Improved Pyrogallol Autoxidation Method: A Reliable and Cheap Superoxide-Scavenging Assay Suitable for All Antioxidants

The original pyrogallol (1,2,3-trihydroxybenzene) method, which was developed specifically for superoxide dismutase, is now widely used for measuring superoxide-scavenging of other antioxidants. However, the strong pH effect has been ignored. In this study, the influencing factors have been systematically investigated for the first time, and a number of experiments have proved that the pH is of major importance. As major antioxidants contain carboxylic acid, ester, or lactone groups, pH 8.2 should be modified to physiological pH 7.4. The improved procedure is as follows. A pyrogallol solution (in 1 M HCl) is thoroughly mixed with pH 7.4 Tris-HCl buffer; A(325 nm) is measured every 30 s for 5 min at 37 °C. As the ΔA(325 nm, control) value reflects the initial concentration of substrate (•)O(2)(-), it should be well controlled to guarantee the accuracy of the method. The improved pyrogallol method is a reliable and cheap superoxide-scavenging assay suitable for all types of antioxidants.