Effects of high hydrostatic pressure on Rhizopus chinensis lipase: II. Intermediate states during unfolding

High hydrostatic pressure (HHP) is currently considered a well-established technology for processing food and biological materials and there is an interest in investigating the changes in the structural and functional properties of these materials after high pressure treatment. Therefore, the changes in the structure of Rhizopus chinensis lipase (RCL) after high hydrostatic pressure treatment were investigated. Far-UV circular dichroism (CD) spectra showed that the secondary structure of RCL is maintained at pressures below 400 MPa and becomes gradually disordered after higher pressures are applied. Near-UV CD spectra showed that the RCL begins to lose its tertiary structure at pressure over 400 MPa. Fluorescence quenching and the binding of 1-anilinonaphthalene-8-sulfonate confirmed that a partially unfolded intermediate, with loosely compacted conformation and hydrophobic regions, is formed at a pressure of 600 MPa. These results also suggest that RCL maintains a native like state at pressures below 400 MPa. Above 500 MPa RCL molecules showed characteristics of being in a molten globule state. Dynamic light scattering (DLS) and atomic force microscopy (AFM) measurements indicated that RCL molecules at these pressures are aggregating. The addition of (NH4)(2)SO4 to the protein solution could prevent the aggregation, and at 600 MPa the molecule had a hydrodynamic radius approximately 8% larger than that observed for the control sample, which was regarded as being in the molten globule state. The observations suggest that at increasing pressures, the unfolding mechanism of RCL follows well-defined steps from a native state via a native-like structure ending in molten globular state or molecular aggregation.