Zymographic approach to determine the intrinsic enzyme specific activity of diamine oxidase in presence of interfering enzymes

In vitro determination of diamine oxidase activity in food matrices by an enzymatic assay coupled to UHPLC-FL

“…Among the range of methodologies described in the literature that challenge the determination of DAO activity, the majority are based on the measurement of the liberation of hydrogen peroxide or the consumption of oxygen occurring along the oxidative deamination reaction [13,21,[24][25][26][27][28][29][30]. Those largely used approaches face an important drawback, as the presence of hydrogen peroxide and dioxygen may be markedly influenced by the concomitant presence of other enzymatic capacities in certain complex biological matrices [34,35]. This is the case of catalase, an enzyme commonly found in plant and animal tissues, which can lead to the underestimation of DAO activity by consuming H2O2 and releasing O2 [34,35].…”

“…Those largely used approaches face an important drawback, as the presence of hydrogen peroxide and dioxygen may be markedly influenced by the concomitant presence of other enzymatic capacities in certain complex biological matrices [34,35]. This is the case of catalase, an enzyme commonly found in plant and animal tissues, which can lead to the underestimation of DAO activity by consuming H2O2 and releasing O2 [34,35]. Therefore, the frequent occurrence of catalase in DAO-positive matrices makes those techniques unadvisable due to major interference effect.…”

“…Therefore, the frequent occurrence of catalase in DAO-positive matrices makes those techniques unadvisable due to major interference effect. In this sense, Ahmadifar et al [35] have recently proposed a zymographic approach consisting in an electrophoretic separation of DAO enzyme followed by its densiometric image analysis capable to evaluate the DAO activity of a sample in the presence of interfering catalase.…”

“…Despite some of these methods may be advantageous in terms of rapidity or automation, they generally have a limited sensitivity, require a laborious experimental set-up or entail a high cost in the correct storage and handling of radioactive waste. Moreover, in those methods in which the DAO activity is estimated through the determination of hydrogen peroxide or dioxygen, the action of other enzymes, such as catalase, may interfere by H2O2 consuming or O2 releasing [34,35]. Additionally, the most extensively used reaction substrates in the methods reported so far are putrescine and cadaverine, which have different affinity or kinetic parameters to histamine [36,37].…”