Adelio Rigo

Formation of α-tocopherol radical and recycling of α-tocopherol by ascorbate during peroxidation of phosphatidylcholine liposomes: An electron paramagnetic resonance study

The events accompanying the inhibitory effect of alpha-tocopherol and/or ascorbate on the peroxidation of soybean L-alpha-phosphatidylcholine liposomes, which are an accepted model of biological membranes, were investigated by electron paramagnetic resonance, optical and polarographic methods. The presence of alpha-tocopherol radical in the concentration range 10(-8)-10(-7) M was detected from its EPR spectrum during the peroxidation of liposomes, catalysed by the Fe3+-triethylenetatramine complex. The alpha-tocopherol radical, generated in the phosphatidylcholine bilayer, is accessible to ascorbic acid, present in the aqueous phase at physiological concentrations. Ascorbic acid regenerates from it the alpha-tocopherol itself. A kinetic rate constant of about 2 X 10(5) M-1 X s-1 was estimated from the reaction as it occurs under the adopted experimental conditions. The scavenging effect of alpha-tocopherol on lipid peroxidation is maintained as long a ascorbic acid is present.

Polarographic determination of superoxide dismutase

Abstract A polarographic procedure is described which allows determination of the catalytic constants for superoxide dismutase-catalyzed reactions. The method presents a single and rapid evaluation of the enzyme concentrations as well as determination of its activity under different conditions; e.g., pH between 9 and 13, presence of urea, guanidine, sodium dodecyl sulphate and inhibitors such as CN − and N 3 − . The results fit very well with data previously obtained with other methods and show that this polarographic procedure can be used under conditions that render the other methods unsuitable for the measurement of the enzyme activity.

An attempt to evaluate the rate of the Haber-Weiss reaction by using OH radical scavengers.

was proposed in 1934 by Haber and Weiss [l] and it was recently suggested by Beauchamp and Fridovich [2] and Peters and Foote [3] to be a source of OH’ in biological systems. This reaction would thus amplify the toxicity of superoxide, because of the high oxidizing power of ‘OH. The value of kl, which would allow one to predict the amount of ‘OH radical formed from O,, is still uncertain. Dainton and Rowbottom [4] gave a value of 3.4 M-’ s-l, while Bray [5], McClune and Fee [6] and Halliwell [7] were unable to show the occurrence of the reaction by various approaches. However a limiting value kl < 10 M-’ s-l can be deduced from their data. This value, although small in comparison to other rate constants of the 0, reactions, is still large enough for reaction (1) to be significant under some conditions. For instance at physiological pH

Competitive inhibition of Cu, Zn superoxide dismutase by monovalent anions.

Abstract Polarographic measurements showed that N3− and halides in hibit the activity of bovine Cu, Zn superoxide dismutase in a competitive fashion, as previously demonstrated for CN− and OH−. All anions increase the spin-lattice nuclear magnetic relaxation time (T1) of aqueous solutions of the enzyme as well, but the stability constants measured from T1 data are lower than those calculated from activity data. The results suggest that substrate and anionic inhibitors bind during the catalytic action at the water coordination position of the enzyme copper, and that these inhibitors may have a greater affinity for the cuprous form of the enzyme which is generated in the catalytic cycle.

Potentiometric detection of formaldehyde in air by an aldehyde dehydrogenase FET

Abstract This paper reports on a system for sampling atmospheric formaldehyde by dissolution in an aqueous solution followed by the monitoring of the aldehyde using an ion-sensitive field-effect transistor (ISFET) in conjunction with an enzyme specific for this pollutant. Formaldehyde dehydrogenase from Pseudomonas putida was chosen on the basis of its characteristics to be coupled to the ISFET transducer. This enzyme, using oxidized nicotinamide adenine dinucleoside (NAD) as cofactor, catalyses the oxidation of a molecule of formaldehyde with the parallel production of two protons, which are sensed by the ISFET. The working conditions were chosen to obtain a linear response of the sensor up to 200 μM formaldehyde. On the basis of the enrichment obtained by the sampling system, the detection limit of 10 μM formaldehyde in aqueous solution, achieved by the ISFET biosensor, corresponds to an atmospheric concentration of the formaldehyde in the ppb range.

