Peroxidase-H2O2 Oxidation of PPDA :Interference by Ascorbic Acid Oxidation of PPDA by HR Per H2O2 results in polymerization to a colored product. Under low
enzyme concentration the rate of color formation is proportional to enzyme concentration and linear with time for at least 5 min (8). Peroxidase in orange juice was assayed by this method (2).
However, substance (s) in the juice interferred with the assay by causing a time lag in formation of the colored product without affecting the rate after the product started to form. Ascorbic acid was shown to be the major and probably the only in terferring substance.
Ascorbic acid also interrupts product formation in the HR Per-H2O2 reaction. It prevents
color development or stops it after the reaction has started. The time lag for color development is proportional to ascorbic acid concentration and inversely related to enzyme activity in the reaction mixture (Figure 1). The proportionality constant
for the lowest enzyme concentration (activity:change of 0.26 Absorbance per min) is equivalent to 1.5 sec per /*g ascorbic acid. These standard curves for ascorbic acid added to orange juice were used to estimate the ascorbic acid concentration of
the original juice. The following values were obtained: 84.4, 82.0, 81.0 and 84.0 fig of ascorbic acid per cuvette, equivalent to 42.2, 41.0, 40.5 and 42.0 mg ascorbic acid per 100 ml original juice. The concentration of ascorbic acid in the juice determined by the 2,6-dichloroindophenol method was 42.0 mg per 100 ml.These results suggested that