Thermosonication inactivation of horseradish peroxidase with different frequency modes: Effect on activity, structure, morphology and mechanisms

This study aimed to investigate the effect of thermosonicaion (TS) with different frequency modes on the horseradish peroxidase (HRP) inactivation from the aspects of ultrasound field and enzyme structural changes. The relationships between the activity and structural modification were also evaluated. A thermal-only treatment was performed to estimate the action of ultrasound in TS treatment. Compared to mono-frequency, especially the 33 kHz, TS with dual- and tri-frequency modes showed an intensive inactivation impact on HRP. Exogenous hydroxyl radical test showed the hydroxyl radical played a vital role in HRP inactivation. Changes in hematin group, tertiary and secondary structures were also confirmed, and the inactivation process followed an all-or-none model. Morphological images indicated the aggregation and deformation of HRP molecules during TS treatment, and roughness values were changed accordingly. Pearson correlation analysis implied that the structural destruction on the spatial conformation of HRP was strongly related to its activity.