Simultaneous determination of superoxide dismutase and catalase in biological materials by polarography

Abstract The polarographic method of catalytic currents applied to a wave of oxygen permits the simultaneous assay of superoxide dismutase and catalase in biological materials with high speed and reproducibility and minimal manipulation of tissues. Washed red blood cells and tissue homogenates give rise to a strong polarographic maximum, apparently due to heme proteins, which interferes with the measurement. This maximum is suppressed by addition of approximately 0.2% plasma. Therefore, the determination of the two enzymes in red blood cells can be carried out by direct addition of whole blood to the polarographic solution. Thirty microliters of blood are enough for optimal determination of both enzymes. The method can determine superoxide dismutase and catalase at concentrations as low as 2 × 10−11 m and 5 × 10−10 m , respectively, and shows a linear correlation between measured activity and enzyme levels. The average values of the two enzymes in human red blood cells was found by this method to be 2.6 × 10−6 m for catalase and 1.8 × 10−6 m for superoxide dismutase, which agree with previously reported values.

A Metabolomic Approach to the Study of Wine Micro-Oxygenation

Wine micro-oxygenation is a globally used treatment and its effects were studied here by analysing by untargeted LC-MS the wine metabolomic fingerprint. Eight different procedural variations, marked by the addition of oxygen (four levels) and iron (two levels) were applied to Sangiovese wine, before and after malolactic fermentation. Data analysis using supervised and unsupervised multivariate methods highlighted some known candidate biomarkers, together with a number of metabolites which had never previously been considered as possible biomarkers for wine micro-oxygenation. Various pigments and tannins were identified among the known candidate biomarkers. Additional new information was obtained suggesting a correlation between oxygen doses and metal contents and changes in the concentration of primary metabolites such as arginine, proline, tryptophan and raffinose, and secondary metabolites such as succinic acid and xanthine. Based on these findings, new hypotheses regarding the formation and reactivity of wine pigment during micro-oxygenation have been proposed. This experiment highlights the feasibility of using unbiased, untargeted metabolomic fingerprinting to improve our understanding of wine chemistry.

Age Dependence of the Level of the Enzymes Involved in the Protection against Active Oxygen Species in the Rat Brain

Abstract Levels of Cu, Zn superoxide dismutase (CuSOD), Mn superoxide dismutase (MnSOD), catalase, and glutathione peroxidase (GPx) were assessed in the rat brain cortex. The concentrations of Cu- and MnSOD were found to increase linearly with the logarithm of the age of the animal from 3 days before birth to 30 months, both in the whole cortex tissue and in its cytoplasmic fraction. Catalase and GPx levels showed different trends; in particular, GPx, which appears to play a key role in detoxification of hydrogen peroxide, after an initial fall increases steadily with age. The enhancement of the levels of SOD and GPx could be related to protection against an increased production of reactive oxygen species in the aging process.

Determination of ascorbic acid with immobilized green zucchini ascorbate oxidase

Ascorbate oxidase from zucchini squash was immobilized onto CH-Sepharose via carbodiimide. The properties of the immobilized enzyme were found to be similar to those of the free ascorbate oxidase. The immobilized enzyme was utilized in a flow-through system equipped with a polarographic detector which monitors the oxygen depletion due to the reaction ascorbic acid + 1/2 O2----dehydroascorbic acid + H2O. This method, the response of which is linear between 3 X 10(-7) and 5 X 10(-4) M ascorbate, was utilized to measure the ascorbic acid in biological samples such as human plasma and fruit juices at a rate of about 60 determinations every hour with a standard deviation lower than 5%.

Effect of ionic strength on the activity of bovine superoxide dismutase.

In studies of activity of superoxide dismutase (SOD), see [l] , two kinds of methods have been used. For routine detection of activity, the inhibition by the enzyme of some 0; dependent reactions has been utilized [2-41. For studies of the catalytic mechanism and when a precise evaluation of rate constants is needed, pulse radiolytic methods have been the only successful ones so far [S--8]. Recently we have applied the polarographic method of the kinetic currents to kinetic studies of the dismutation of O;, which allows the determination of the rate constants by a very simple procedure [9]. Moreover it presents some advantages with respect to pulse radiolysis in so far that the composition of the solution under study can be varied in a way which would disturb straightforward pulse radiolysis experiments. For example, variation of ionic strength and type of ions of the solution, can be somewhat limited in pulse radiolysis, because of the possible production of secondary radicals. In this report we present results on the effect of ionic strength and different species of anions and cations on the activity of SOD as a part a program.