Varda Kahn

Inhibition of mushroom tyrosinase by tropolone

Abstract Tropolone inhibits both mono- and o-dihydroxyphenolase activity of mushroom tyrosinase. Most of the inhibition exerted by tropolone was reversed by dialysis or by excess CU2+. The data indicate that tropolone and o-dihydroxyphenols compete for binding to the copper at the active site of the enzyme. Comparison between the effectiveness of various copper chelators showed that tropolone is one of the most potent inhibitors of mushroom tyrosinase; 50% inhibition was observed with 0.4 × 10−6 M tropolone.

Catechol oxidase from green olives: Properties and partial purification

Abstract Catechol oxidase was extracted from an acetone powder prepared from green olive. The enzyme was purified 240-fold by ammonium sulphate fractionation followed by ion exchange chromatography and gel filtration. The enzyme was characterized by substrate specificity and response to inhibitors. Between 7 and 9 bands having catechol oxidase activity could be detected by gel electrophoresis and electrofocusing. The purified enzyme had an estimated MW of 42 000. The enzyme was strongly inhibited by diethyldithiocarbamate. Inhibition by chloride was strongly dependent on pH. The enzyme did not oxidise monophenols.

Tropolone—a compound that can aid in differentiating between tyrosinase and peroxidase

Abstract In studies dealing with melanogenesis in mammalian tissues, ultrastructural localization of enzymes, identification of subcellular organelles, differentiation and lignification in plant tissues, it is important to have means to differentiate between tyrosinase and peroxidase activities. For a variety of reasons, established criteria used for this purpose are not always reliable. We suggest that tropolone can aid in differentiating between tyrosinase and peroxidase activities since: (a) it is a very effective inhibitor of tyrosinase; (b) in the presence of hydrogen peroxide it can serve as a substrate for peroxidase; (c) at concentrations that inhibit tyrosinase, it does not inhibit peroxidase activity; and (d) it inhibits tyrosinase activity even in the presence of hydrogen peroxide and peroxidase. In a system containing a mixture of tyrosinase and peroxidase, tropolone can differentiate reliably between peroxidase and monohydroxyphenolase or o-dihydroxyphenolase activities of tyrosinase. Moreover, tropolone can differentiate reliably between peroxidase and tyrosinase activities using slices or crude dialysed extracts of various plant tissues.

Polyphenol oxidase isoenzymes in avocado

Abstract Avocado polyphenol oxidase (PPO) was precipitated mainly in the 30–90% saturated ammonium sulfate fraction. The 40–75% saturated ammonium sulfate fraction (the partially purified enzyme) had the highest specific activity in the cultivars Lerman, Horeshim and Fuerte. The PPO was active towards o -dihydroxyphenols. Six active enzymes (a–f) were detected with D,L -DOPA, 4-methylcatechol, catechol, caffeic acid or chlorogenic acid. Band e was the most active in all cases. More isoenzyme bands (fast-moving) were observed with caffeic acid than with 4-methylcatechol. Furthermore, the isoenzyme patterns of the partially purified extracts of the cultivars could be distinguished with respect to caffeic acid.

Relation Between Structure of Polyphenol Oxidase and Prevention of Browning

Polyphenol oxidase (monophenol, dihydroxyphenylalanine:oxygen oxidoreductase, E.C. 1.14.18.1), often called tyrosinase, catalyzes two distinctly different chemical reactions. One reaction is the orthohydroxylation of monophenols; the second reaction is dehydrogenation of orthodihydroxyphenols. These reactions are shown in Equations 1 and 2.

Respiration, ATP level, and sugar accumulation in potato tubers during storage at 4°

Abstract A kinetic study was made of the relationship between respiration rate, sugar content and ATP levels, in fresh and aged potato tubers stored at 4°. The ATP content in tubers rose rapidly immediately after the chilling stress, while respiration rate decreased below the initial rate and sugar accumulation was not detected. After 4 days of storage, the ATP level declined and the sugars started to accumulate. The typical increase in respiration rate that usually follows chilling stress, appeared only in fresh tubers (at about the 6th day of storage). In dinitrophenol-treated tubers, the ATP level remained below the initial level and sugar accumulation was blocked completely. The evidence presented suggests that ATP elevation is not generated by the respiration burst.

