1997/12/09 by Roman V. Rariy, Alexander M. Klibanov · 146 citations
Biochemistry, Genetics and Molecular Biology · Chemistry · Materials Science · #Anhydrous #Aqueous solution #Biochemistry #Chemistry #Chromatography #Dimethyl sulfoxide #Enzyme Structure and Function #Glycerol #Hemoglobin structure and function #Lysozyme #Materials science #Organic chemistry #Oxidizing agent #Protein Structure and Dynamics #Protein folding #Solvent #Yield (engineering)
paper · open access · doi:10.1073/pnas.94.25.13520
published in Proceedings of the National Academy of Sciences 94(25), 13520-13523 (National Academy of Sciences)
openalex publication_date 1997/12/09 · openalex created_date 2025/10/10 · openalex updated_date 2026/07/26
Water is the natural medium for protein folding, which is also used in all in vitro studies. In the present work, we posed, and answered affirmatively, a question of whether it is possible to fold correctly a typical protein in a nonaqueous solvent. To this end, unfolded and reduced hen egg-white lysozyme was refolded and reoxidized in glycerol containing varying amounts of water. The unfolded/reduced enzyme was found to regain spontaneously substantial catalytic activity even in the nearly anhydrous solvent; for example, the refolding yield in 99% glycerol was still some one-third of that in pure water, and one-half of that was regained even in 99.8% glycerol. The less than full recovery of the enzymatic activity in glycerol is, as in water, because of competing protein aggregation during the refolding. Lysozyme reoxidation in glycerol was successfully mediated by two dissimilar oxidizing systems, and the refolding yield was markedly affected by the pH of the last aqueous solution before the transfer into glycerol. No recovery of the lysozyme activity was observed when the refolding/reoxidation reaction was carried out in the denaturing solvent dimethyl sulfoxide. This study paves the way for a systematic investigation of the solvent effect on protein folding and demonstrates that water is not a unique milieu for this process.