Oxidation Of Chlorogenic Acid Research Articles

Cysteine Inhibition of Enzymatic Blackening with Polyphenol Oxidase from Potatoes

SUMMARYThe possible sites of cysteine inhibition of enzymatic blackening using polyphenol oxidase from potatoes were studied. The initial site of cysteine inhibition of enzymatic blackening caused by tyrosine oxidation occurred at the oxidation of tyrosine to 3,4‐dihydroxyphenylalanine (dopa). Following the initial induction period, the oxygen uptake paralleled those treatments with no cysteine. Concentrations of cysteine (9.5 × 10‐3M) which inhibited tyrosine oxidation for 100 min did not inhibit dopa oxidation significantly. However, higher concentrations (1.9 × 10‐2M) inhibited dopa oxidation. Cysteine did not inhibit chlorogenic acid oxidation under the conditions of our study. Oxygen uptake with chlorogenic acid plus cysteine was higher than in the absence of cysteine. Cysteine concentrations which effectively inhibited tyrosine oxidation did not inhibit oxidation in the presence of tyrosine plus chlorogenic acid.

Biochemical characteristics of tobacco leaves during flue-curing

During normal flue-curing of ripe tobacco leaves, polyphenol oxidase and peroxidase activities rose rapidly during the first few days of curing, and then decreased sharply so that in the fully cured leaf no polyphenol oxidase activity and very little peroxidase activity remained. The ascorbic acid content decreased steadily during the cure, leading to a transient rise in dehydro-ascorbic acid, although very little of either remained in the cured leaves. The chlorogenic acid content rose during the first one or two days of curing, and later fell slightly. Detached tobacco leaves were fed with water, with ascorbic acid or with chlorogenic acid and flue-cured with either a normal or an extended yellowing period. There was very little browning in normal cures, and the colour of the leaves was found to be unrelated to either the normal or additional levels of ascorbic acid or chlorogenic acid. With extended yellowing, however, there was a greater production of pigment, which was related to the chlorogenic acid content of the leaves but which was unaffected by ascorbic add. There was a great excess of enzyme available for the oxidation of the chlorogenic acid present. Ascorbic add was probably of little or no significance in preventing oxidation of chlorogenic acid, but in both normal and extended cures, the ascorbic acid content was related to the lustre of the leaves.

Substrates and inhibitors of potato tuber phenolase

The substrate specificity of and effect of inhibitors on potato tuber phenolase are described. For a particular inhibitor, K i depends upon whether an o-dipbenol or monophenol is used as substrate, and thus it is proposed that different protein molecules are responsible for the oxidation of o-diphenols and the hydroxylation of monophenols. A study of the substrate specificity of the enzyme suggests that the monophenol hydroxylase activity has no biosynthetic significance and that the main function of the enzyme is to provide a defence against wounding and infection by the oxidation of chlorogenic acid.

Possible interaction of chlorogenic acid, tyrosine, and bisulfite in enzymatic blackening of potatoes

Warburg manometric experiments were performed to study the interaction of crude polyphenol oxidase from potatoes with chlorogenic acid, tyrosine, and sodium bisulfite. Bisulfite concentrations of 5.4×10−4 to 1×10−3M which inhibited tyrosine oxidation for 30–40 min. did not inhibit chlorogenic acid oxidation. When chlorogenic acid, tyrosine, and bisulfite are mixed together the enzymatic oxidation of chlorogenic acid will reduce the bisulfite concentration so that tyrosine oxidation occurs rapidly.

The purification and some properties of the polyphenol oxidase from tea (Camellia sinensis L.)

1. Polyphenol oxidase (EC 1. 10. 3.-) from the shoots of the tea plant was purified about 5000-fold on a dry-weight basis. 2. At an intermediate stage of purification four soluble yellow fractions were obtained. They are believed to represent complexes of a basic enzyme protein with acidic phenolic oxidation products and nucleic acids. After removal of the complex-forming materials the fractions were blue and similar to each other. About 40% of the activity could not be extracted from the acetone-dried powder. 3. Each of the four blue fractions was resolved further into two species, A and B. The following results refer to species A. 4. The enzyme showed absorption maxima at 279mmu (E(1%) (1cm.), 13.5) and 611mmu (E(1%) (1cm.), 0.84) with a shoulder at 330mmu. The enzyme was bleached by substrate under anaerobic conditions and the colour was restored by oxygen. 5. The molecular weight measured by sedimentation and diffusion was 144000+/-16000. The copper content was 0.32% (w/w). 6. Kinetic constants are given for a number of substrates and inhibitors, including the natural substrates of the tea leaf. The specific activity towards pyrogallol was 373 units/mg. at 30 degrees . 7. The best substrates were o-dihydric phenols. Quinol and p-phenylenediamine were slowly oxidized. Monohydric phenols and ascorbic acid were not oxidized. 8. The kinetics of oxidation of most substrates are consistent with a mechanism in which oxidized and reduced forms of the enzyme form binary complexes with phenol and oxygen respectively. A modified mechanism is postulated for the oxidation of chlorogenic acid. 9. The relation of the results to the mechanism of tea fermentation is discussed.

