Effects of site-specific phosphorylation on the mechanical properties of ovalbumin-based hydrogels

An efficient one-step grafting approach was developed to modify ovalbumin (OVA) by phosphorylation through selective reaction with the hydroxyl group of Ser and Thr residues present in OVA. The site-specific phosphorylated conjugates were characterized by Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF/MS) and the results indicated that the Ser residue could be more readily phosphorylated, and the typical phosphopeptides ²⁶⁴LTEWTSSNVMEER²⁷⁶ and ³⁴⁰EVVGSAEAGVDAASVSEEFR³⁵⁹ demonstrated the formation of monophosphoester. Moreover, ¹³C NMR analysis showed that the βCH₂ of Ser acted as a hydroxyl donor to react with sodium tripolyphosphate (STPP), and the conjugates with variable phosphorylation sites could improve the weak network and the resulting poor mechanical properties of ovalbumin-based hydrogels. Furthermore, small-amplitude oscillatory measurements, creep recovery tests and texture profile analysis of hardness and stickiness indicated that phosphorylation can strengthen the intermolecular cross-linking of protein molecules and produce significant influence on the rheological behavior and texture properties, suggesting that a suitable conjugation site is essential for the best gelation properties at a different pH. The integrated results indicate that phosphorylation change significantly modify the viscoelastic and mechanical properties of OVA-based hydrogels by changing molecular dynamics upon heating.