Arnao M B, Cano A, Hernández-Ruiz J, García-Cánovas F, Acosta M
Departamento de Biología Vegetal, Facultad de Biología, Universidad de Murcia, Spain.
Anal Biochem. 1996 May 1;236(2):255-61. doi: 10.1006/abio.1996.0164.
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.
在反应介质中，L-抗坏血酸的存在可抑制过氧化物酶催化的2,2'-联氮-双(3-乙基苯并噻唑啉-6-磺酸)(ABTS)自由基的积累，这种抑制作用会延迟ABTS自由基的积累并产生一个滞后时间。本文提出了一种解释该滞后时间的动力学方法，该方法还能够测定反应介质中L-抗坏血酸的含量。该体系的化学计量关系确定为1摩尔L-抗坏血酸还原2摩尔ABTS自由基。L-抗坏血酸并非唯一具有这种能力的化合物，因为其他抗氧化化合物也能与ABTS自由基发生反应。我们研究了在L-抗坏血酸和其他抗氧化化合物存在下的ABTS/H₂O₂/辣根过氧化物酶体系。研究了pH、酶浓度和L-抗坏血酸浓度等因素的影响。观察到滞后时间与介质中存在的L-抗坏血酸之间具有良好的相关性，在最佳条件下，该方法能够测定低至0.65纳摩尔的L-抗坏血酸。基于我们的研究结果，我们提出了一种测量与L-抗坏血酸相关的不同化合物总抗氧化活性的方法，并将该方法应用于测定果汁中存在的总抗氧化活性。
