The room-temperature liquid salt, ethylammonium nitrate (EAN), has been used to enhance the recovery of denatured-reduced hen egg white lysozyme (HEWL). Our results show that EAN has the ability to prevent aggregation of the denatured protein. The use of EAN as a refolding additive is advantageous because the renaturation is a one-step process. When HEWL was denatured reduced using routine procedures and renatured using EAN as an additive, HEWL was found to regain 75% of its activity. When HEWL was denatured and reduced in neat EAN, dilution resulted in over 90% recovery of active protein. An important aspect of this process is that renaturation of HEWL occurs at concentrations of 1.6 mg/mL, whereas other renaturation processes occur at significantly lower protein concentrations. Additionally, the refolded-active protein can be separated from the molten salt by simple desalting methods. Although the use of a low-temperature molten salt in protein renaturation is unconventional, the power of this approach lies in its simplicity and utility.

By means of a kinetic test, it was demonstrated that one of the folding intermediates (Iα) of hen lysozyme with α-domain folded and β-domain unfolded is on the folding pathway under the classical definition. Iα folds to the native (N) state directly (unfolded (U) [hArr ] Iα [hArr ] N) without having to unfold to U and then refold to N through alternative folding pathways as in Iα [hArr ] U [hArr ] N.