Effect of Ascorbic Acid, Sodium Bisulfite, and Thiol Compounds on Mushroom Polyphenol Oxidase

The effect of ascorbic acid, sodium bisulfite, glutathione (reduced), and dithiothreitol on the observed activity of mushroom polyphenol oxidase (PPO) was determined by spectrophotometry (color formation) and by polarography (O₂ uptake). By polarography, O₂ uptake began immediately on adding enzyme. Ascorbic acid and sodium bisulfite had little effect on the initial velocity while dithiothreitol and glutathione decreased the initial velocity by 35%, with no further decrease at higher concentrations. By spectrophotometry, there was an initial lag in the absorbance change followed by a slower increase in absorbance than for the control at zero time. By spectrophotometry, the I₅₀ values were as follows: dithiothreitol, 0.06 mM; glutathione, 0.17 mM; sodium bisulfite, 0.20 mM; ascorbic acid, 0.24 mM. The direct effect of the reductants was determined by (1) incubation of the reductant with PPO and then measuring the remaining activity by polarography and (2) gel electrophoresis. At 0.1 mM, dithiothreitol caused a complete loss of activity after 70 min at 25 °C (t_1/2 = 8 min) while sodium bisulfite, glutathione, and ascorbic acid caused 50% inactivation after 28, 106, and 130 min, respectively, at 5 mM.