Refolding of urea‐denatured α ‐chymotrypsin by protein‐folding liquid chromatography

An approach for re‐folding denatured proteins during proteome research by protein folding liquid chromatography (PFLC) is presented. Standard protein, α ‐chymotrypsin ( α ‐Chy), was selected as a model protein and hydrophobic interaction chromatography was performed as a typical PFLC; the three different α ‐Chy states – urea‐denatured (U state), its folded intermediates (M state) and nature state (N state) – were studied during protein folding. Based on the test by matrix‐assisted laser desorption/ionization time of flight mass spectrometry and bioactivity, only one stable M state of the α ‐Chy was identified and then it was prepared for further investigation. The specific bioactivity of the refolded α ‐Chy was found to be higher than that of commercial α ‐Chy as the urea concentration in the sample solution ranged from 1.0 to 3.0  m ; the highest specific bioactivity at urea concentration was 1.0  m , indicating the possibility for re‐folding some proteins that have partially or completely lost their bioactivity, as a dilute urea solution was employed for dissolving the sample. The experiment showed that the peak height of its M state increased with increasing urea concentration, and correspondingly decreased in the amount of the refolded α ‐Chy. When the urea concentration reached 6.0  m , the unfolded α ‐Chy could not be refolded at all. Copyright © 2012 John Wiley & Sons, Ltd.