Probing the change of enzymatic activity of horseradish peroxidase induced by membrane permeation using tryptophan fluorescence

Changes in the molecular structure of horseradish peroxidase (HRP-4C) induced by membrane ultrafiltration, as well as its impact on the enzymatic activity, were monitored using three complementary fluorescence techniques: steady-state fluorescence, fluorescence anisotropy and picosecond time-resolved fluorescence. Ultrafiltration experiments were performed using membranes of polyethersulfone (PES) with 30 and 100 kDa and membranes of regenerated cellulose (RC) with 10 and 30 kDa. The results obtained clearly shows HRP-4C structural changes during ultrafiltration, which were essentially controlled by hydrophobic and electrostatic protein-membrane interactions. The structural changes observed depend on the affinity of the membrane material to ions, such as Fe³⁺ and Ca²⁺, which have a substantial structural and functional relevance to this enzyme. Additionally, it was found that the impact of the ultrafiltration process on the enzymatic activity was a direct consequence of the depletion of iron from the protein structure, causing irreversible structural alterations on HRP-4C, and thus inducing severe changes in its enzymatic activity.