A fluorescence approach to the unfolding thermodynamics of horseradish peroxidase based on heme degradation by hydrogen peroxide

Highlights: • This work proposed a new approach to assessment of unfolding of HRP. • Unfolding of HRP could be probed by emission of degraded heme induced by H₂O₂. • The unfolding parameters of HRP can be quantitatively assessed by the new method. Horseradish peroxidase (HRP) is a classical heme-containing protein which has been applied in many fields. The prosthetic group heme in HRP, especially in unfolded state, can react with hydrogen peroxide (H₂O₂) to produce a fluorescent product with the maximum emission wavelength at 450 nm. Utilizing this emission band as a fluorescence probe, the unfolding process of HRP in urea can be assessed quantitatively, and the calculated thermodynamic parameters are consistent with those determined by circular dichroism (CD) at 222 nm and steady-state tryptophan (Trp) fluorescence methods.