ascorbic-acid and cupric-acetate

The catalytic system Cu(AcO)2-pyridine 1:4 mol% in methanol, slowly catalyses the air oxidation of ascorbic acid to the 2-methyl hemi-ketal of dehydroascorbic acid 5, and hydrogen peroxide. However, with Cu(AcO)2-pyridine 3:4 mol% the air oxidation is quite fast and no hydrogen peroxide is present at the end of the reaction. Removal of the catalyst and refluxing the foamy 5 in MeCN gives the oxidized, dimeric, dehydroascorbic acid in very good yields (approximately 70%) contaminated by approximately 1-2% MeCN.

Topics: Ascorbic Acid; Catalysis; Crystallization; Dehydroascorbic Acid; Dimerization; Molecular Structure; Organometallic Compounds; Oxidation-Reduction; Pyridines

Incubation of papain (EC 3.4.22.2) with ascorbic acid (AsA) and Cu2+ in acetate buffer (pH 5.6) results in an irreversible loss of enzyme activity by site-specific generation of free radicals [H. Kanazawa, S. Fujimoto, A. Ohara, Biol. Pharm.Bull., 16, 11 (1993)]. In this study, the effect of some compounds, known free radical scavengers, on the relationship between the inactivation of papain by the Cu(2+)-AsA system and the oxidation of AsA was investigated. Catalase completely protected the enzyme from inactivation by the Cu(2+)-AsA system, although hydrogen peroxide (H2O2) by itself, known to be generated during the autoxidation of AsA, did not inactivate the enzyme. The oxidation of AsA was unaffected by catalase. Both thiourea and sodium thiocyanate completely protected the enzyme from inactivation, while AsA was partially oxidized only in the initial stage. In the presence of potassium iodide, both the inactivation of the enzyme and the oxidation of AsA were characterized by a rapid initial phase followed by a stable phase where no reaction took place and, subsequently, a slower phase. Histidine partially prevented the inactivation of the enzyme and the oxidation of AsA. The present results suggest that H2O2 serves as a source of secondary, highly reactive species, probably hydroxyl radicals, which are responsible for the inactivation, and that the protection from inactivation by some radical scavengers, such as thiourea, sodium thiocyanate, potassium iodide, and histidine, is based on the removal of metal ions (Cu2+ or Cu+) at the specific site of inactivation.

Topics: Ascorbic Acid; Benzoylarginine Nitroanilide; Catalase; Free Radical Scavengers; Histidine; Hydrogen Peroxide; Organometallic Compounds; Oxidation-Reduction; Papain; Potassium Iodide; Reactive Oxygen Species; Spectrophotometry, Ultraviolet; Substrate Specificity; Thiocyanates; Thiourea

The mechanism of inactivation of papain (EC 3.4.22.2) by ascorbic acid (AsA) in the presence of cupric ions (Cu2+) was investigated. The aerobic combination of Cu2+ and AsA resulted in an irreversible loss of enzyme activity. The inactivation was found to be an apparent first order reaction. The prior mixing of Cu2+ and AsA caused the complete disappearance of the inactivation. The addition of iron ions led to significant suppression against the inactivation. Cu2+ was bound to the enzyme in a molar ratio of 1:1. At lower concentrations of Cu2+ (molar ratio of enzyme to Cu2+ of 1: < 1), the extent of inactivation showed the same dependence against the extent of oxidation of AsA. The rate of inactivation increased as the concentration of AsA was increased. Saturation kinetics were observed with respect to the concentration of AsA. Changes in the concentration of Cu2+ had no effect on the dissociation constant of the enzyme-AsA complex (KI), though the rate constant of inactivation (k2) showed a linear relationship with the concentration of Cu2+. At various pH values tested, no change of k2 was found, whereas the value of KI increased when the pH bacame lower. At higher concentrations of Cu2+, the rate of inactivation fell beyond a certain concentration of AsA. The present results suggest that both Cu2+ and AsA bind to the enzyme to form a ternary complex and that free radicals are site-specifically formed and react preferentially with the enzyme, at the site of their formation, impairing its activity.

Topics: Ascorbic Acid; Copper; Free Radicals; Iron; Kinetics; Organometallic Compounds; Oxidation-Reduction; Papain; Spectrophotometry, Ultraviolet

Rats, sheep, and normal humans displayed a comparable sensitivity to copper acetate (3 mM)-induced changes in reduced glutathione (GSH) levels in vitro. However, the human erythrocytes were more sensitive than either animal to methemoglobin (METHB) formation with the rat being least sensitive. Ascorbic acid incubation markedly enhanced the occurrence of copper acetate-induced increases in METHB and decreases in GSH in the sheep and humans. However, ascorbic acid incubation reduced the occurrence of copper acetate-induced increases in METHB, while not effecting changes in GSH in rats.

Topics: Animals; Ascorbic Acid; Copper; Drug Interactions; Erythrocytes; Female; Glutathione; Humans; In Vitro Techniques; Male; Methemoglobin; Organometallic Compounds; Oxidation-Reduction; Rats; Rats, Inbred Strains; Sheep; Species Specificity

Topics: Ascorbic Acid; Copper; Erythrocytes; Humans; In Vitro Techniques; Methemoglobin; Organometallic Compounds; Oxidation-Reduction