Interference by ascorbic acid in test systems involving peroxidase. II. Redox-coupled indicator systems.

Ascorbic acid hampers some test systems based on use of peroxidase (EC 1.11.1.7) and redox indicators, by producing a lag time in color development. With reversible indicators, no color development occurs during the ascorbic acid lag time. With oxidatively coupled indicator systems, such as 3-methyl-2-benzothiazolinone hydrazone (MBTH) and a suitable coupler such as chromotropic acid (CTA; 4,5-dihydroxynaphthalene-2,7-disulfonic acid), ascorbic acid diminishes the rate of color development, but does not abolish it. The effect of ascorbic acid strongly depends on the reaction pH as well as the nature of the coupler used. The ascorbate-elicited reduction (or lag) in color development was inversely proportional to the concentrations of MBTH and virtually unaffected by changes in CTA coupler concentration. The rate of color development following the lag was directly proportional to the concentration of MBTH but unaffected by the CTA. These observations suggest that peroxidase with H2O2 catalyzes the oxidation and activation of MBTH to an oxidized species (MBTHox). This species is reduced by ascorbic acid and at the same time couples oxidatively with CTA. Thus, the activity during the ascorbate-induced lag time reflects this competition of ascorbic acid and coupler for MBTHox. This study of peroxidase/ascorbate lag time with the redox coupled indicator system has led to the selection of fast couplers that are highly resistant to interference by ascorbic acid. Suitable resistant couplers (e.g., chromotropic acid, Chicago acid, and H acid) appear to be aromatic ring systems with highly activating substituents and directing toward electrophilic aromatic substitution at the ortho and para positions.