Study on the Characteristics of \(\alpha\)-Chymotrypsin Folding Intermediates by Hydrophobic Interaction Chromatography (HIC)

Abstract

The investigation of protein folding intermediates is of great significance for exploring the folding mechanism of denatured proteins and improving the folding efficiency of protein. In the present research, by taking some of linear parameters of stoichiometric displacement theory of retention of solute (SDT-R) of hydrophobic interaction chromatography (HIC), a new approach to characterize the intermediate of urea-denatured \(\alpha\)-chymotrypsin ( \(\alpha\)-Chy) was developed. The contact surface region (Z, S), affinity (logI), and the character of interaction force (j) of the \(\alpha\)-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 (C_urea) changes. With the changes in C_urea, a linear relationship between logI  and Z was found to exist only for its N state, rather than for M state, indicating the interaction force between \(\alpha\)-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.