## 3 Results and Discussion
It should be emphasized that very low Syn aggregation was observed during centrifugation of protein solutions. By DLS, the Syn aggregated species have numbers <0.01% of the Syn species total population. Furthermore, UV absorption spectroscopy was revealed to be sensitive to this very low Syn aggregation detected, which was referred to. Due to the very low amounts of Syn aggregated species, we can infer that the equilibrium conditions of the present system were not much affected. Figure 3: _Cont._  
The UV absorption spectral characterization of Syn solutions submitted to stress-induced conditions by using centrifugal filters with the decrease in their cut-off, under centrifugation conditions, revealed a spectra perturbation that was linked to the disruption of intramolecular interactions between protein tyrosl groups and protein peptide bonds. Moreover, the concomitant increase in Syn intermolecular interactions with the decrease in the membrane centrifugal filters pore size was predicted. In order to determine the Syn tyrosl groups exposure in such developed protein intermolecular interactions, we decided to record the first 1000 s of the Syn aggregation reaction at pH 7, by determining the intrinsic fluorescence intensity (_FI_) variations of the Syn solutions (Figure 5). The Syn protein contains four tyrosine residues (Y39, Y125, Y133 and Y136) and no tryptophan residue and the collected fluorescence intensity in the Syn protein should reflect the environment that the protein tyrosine residues display in the experiment. As such, if a protein tyrosine residue or protein tyrosine residues experience a more hydrophobic environment, an increase in the fluorescence quantum yield should be expected [22]. Contrary to tyrosine absorption, which is influenced by the polarity of the solvent, the fluorescence of tyrosine does not depend on the polarity of the solvent and just an increase in the fluorescence intensity is observed, as mentioned in [22]. In Figure 5, an increase in the fluorescence intensity of the Syn solutions subjected to centrifugation was noticed with increasing the membrane centrifugal filter pore size. This observation affords for the fact that with decreasing the membrane centrifugal filters pore size, there is an increase in the Syn tyrosl groups exposure to a more hydrophilic environment. Figure 3: (**A,C**) Normalized UV absorption spectra of Syn solutions (10 mM tris-HCl, pH 7) with no filtration by centrifugation and with centrifugal filtration using different membrane centrifugal filters with different cut-off of 200 nm, 100 kDa, 50 kDa and 30 kDa. (**B,D**) Normalized far UV absorption spectra of Syn solutions (10 mM tris-HCl, pH 7) submitted to filtration by centrifugation using different membrane centrifugal filters with different cut-off of 200 nm, 100 kDa, 50 kDa and 30 kDa. (**E–G**) Calculated difference spectra from the UV absorption spectra shown in (**A,C**). The mentioned UV absorption spectra were collected 5 min after filtration by centrifugation. For more information regarding this figure legend, please see the text. Figure 4: (**A**) Average of normalized volume distributions obtained by DLS (measurements obtained during the first ca. 30 min, \(N\geq 4\)) as a function of the particle size (nm) for Syn solutions subjected to centrifugation using membrane centrifugal filters with the 200 nm, 100 kDa, 50 kDa and 30 kDa cut-off. (**B**) Volume peak area obtained from the volume distributions in (**A**) as a function of the membrane centrifugal filters pore size (nm) [21]. Figure 5: Protein intrinsic fluorescence intensity (\(\lambda_{\text{excitation}}=275\) nm and \(\lambda_{\text{emission}}=305\) nm) as a function of time for 33.5 \(\upmu\)M Syn solutions (\(A_{275\text{nm}}=0.2\)) (10 mM tris-HCl) subjected to centrifugation with different membrane centrifugal filters with cut-off of 200 nm, 100 kDa, 50 kDa and 30 kDa, at pH 7. Intrinsic fluorescence intensity traces were performed in duplicate.