A New Approach for Characterizing the Intermediate Feature of α-Chymotrypsin Refolding by Hydrophobic Interaction Chromatography

• Main Authors: Zhenling Liu, Jianjun Li, Congyu Ke, Xindu Geng
• Format: Article
• Language: English
• Published: MDPI AG 2009-02-01
• Series: International Journal of Molecular Sciences
• Subjects: Protein folding; protein drugs; misfolding; intermediates; protein folding liquid chromatography; characterization; hydrophobic interaction chromatography; α-Chymotrypsin; stoichiometric displacement theory
• Online Access: http://www.mdpi.com/1422-0067/10/2/616/

A new approach for characterizing the intermediate of urea-denatured α-chymotrypsin (α-Chy) by hydrophobic interaction chromatography (HIC) is presented. The contact surface region (Z, S), affinity (logI), and the character of interaction force (j) of the α-Chy to the stationary phase of HIC (STHIC) between the intermediate (M) and native (N) states were found to be quite different as urea concentration (Curea) changes. With the changes in Curea, a linear relationship between logI and Z was found to exist only for its N state, not for M state, indicating the interaction force between α-Chy in N state to the STHIC to be non-selective, but selective one for its M state. Also, the measured magnitude of both logI and Z in M state is only a fifth of that in N state. All three parameters were employed to distinguish protein in the N state from that in the M state. It would be expected that this result could be employed to distinguish any kind of non-functional protein having correct three-, or four-dimensional molecular structure from their stable M state of any kinds of proteins, and/or other proteins in proteome investigation, separation process of protein, and intensively understanding the intrinsic rule of protein folding in molecular biology.