An unexpected side reaction in the guaiacol assay for peroxidase

In routine guaiacol assays for thyroid peroxidase and lactoperoxidase employing a newly purchased bottle of guaiacol from Aldrich Chemical Co., we were surprised to find the formation of a blue color instead of the expected amber color classically associated with this assay. This was observed also with horseradish, myelo-, and cytochrome c peroxidase. The blue color (Amax approximately 650 nm) was not formed with guaiacol reagents obtained from two other chemical companies, nor was it seen with a bottle of old Aldrich guaiacol that had been in use in the laboratory for more than 10 years. In the present investigation we provide evidence that formation of the blue color is closely associated with the presence of a low concentration of catechol (approximately 0.5 mol%) in the new Aldrich guaiacol reagent. Catechol itself, even in much higher concentration, is a very weak donor for peroxidase, forming a light pink color. The blue color in Aldrich new guaiacol is not formed to the exclusion of 470-nm-absorbing product(s). Formation of the latter is, however, inhibited, and use of Aldrich new guaiacol for assay leads to low values for peroxidase activity. Other dihydroxyphenols (resorcinol and hydroquinone) do not mimic the action of catechol in formation of the blue color. Resorcinol is a very potent inhibitor of peroxidation of guaiacol. Possible schemes are proposed for formation of the products that may be associated with the amber and blue colors.