Inhibitors in Commercially Available 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonate) Affect Enzymatic Assays

ABTS, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate), is a common chromogenic substrate for peroxidase enzymes, which are widely used in biochemical research and diagnostic tests. We discovered that impurities in the commercially available ABTS significantly affect the results of peroxidase activity assays. We show that the impurities inhibit the activity of the peroxidases and the influence varies for different batches of ABTS from the same source. The inhibition of horseradish peroxidase (HRP) is uncompetitive for the substrate H₂O₂ while it is competitive for the substrate ABTS. By using high-resolution mass spectrometry, potential inhibitors were identified to be precursors or analogs of ABTS. The inhibitors are also capable of inhibiting the GOx-catalyzed reduction of the ABTS radical cation by glucose in anaerobic conditions. As the inhibition is found to be pH-dependent, diagnostic applications, such as ELISA tests based on the peroxidase-H₂O₂-ABTS system, should be carried out at pH 4.4 to minimize the inhibitory effect of potentially present impurities.