## 3 Results and Discussion
A correlation between the area of the absorption of the tyrosl group band with a maximum at 275 nm and the area of the absorption of the partially resolved peptide bond band, when increasing the NAYA concentration, exists (Figure 1C); thus meaning that there is evidence for the formation of an intramolecular interaction between the NAYA tyrosl group and the NAYA peptide bond, for NAYA solutions in the aqueous buffer 10 mM tris-HCl (pH 7). When centrifuging NAYA solutions, a UV spectral perturbation at 290-320 nm occurs, i.e., in the NAYA tyrosl group absorption band with the maximum at 275 nm (Figure 2A). This UV spectral perturbation occurs, particularly when membrane centrifugal filters with 100 kDa, 50 kDa and 30 kDa cut-off are used, under centrifugation conditions (Figure 2A). It might be the case that, when using the referred membrane centrifugal filters with smaller pore size, the strained intramolecular interaction between the NAYA tyrosl group and the NAYA peptide bond become evidenced. Further inspection of Figure 2A revealed that the UV spectral perturbation at 290-320 nm decreases when decreasing the membrane centrifugal filters pore size used. This indicates the progressive disruption of the intramolecular contacts in the NAYA molecular structure. Although no NAYA molecular association is expected to occur. In Figure 2B, the normalized far UV absorption spectra of NAYA compound solutions by using membrane centrifugal filters with the cut-off of 200 nm, 100 kDa, 50 kDa and 30 kDa, under centrifugation conditions, is shown. These normalized far UV spectra revealed a quasi-imperceptible blue shift (<1 nm) of the peptide bond when the NAYA compound solutions were submitted to the referred membrane centrifugal filters with decreasing cut-off. For comparative purposes, in Figure 2C the far UV absorption spectra for the NAYA compound aqueous solutions containing the 1,4-dioxane (Dx) nonpolar aprotic and water miscible compound are shown. In the latter representation, it was not possible to normalize the absorbance values at 205 nm or so, i.e., corresponding to the peptide bond absorption, due to the high absorption at the referred wavelengths but, it can still be inferred that when decreasing the Dx content (% \(v/v\)), the partial absorption band presents a blue shift (Figure 2C). Meaning, the quasi-imperceptible blue shift observed in Figure 2B indicates that the peptide bond in the NAYA compound is sensing a more polar environment when NAYA solutions are subjected to membrane centrifugal filters with decreasing cut-off. This is in agreement with the progressive disruption of the intramolecular interactions between the NAYA tyrosl group and the NAYA peptide bond. In Figure 2D, the calculated absorbance of different spectra of the UV spectra presented in Figure 2A is represented, when several membrane centrifugal filters of 200 nm, 100 kDa,50 kDa and 30 kDa cut-off were used, in comparison to when no membrane centrifugal filters were used. Inspection of Figure 2D reveals bands with maximums at ca. 291 nm. Figure 1: (**A**) UV absorption spectra of the NAYA compound when increasing its concentration in solution (10 mM tris-HCl, pH 7). (**B**) Representation of the variations of the peptide bond band and of the tyrosyl group bands absorbance maximums as a function of the NAYA concentration (10 mM tris-HCl, pH 7). (**C**) Absorption band area determined for the partially resolved peptide bond and for the tyrosyl group bands (bands shown in the UV absorption spectra displayed in (**A**)) as a function of the NAYA concentration (10 mM tris-HCl, pH 7). Figure 2: _Cont._  
Understanding the UV absorption spectral characteristics of NAYA solutions with no filtration and with filtration using membrane centrifugal filters with different cut-off enabled us to discuss UV absorption spectral characteristics of Syn solutions, under the same conditions.