Protein Analysis by 31P NMR Spectroscopy in Ionic Liquid: Quantitative Determination of Enzymatically Created Cross-Links

Cross-linking of beta-casein by Trichoderma reesei tyrosinase (TrTyr) and Streptoverticillium mobaraense transglutaminase (Tgase) was analyzed by P-31 nuclear magnetic resonance (NMR) spectroscopy in ionic liquid (IL). According to P-31 NMR, 91% of the tyrosine side chains were cross-linked by TrTyr at high dosages. When Tgase was used, no changes were observed because a different cross-linking mechanism was operational. However, this verified the success of the phosphitylation of phenolics within the protein matrix in the IL. Atomic force microscopy (AFM) in solid state showed that disk-shaped nanoparticles were formed in the reactions with average diameters of 80 and 20 nm for TrTyr and Tgase, respectively. These data further advance the current understanding of the action of tyrosinases on proteins on molecular and chemical bond levels. Quantitative P-31 NMR in IL was shown to be a simple and efficient method for the study of protein modification.