title: Refolding of urea-denatured α-chymotrypsin by protein-folding liquid chromatography.
abstract: 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.
authors.affiliation: College of Chemistry & Chemical Engineering, Xi' An ShiYou University, Xi'an, China.
authors.forename: Ke
authors.initials: K
authors.lastname: Congyu
authors.forename: Sun
authors.initials: S
authors.lastname: Wujuan
authors.forename: Zhang
authors.initials: Z
authors.lastname: Qunzheng
authors.forename: Geng
authors.initials: G
authors.lastname: Xindu
mesh_terms: D002853:Chromatography, Liquid; D002918:Chymotrypsin / Q000737:chemistry*; D057927:Hydrophobic and Hydrophilic Interactions; D011489:Protein Denaturation; D058849:Protein Refolding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014508:Urea / Q000737:chemistry*
chemical_list: D014508:Urea; D002918:Chymotrypsin; C117608:alpha-chymotrypsin