The OH initiated oxidation of aliphatic hydrocarbons by the simultaneous electrochemical reductio... more The OH initiated oxidation of aliphatic hydrocarbons by the simultaneous electrochemical reduction of 02 and of Fe(III) at controlled potential was investigated in the liquid phase over a Fe(III) concentration range 0.5-5 mM. OH radicals were generated by the reaction: Fe(II) + H202 r-Fe(III) + "OH + OH-The compounds studied were the linear alkane hydrocarbons from Cs to C10 and 3-methyl pentane. The results showed that the ketones are the only reaction products and that the yields decrease with increasing number of carbonium atoms of the hydrocarbon. Decreasing yields were also observed with increasing Fe(III) concentration.
Forty-one apple samples, representing eight of the most widely cultivated varieties in western Eu... more Forty-one apple samples, representing eight of the most widely cultivated varieties in western Europe, were collected in Trentino, Italy. Samples were extracted from fresh fruit with a mixture of acetone/ water to achieve a good extraction of polyphenols, including proanthocyanidin oligomers which were analyzed by normal-phase HPLC. Up to 20 compounds including catechin, epicatechin, B 2 procyanidin, hydroxycinnamates, flavonols, anthocyanins, and dihydrochalcones were analyzed by reversed-phase HPLC and LC-MS. Total polyphenol content was independently measured with an optimized Folin-Ciocalteu assay. The mean content of total polyphenols lay between 66.2 and 211.9 mg/100 g of FW depending on the variety. With chromatographic analysis, it was possible to explain the whole amount of total polyphenols measured by the FC assay. Flavanols (catechin and proanthocyanidins) are the major class of apple polyphenols (71-90%), followed by hydroxycinnamates (4-18%), flavonols (1-11%), dihydrochalcones (2-6%), and in red apples anthocyanins (1-3%).
Accurate ab initio CASSCF, CASFT2, and DFT computations have been performed on three different mo... more Accurate ab initio CASSCF, CASFT2, and DFT computations have been performed on three different model systems which emulate the oxygenated active site of hemocyanin (a Cu+-Cu+ dimer that binds oxygen as peroxide to form oxyhemocyanin). The three models differ in the number of the ammonia molecules (0,4, and 6 molecules, respectively) which emulate the real histidine metal ligands of the protein matrix. While the CASSCF computations indicate that the ground state wave function of the oxyhemocianin active site is in all cases a singlet, the CASPT2 and the DFT approaches provide a significantly different description and suggest that the greater stability of the singlet versus the triplet state (experimentally observed) is not an intrinsic property of the oxygenated form of the hemocyanin active site but depends on the presence of ligands on copper atoms. These results indicate that the dynamic correlation contributions (included in the CASPT2 and DFT methods) are essential to obtain a proper description of these systems that cannot be correctly emutated using models where metal ligands are not included.
Neutrophils are the first barrier against infections. Aged neutrophils display impaired oxidative... more Neutrophils are the first barrier against infections. Aged neutrophils display impaired oxidative burst and phagocytosis with subsequent less capability to destroy bacteria. In successful ageing (nonagenarians), neutrophil efficiency (phagocytosis) increases. After ingested microbes, aged neutrophils are less prone to undergo apoptosis favouring chronic inflammation. Moreover, the superoxide dismutase (SOD) activity, which is necessary in avoiding ROS produced by oxidative burst, is limited in ageing. The mechanisms of age-related changes in neutrophil function are not fully understood, taking also into account that nonagenarians escape infections in comparison with elderly. Zinc pool may be involved because it is pivotal for neutrophil efficiency and SOD activity. Since zinc also controls the inflammation, via IL-6 and soluble factor of gp130 (sgp130), we have assessed the possible interrelationship among oxidative burst, apoptosis, inflammation, SOD, adhesion molecule Mac-1 and zinc pool in elderly and in nonagenarians. The oxidative burst and the capacity to increase Mac-1 after PMA stimulation decrease both in elderly and nonagenarians, but the latter display a slight increased neutrophil induced apoptosis, decreased sgp130, increased SOD, and more neutrophil zinc content, as it occurs in young-adults. Significant correlation exists between sgp130 and zinc pool in very old age. These findings suggest lower chronic inflammation in nonagenarians, via more zinc available, with subsequent long-life survival. Therefore, a more correct interrelationship among neutrophil efficiency, inflammation, antioxidant activity and zinc pool exists in successful ageing with subsequent more effectiveness to control the inflammatory response to pathogens.