Multiple effects of hydrogen peroxide on the activity of avocado polyphenol oxidase

Abstract Hydrogen peroxide (H2O2) affects polyphenol oxidase (PPO) of avocado mesocarp in several ways. In the absence of an exogenous hydrogen donor (AH2), H2O2—at different concentrations (3.6–364 mM)—shortens the lag period of tyrosine hydroxylation and this effect was abolished when catalase was included in the reaction mixture. DOPA oxidation by avocado PPO is slightly increased by a relatively low concentration of H2O2 (3.3–30 mM), while higher concentrations of H2O2 decrease both the rate and the final level of dopachrome formed. Dopachrome and melanins were bleached in the presence of relatively high concentrations of H2O2 (50–500 mM). Preincubation of avocado PPO with H2O2 in the absence of a substrate resulted in the gradual loss of enzymatic activity, with the rate of loss of monophenolase activity being faster than that of o-dihydroxyphenolase activity. The possibility that relatively low in situ concentrations of H2O2 contribute indirectly to the low or high browning potential of avocado mesocarp is discussed.

Tropolone as a substrate for horseradish peroxidase

Abstract Tropolone (2,4,6-cycloheptatrien-1-one), in the presence of hydrogen peroxide but not in its absence, can serve as a donor for the horseradish peroxidase catalysed reaction. The product formed is yellow and is characterized by a new peak at 418 nm. The relationship between the rate of oxidation of tropolone (Δ A at 418 nm/min) and various concentrations of horseradish peroxidase, tropolone and hydrogen peroxide is described. The yellow product obtained by the oxidation of tropolone by horseradish peroxidase in the presence of hydrogen peroxide was purified by chromatography on Sephadex G-10 and its spectral properties at different pHs are presented. The M , of the yellow product was estimated to be ca 500, suggesting that tropolone, in the presence of horseradish peroxidase and hydrogen peroxide is converted to a tetratropolone.

Multiple effect of hydroxylamine on mushroom tyrosinase

Abstract Mushroom tyrosinase is affected by hydroxylamine (NH 2 OH) in several ways. At relatively low concentrations (up to 33 mM) NH 2 OH shortens the lag period of tyrosine hydroxylation. The o -dihydroxyphenolase activity of mushroom tyrosinase is slightly stimulated by short exposure to relatively low concentrations ofNH 2 OH (1.5 mM). Relatively high concentrations ofNH 2 OH (above 20 mM) inhibit the o -dihydroxyphenolase activity of the enzyme and lowers the extent of final pigment production. Preincubation of mushroom tyrosinase with different concentrations ofNH 2 OH for different times results in the inactivation of the enzyme. The rate of inactivation occurred much faster under anaerobic than under aerobic conditions. It was also found that NH 2 OH changes the spectra of o -quinones prepared chemically or of products formed during the oxidation of o -dihydroxyphenols by mushroom tyrosinase. These spectral changes were attributed to the formation of oximes (mono- or dioximes) as a result of an interaction between o -quinones and NH 2 OH. The apparent inhibition exerted by NH 2 OH on the o -dihydroxyphenolase activity of mushroom tyrosinase is, in part, due to spectral changes in pigmented product formation and, in part, due to the inactivation of the enzyme by NH 2 OH.

Tiron as a substrate for mushroom tyrosinase

Abstract Tiron (4,5-dihydroxy-1,3-benzene disulphonic acid) can serve as a substrate for mushroom tyrosinase with a K m value of 34 mM. The product(s) formed is yellow (λ max 435 nm) and is stable with time. An intermediate product, having significant absorbance at 3685 nm (probably tiron- o -quinone), was detected and the kinetics of its conversion to the final yellow product(s) was studied. Hydrogen peroxide (at 0.03–1.3 mM) accelerated the conversion of the intermediate compound to the yellow product(s). Tiron-semiquinone was detected by EPR spectroscopy during the initial phase of Tiron oxidation by mushroom tyrosinase. Maximum EPR signal intensity due to Tiron-semiquinone and the time required to reach maximum intensity were dependent on the amount of mushroom tyrosinase but not on the presence or absence of hydrogen peroxide (3.3 mM). In view of separate studies showing that the yellow product(s) is mainly a low M , polymerized Tiron-quinone, suggestions as to possible pathways by which such a product(s) is formed are discussed.