Ppo Analysis

Qualitative and quantitative colorimetric tests and man-ometric determinations have been used to detect and measure enzyme activity. Because PPOs use molecular oxygen to oxidize phenolic substrates, their activity can be determined by measuring the rate of substrate disappearance or the rate of product formation. When based on the rate of substrate disappearance, oxygen absorption is usually measured either manometrically in a Warburg respiro-meter or polarographically with an oxygen electrode. The most popular method of assay is to follow the initial rate of formation of the quinone spectrophotometrically by measuring the optical density. Care must be taken to restrict measurement to the initial phase of the oxidation, because the reaction soon slows down. Some researchers object to the use of spectrophotometric analysis because it measures the secondary reaction products of PPO, and the secondary reactions are influenced by many factors difficult to control. Thus the presence of ascorbic acid creates a lag phase and lowers the values obtained, whereas autooxidation products of polyphenols may increase the levels of enzyme activity. It is important to note that the reaction changes with time and temperature and depends on the substrate type and concentration, on the pH of the reaction mixture, and on the buffer used (22,23). A wide variety of substrates can be used with spectrophotometric methods: catechol (24), pyrogallol (25), or chlorogenic acid (26). It has to be taken into account that the reaction products formed from the oxidation of various phenols have absorption maxima at different wavelengths, that the substrate may undergo autooxidation, and that an excess of some substrates causes strong inhibition of the enzyme (26). Other methods for PPO activity are based on disappearance of ascorbic acid, a reaction that is directly proportional to enzyme activity (27) and spectrophotometric methods using Bes-thorn's hydrazone (28), or 2-nitro-5-thiobenzoic acid (29).

It is important to differentiate between catechol oxidase and lacease on the one hand and peroxidase on the other. Because peroxidative oxidation of phenols is often mistaken for PPO or lacease, peroxides should be removed from the reaction mixture by addition of catalase and alcohol to prevent any oxidation of phenols (30). For the determination of laccase, syringaldehyde (31) and 2,6-dimethoxyphenol (32) have been used as substrates of laccase. The PPO and laccase activities can be differentiated by the use of cinnamic acid derivatives to inhibit PPO and cationic detergents to inhibit laccase (33).

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