Modification of Cysteine S sulfonated keratin properties through pH variations

This study aimed to analyze the effect of pH variations on the properties of lyophilized cysteine-S-sulfonate keratin, such as secondary structure contents, cell viability during in vitro tests, thermal and antibacterial properties. Cysteine-S-sulfonated keratin was extracted from wool fibers by sulfitolysis and it was subjected to pH variations between 3.0 and 8.0 when the protein was still within extraction solution (pH 6.5). After extraction, the pH of extraction solution with keratin was modified using NaOH (pH > 6.5) and four different acids (pH<6.5) – HCl, formic acid, acetic acid and citric acid. Afterward, the protein was purified by dialysis for three days, and then the purified protein solutions were frozen and lyophilized for seven days to remove water content. It is important to recall that after extraction the solution consists of urea, sodium dodecyl sulfate and ionic species (citrate, acetate, formate, Na, Cl, H and OH). Their presence avoids the complete flocculation of protein during titration, namely when the pH value was within the isoelectric point of keratin type I (4.75 – 5.40) and keratin type II (5.00 – 6.7). Nevertheless, these species are removed during dialysis, which causes the protein to aggregate and precipitate to some extent. Therefore, a considerable rise in standard deviation was taken into account like an increase of heterogeneous protein. The FTIR spectrums confirmed protein protonation for acid titrations with a peak above 1700 cm-1 associated with RCOOH groups and protein deprotonation for alkaline titration with a peak at 1400 cm-1 associated with RCOO groups. Furthermore, antibacterial tests against Gram-positive Staphylococcus aureus ATCC 6538 showed a complete biocidal activity for protonated cysteine-S-sulfonated keratin (positively charged polyelectrolyte) and an exponential growth of S. aureus colonies for negatively charged protein (deprotonated keratin). DSC analyses showed a rise in the temperature of denaturation peak for cysteine-S-sulfonated keratin, about 50°C when the pH value was below 4.5. This increasing thermostability of keratin is a well-known consequence of protein-ligand interactions between protonated proteins and anionic ligands (bisulfite, citrate, formate and acetate ions). As a result, removing anionic ligands from the keratin structure requires more energy and it increases the irreversible denaturation temperature of keratin. Finally, a relation was observed between the secondary structure content of intramolecular sheet and the growth of human mesenchymal stem cells (hMSCs) during in vitro tests. The greatest values of cell viability corresponded to the smallest values of intramolecular sheet; when the content of this secondary structure increased, the growth of hMSCs decreased as well. The same result was also noticed for the negatively charged keratin from alkali titration. All these results demonstrated the great potential of cysteine-S-sulfonated keratin and the importance of pH on stimuli-responsive keratin materials, for example hydrogels for cell culture and scaffolds for wound healing.