorbance, which can be accounted for by some influence of the tyrosl group absorption in the absorption of the peptide bond, in the Syn protein molecular structure. Therefore, it is of note that some influence of the protein tyrosl groups absorption in the absorption of the protein peptide bond arises when  
Figure 2: **(A)** Normalized UV absorption spectra of NAYA compound solutions (10 mM tris-HCl, pH 7) with no filtration and subjected to membrane centrifugal filters with different cut-off (200 nm, 100 kDa, 50 kDa and 30 kDa). In the latter representation, the NAYA solutions possessed optical densities at 275 nm of ca. 0.1. **(B)** Normalized far UV absorption spectra of the NAYA compound solutions (10 mM tris-HCl, pH 7) with no filtration and subjected to membrane centrifugal filters with different cut-off (200 nm, 100 kDa, 50 kDa and 30 kDa). In the latter representation, the NAYA solutions possessed optical densities at 275 nm of ca. 0.1. **(C)** Normalized far UV absorption spectra of NAYA aqueous solutions containing 1,4-dioxane (Dx) (% \(v/v\)). In this representation, NAYA solutions possessed optical densities at 275 nm of ca. 0.1. **(D)** Calculated absorption difference spectra of NAYA solutions referred to in **(A)**. the protein solutions are submitted to the centrifugation procedure by using membrane centrifugal filters with 200 nm, 100 kDa and 50 kDa cut-off, but not when the membrane centrifugal filter with the 30 kDa cut-off is used (Figure 3B). This result is intriguing, and it affords for the fact that there is minor or no influence of protein tyrosl groups absorption in the absorption of the peptide bond in this latter situation. It deserves, therefore, to be said that negligible or no interactions are established with protein tyrosl groups when the membrane centrifugal filter with the 30 kDa cut-off is used. To note that Syn monomeric protein has a molecular weight of about 14.46 kDa and, therefore, below the membrane centrifugal filters cut-off used in this study. In Figure 3C, the normalized UV absorption spectra of Syn solutions are shown, similarly as presented in Figure 3A, but the Syn solutions have here similar and low optical densities at 275 nm, of ca. 0.1.