Monitoring the heme iron state in horseradish peroxidase to detect ultratrace amounts of hydrogen peroxide in alcohols

Abstract

Despite the importance of hydrogen peroxide (H₂O₂) in initiating oxidative damage and its connection to various diseases, the detection of low concentrations of H₂O₂ (<10 μM) is still limited using current methods, particularly in non-aqueous systems. One of the most common methods is based on examining the color change of a reducing substrate upon oxidation using UV/Vis spectrophotometry, fluorophotometry and/or paper test strips. In this study, we show that this method encounters low efficiency and sensitivity for detection of ultratrace amounts of H₂O₂ in non-aqueous media. Thus, we have developed a simple, fast, accurate and inexpensive method based on UV/Vis spectrophotometry to detect H₂O₂ in non-aqueous systems, such as alcohols. In this regard, we demonstrate that monitoring the Soret and Q-band regions of high-valent iron-oxo (ferryl heme) intermediates in horseradish peroxidase (HRP) is well suited to detect ultratrace amounts of H₂O₂ impurities in alcohols in the range of 0.001–1000 μM using UV/Vis spectrophotometry. We monitor the optical spectra of HRP solution for the red shift in the Soret and Q-band regions upon the addition of alcohols with H₂O₂ impurity. We also monitor the reversibility of this shift to the original wavelength over time to check the spontaneous decay of ferryl intermediates to the ferric state. Thus, we have found that the ferryl intermediates of HRP can be used for the detection of H₂O₂ in alcohols at μg L⁻¹ levels through via UV/Vis spectrophotometric method.