The enzymic oxidation of chlorogenic acid and some reactions of the quinone produced.

1. Partially purified preparations of tobacco-leaf o-diphenol oxidase (o-quinol-oxygen oxidoreductase; EC 1.10.3.1) oxidize chlorogenic acid to brown products, absorbing, on average, 1.6atoms of oxygen/mol. oxidized, and evolving a little carbon dioxide. 2. The effect of benzenesulphinic acid on the oxidation suggests that the first stage is the formation of a quinone; the solution does not go brown, oxygen uptake is restricted to 1 atom/mol. oxidized, and a compound is produced whose composition corresponds to that of a sulphone of the quinone derived from chlorogenic acid. 3. Several other compounds that react with quinones affect the oxidation of chlorogenic acid. The colour of the products formed and the oxygen absorbed in their formation suggest that the quinone formed in the oxidation reacts with these compounds in the same way as do simpler quinones. 4. Some compounds that are often used to prevent the oxidation of polyphenols were tested to see if they act by inhibiting o-diphenol oxidase, by reacting with quinone intermediates, or both. 5. Ascorbate inhibits the enzyme and also reduces the quinone. 6. Potassium ethyl xanthate, diethyldithiocarbamate and cysteine inhibit the enzyme to different extents, and also react with the quinone. The nature of the reaction depends on the relative concentrations of inhibitor and chlorogenic acid. Excess of inhibitor prevents the solution from turning brown and restricts oxygen uptake to 1 atom/mol. of chlorogenic acid oxidized; smaller amounts do not prevent browning and slightly increase oxygen uptake. 7. 2-Mercaptobenzothiazole inhibits the enzyme, and also probably reacts with the quinone; inhibited enzyme is reactivated as if the inhibitor is removed as traces of quinone are produced. 8. Thioglycollate and polyvinylpyrrolidone inhibit the enzyme. Thioglycollate probably reduces the quinone to a small extent.

Soluble browning reaction pigments of aged Burley tobacco—II. : The dialysable fraction

The major phenolics of air-cured tobacco exert a pronounced influence on the color of cured leaf, the maximum effect of which is revealed in the soluble and insoluble brown pigments produced by enzymic oxidation of chlorogenic acid and rutin. The soluble brown pigments may be classified into two fractions: the dialysable and the nondialysable. Brown pigments contained in concentrated dialysate fractions of aqueous extracts were separated by gel filtration on a column of Sephadex G-25. Constituent pigments so obtained were, in the order of decreasing molecular weight, a major dark brown pigment and two minor reddish brown pigments. The major pigment was characterized as a polymericiron—protein—chlorogenic acid complex with a molecular weight estimated to be in the order of 4000. The minor pigments were each a polymeric protein—chlorogenic acid complex. Neither of the minor pigments contained iron. There was no evidence of the involvement of rutin in the compositions of the dialysable pigments, in distinction to that of a soluble nondialysable pigment previously isolated and characterized as an iron—protein—chlorogenic acid—rutin complex with a molecular weight of the order of 20,000–30,000. The amount of dialysable pigments so isolated corresponded to 0·2% of dry leaf weight. Methods of isolation and characterization are described, and some of the more significant biochemical aspects of the pigments are considered.

ELECTROPHORETIC SEPARATION OF THE PHENOLASES FROM POTATO TUBERS.

EARLY investigations of the properties of polyphenol oxidase from plants showed that most preparations catalysed the oxidation of several polyphenolic substrates. In the past few years it has been demonstrated that crude polyphenol oxidase preparations actually are mixtures of several phenol oxidase proteins, each exhibiting unique catalytic and physical properties. Smith and Krueger2 used a column of hydroxyl apatite and chromatographically separated crude extracts of mushrooms into a series of purified fractions. These contained several enzyme types, including the classical catecholase and cresolase enzymes. Sisler and Evans3 showed that chlorogenic acid was a better substrate for the crude polyphenol oxidase from tobacco than for a comparable preparation from mushrooms. The ratios of rates of oxidation of chlorogenic acid to rates of oxidation of catechol as catalysed by each of a series of purified extracts from tobacco varied widely, indicating the presence of more than one type of polyphenol oxidase enzyme. Uritani4 used starch-gel electrophoresis and separated crude extracts of sweet potato roots into three distinct bands each showing a capacity to catalyse the oxidation of chlorogenic acid. From these experiments it seems clear that organisms contain several types of polyphenol oxidase enzymes.

