Polyphenol oxidase (PPO; EC 1.14.18.1) catalyzes the hydroxylation of monophenols to o-diphenols (cresolase activity) and the oxidation of o-diphenols to o-quinones (catecholase activity), leading to browning in plants and produce. Further interest in the enzyme has been triggered by the active role that it plays in plant defense systems. PPO can be found in latent forms and is activated in vitro by various agents including urea, detergents, and proteases. The activation of PPO from several sources by sodium dodecyl sulfate (SDS) has been extensively investigated, but reports on the effect of other detergents or on the differential effect of detergents on each of PPO's activities are scarce. In addition, investigations on the enzyme in other plant parts besides fruits and vegetables are also scarce. Here, the effect of various detergents and chaotropic agents on PPO from dormant saffron (Crocus sativus L.) corm extract was investigated. SDS and sarkosyl activated the cresolase activity, while only SDS activated the catecholase activity. All other detergents tested, in milli- or micromolar concentrations, inhibited the cresolase activity but barely affected the catecholase activity. In contrast, urea and guanidine-HCl drastically inhibited the catecholase activity but moderately inhibited the cresolase activity. The same effects were obtained on the partially purified enzyme. Results identified a PPO, present in dormant corms, which was activated only by anionic detergents and was inhibited by other reputed activating agents such as urea. Results also emphasized the differences in structure and accessibility of the active sites for cresolase and catecholase activities.
Peroxidase (EC 1.11.1.7; donor: hydrogen-peroxide oxidoreductase, POD) is one of the key enzymes controlling plant growth, differentiation and development. The enzyme participates in construction, rigidification and eventual lignification of cell walls, biosynthesis of ethylene from 1-aminocyclopropane-1-carboxylic acid and H 2 O 2 , regulation of auxin level through auxin catabolism, protection of tissue from damage and infection by pathogenic microorganisms, the oxidation of indoleacetic acid. For peroxidase activity in wild pears extract one pH optimum was observed at 6.5 that probably belong to atleast one isoenzyme. Activity of peroxidase in presence of guaiacol and H 2 O 2 was optimum after incubation at 40 °C. Maximum activity of peroxidase is 300 % .Activity increased to 240 %, 300 %, 70 % and 10 % after 60 minute incubation at 30, 40, 45 and 60 °C for peroxidase. Incubation at high temperature (70 °C) was accompanied with decrease of activity to 10 % peroxidase activity.
The interaction of myelin basic protein (MBP) from bovine central nervous system with divalent zinc ion was studied by UV‐Vis titration spectrophotometry and isothermal titration calorimetry techniques at 27°C in Tris buffer solution at pH = 7.2. MBP has one binding site for a zinc ion. The binding of a zinc ion is endothermic (ΔH = + 159 kJ mol−1) with a dissociation binding constant of 0.445 μM. The results obtained by two previous methods of isothermal titration spectrophotometry and calorimetry are similar and consistent with the result obtained from a new calculation method of calorimetric data analysis according to the Scatchard plot.
Polyphenol oxidase (PPO) from hawthorn was extracted and partially purified through (NH4)2SO4 precipitation, dialysis and ion exchange chromatography. The activity of polyphenol oxidase was investigated in Crataegus spp. Spectrophotometric method was used to assay the enzyme activity and the kinetic constants - maximum enzyme velocity (Vmax) and Michealis - Menten constant (Km). Of the substrates tested, catechol was the best substrate for PPO with a Km value of 2.2 mM. The optimum pH for PPO activity was found to be 7. The enzyme showed high activity over a broad pH range of 4 - 8. The optimal pH and temperature for enzyme activity were found to be 7 and 40-45 °C, respectively. km value for hawthorn PPO is calculated 22 mM for catechol and 6.7 mM for pyrogallol and 9.7 mM for L-dopa. As can be seen, affinity of PPOs for various substrates varies widely. The enzyme showed a broad activity over a broad pH and temperature range. The thermal inactivation studies showed that the enzyme is heat resistant. The enzyme showed the highest activity toward pyrogallol and no activity toward tyrosine. Of the inhibitors tested, the most potent inhibitors were kojic acid, cysteine and glycine , respectively
Abstract-Polyphenol oxidase (EC.1.14.18.1) catalyzes the hydroxylation and oxidation of phenols to o-quinones leading to browning in plants. For polyphenol oxidase activity two pH optimum were observed, respectively at 6.7 and 8, that probably belong to at least two isoenzyme, so we named them ISOIPPO and ISOIIPPO respectively. These isoenzymes have different kinetics properties. Activation of PPO by SDS occurred below 1.3 mM SDS. ISOIPPO was much more active (86 times) with pyrogallol as substrate (4.9 unit/mg protein) in comparison of catechol (0.057 unit/mg protein) in presence of SDS. This comparison is 143 times in absence of SDS (3.3 and 0.023 unit/mg protein for pyrogallol and catechol, respectively). SDS activation probably alters both its enzymatic and physical characteristics. ISOIIPPO activity gradually increased during development of tomatoes from green to ripe, but ISOIPPO activity is nearly constant during development. PPO are believed to play physiological roles both in preventing insects and microorganisms from attacking plant and as part of the wound response of plant by its phenolic content, so these roles are more important for ISOIIPPO than ISOIPPO during changes from green to red tomatoes.
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