Inhibition by L-ascorbic acid and other antioxidants of the 2.2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) oxidation catalyzed by peroxidase: a new approach for determining total antioxidant status of foods

Anal Biochem. 1996 May 1;236(2):255-61. doi: 10.1006/abio.1996.0164.

Abstract

The accumulation of 2.2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical catalyzed by peroxidase can be inhibited by the presence of L-ascorbic acid in the reaction medium, this inhibition delaying the accumulation of the ABTS radical and giving rise to a lag time. A kinetic approach to explain this lag time is presented, which also makes it possible to determine the amount of L-ascorbic acid in the reaction medium. The stoichiometry of the system was determined as 1 mol of L-ascorbic reducing 2 mol of ABTS radicals. L-Ascorbic acid is not the only compound to have this ability, since other antioxidant compounds also react with the ABTS radical. We studied the ABTS/H2O2/horseradish peroxidase system in the presence of L-ascorbic acid and other antioxidant compounds. The influence of such factors as pH, enzyme concentration, and L-ascorbic acid concentration was studied. A good correlation between the lag time and the L-ascorbic acid present in the medium was observed, and under optimal conditions, the method could determine as little as 0.65 nmol of L-ascorbic acid. Based on our findings, we propose a method to measure the total antioxidant activity of different compounds related to L-ascorbic acid and apply this method to determining the total antioxidant activity present in fruit juices.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antioxidants / pharmacology*
  • Ascorbic Acid / pharmacology*
  • Benzothiazoles
  • Calibration
  • Catalysis
  • Enzyme Inhibitors / pharmacology*
  • Food Analysis / methods*
  • Free Radicals
  • Horseradish Peroxidase / antagonists & inhibitors*
  • Hydrogen-Ion Concentration
  • Kinetics
  • Metmyoglobin
  • Oxidation-Reduction
  • Spectrophotometry
  • Sulfonic Acids / chemistry*

Substances

  • Antioxidants
  • Benzothiazoles
  • Enzyme Inhibitors
  • Free Radicals
  • Sulfonic Acids
  • Metmyoglobin
  • 2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid
  • Horseradish Peroxidase
  • Ascorbic Acid