Untersuchungen über die Beteiligung des Sauerstoffs in photosynthetischen Reaktionen mit Hilfe von Hemmstoffen

Hydrogenperoxide formation in the light in isolated chloroplasts is markedly increased upon the addition of KCN, diethyldithiocarbamate and 3-amino-1.2.4-triazol, whereas photophosphorylation is not influenced by these compounds. Catalase activity of chloroplasts is inhibited by KCN, diethyldithiocarbamate and aminotriazol. Since furthermore the oxidation of chlorogenic acid requires light, we assume that the reduced cofactor in oxygen catalyzed photophosphorylation is not reoxidized by phenoloxidase but by a chlorophyll sensitized photoöxidation accompanied by H2O2-formation. In the absence of inhibitors the H2O2 is decomposed by catalase. The influence of dichlorphenyldimethylurea and o-phenanthroline on cyclic photophosphorylation in the absence of oxygen has been investigated. Photophosphorylation catalyzed by Vitamin K or dichlorphenolindophenol is not inhibited provided that the cofactor is in the reduced state at the beginning of the reaction. The finding that indophenols are catalysts of cyclic photophosphorylation without the participation of oxygen explains former results hitherto hard to fit into the known schemes of electrontransport in chloroplasts.

Sulla azione di alcuni chinoni naturali sulla attività glucosio — 6 — P Deidrogenasica

Abstract INHIBITION OF GLUCOSE-6-P DEYDROGENASE BY OXIDATION PRODUCTS OF NATURAL PHENOLS. — The inhibition of Gl-6-P dehydrogenase activity found in highly concentrated homogenates from potato tuber tissue, seems due to natural occurring orto-di-quinones, such those arising from polyphenoloxidase catalized oxidation of chlorogenic acid, caffeic acid, and tyrosine. These compounds strongly inhibit Gl-6-P dehydrogenase activity of the potato tuber homogenate as well as that of purified Gl-6-P dehydrogenase from yeast. The inhibition effect appears much stronger when the diphenols are oxidized in the presence of the Gl-6-P dehydrogenase. Gl-6-P and TPN efficently protect the enzyme from the inhibition. Ascorbic acid prevents the inhibition by mantaining the diphenols in the reduced form, but is ineffective in reversing the inhibition.

A Comparison of Chlorogenic Acid and Catechol as Substrates for the Polyphenol Oxidase from Tobacco and Mushroom.

It is reported that chlorogenic acid is widely distributed in plants (7) and is the principle poly phenol in tobacco leaves (8). When the tissues of the tobacco plant are injured, the polyphenols are oxidized and then polymerize, forming a brown pigment. This oxidation is reported to be catalyzed by polyphenol oxidase. Polyphenol oxidase has been identified in many species of plants and the enzyme from potato (3) and mushroom (2) has been investigated in some detail. In most cases the activity of the enzyme has been assayed by determination of the rate of oxidation of catechol using direct or indirect methods. Dawson et al (1) have presented curves indicating that rates of oxygen uptake in the presence of tobacco leaf extracts were greater when chlorogenic acid was used as a substrate than when either catechol or caffeic acid were utilized. These workers, however, did not indicate the concentrations of catechol or caffeic acid used. During the course of this investigation, it was found that the rate of oxidation of chlorogenic acid in the presence of a crude tobacco root extract was about 5-fold greater than the rate of oxidation of catechol. Conversely, the rate of oxidation of catechol catalyzed by a crude mushroom extract was about 6.5-fold greater than that with chlorogenic acid. The enzymes from both sources have been partially purified to determine whether or not more than one polyphenol oxidase was present in each of the extracts.

The enzymic oxidation of chlorogenic and caffeic acids in the presence of nornicotine

The enzymic oxidation of chlorogenic acid alone and in the presence of nornicotine, nicotine, glycine, and proline under the influence of tobacco polyphenoloxidase has been studied in model experiments. Nornicotine leads to the formation of a characteristic red color during the reaction, whereas nicotine and the two amino acids do not substantially influence the color changes as observed with chlorogenic acid alone. Caffeic acid also produces a red color on enzymic oxidation but only in the presence of nornicotine. No additional oxygen consumption was observed in the presence of nornicotine in comparison with the controls. The chemical nature of the red pigments formed is still obscure and calls for further investigation.