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.