Kinetic and spectroscopic studies were carried out to study the role of hydrophobic effect on the... more Kinetic and spectroscopic studies were carried out to study the role of hydrophobic effect on the activity of bovine serum amine oxidase (BSAO). Increasing the chain length of the substrates (linear aliphatic primary monoamines), the affinity for the active site increases while the catalytic constant decreases in accordance with a relative low value of dielectric constant (about 10) estimated for the microenvironment of BSAO active site using a fluorescent probe sensitive to solvent polarity. The aliphatic chain of 1-aminononane induces a shift in the pK(a) of the product Schiff base, the hydrolysis of which appears to be a rate-determining step of the reaction. Furthermore, circular dichroism studies highlighted the "flexibility" of BSAO secondary structure that can explain the wide substrate specificity of this enzyme. These results should be useful to elucidate the substrate/inhibitor preferences of CuAOs, in particular of the human enzyme.
Bovine serum amine oxidase (BSAO) 2 is a copper en-The effect of incubation temperature on the ac... more Bovine serum amine oxidase (BSAO) 2 is a copper en-The effect of incubation temperature on the activity zyme which catalyzes the oxidative deamination of bioof bovine serum amine oxidase was studied using natu-genic spermine and spermidine according to the genral substrates of this enzyme, namely spermine and eral scheme [2] spermidine. The activity behavior was found to be rather complex and different from that observed using benzylamine as substrate. The enzyme is fully active RCH 2 NH 2 / O 2 / H 2 O r BSAO after 3 min incubation at temperatures up to 60ЊC, RCHO / NH 3 / H 2 O 2. [1] while at higher temperatures it shows an S-shaped irreversible decrease of the activity with a T 50 of about 70ЊC. The dependence of the kinetic parameters k cat This enzyme contains an organic cofactor, 6-hydroxyand K m on temperature was also studied in the range dopa (3), characterized by the presence of carbonyl 5-66ЊC, measuring the initial rates of oxidation of spergroups which have been demonstrated to be directly midine. The Arrhenius plot of k cat shows a continuous involved in the formation of a Schiff base. Reaction bend in the thermal stability range of the enzyme. This [1] can be tentatively written as a sequence of partial nonlinearity is due to the dramatic change of k cat actireactions involving the following steps: (a) formation vation entropy (from /11 eu at 8ЊC, to 031 eu at 50ЊC) of a molecular complex between the enzyme and the and indicates that k cat is a composite constant, involvpolyamine, (b) condensation between a terminal amino ing two or more successive steps characterized by difgroup of the polyamine and a carbonyl group of the ferent kinetic parameters. In the case of the ratio k cat / cofactor with the generation of an imine (Schiff base), K m , which for natural substrates of bovine serum (c) isomerization of the imine with the parallel reducamine oxidase was shown that its dependence on ionic tion of the cofactor, (d) hydrolysis of the isomerized strength gives information on the influence of electroimine, and (e) oxidation of the cofactor by molecular static field on k 1 (Stevanato et al. (1994) Biochem. J. oxygen (1, 4). 299, 317-320), where k 1 is the kinetic constant of inter-In a previous paper on the dependence of benzylaction between the polyamine and the enzyme, the Aramine oxidase activity of BSAO on temperature, it has rhenius plot is linear in the range 12-45ЊC, and an activation entropy of 18.8 eu was calculated at 37ЊC. This been found that the enzyme shows a nonstrict Arrhenvalue is in accord with a mechanism involving the for-ius behavior (5). Furthermore, conflicting hypotheses mation of a polyamine-enzyme complex which is acabout the protonation state of the substrate in the first companied by neutralization of charges between the steps of catalytic process have been reported (2). In polycationic substrate and negatively charged groups fact, at physiological pH values, the natural substrates of the enzyme.
A rotating disk electrode coated with a thin mercury film is described. It is suitable for electr... more A rotating disk electrode coated with a thin mercury film is described. It is suitable for electrochemical determinations of the catalytic constant of superoxide dismutase in a much wider pH range than that accessible to polarographic methods. In particular this is the only direct method, except for pulse radiolysis, that allows mechanistic studies of the enzyme in the physiological pH range. Furthermore, the continuous response of this electrode allows enzyme activity changes characterized by half-lives in the range from a few to 10(3) s. to be followed.
The OH initiated oxidation of aliphatic hydrocarbons by the simultaneous electrochemical reductio... more The OH initiated oxidation of aliphatic hydrocarbons by the simultaneous electrochemical reduction of 02 and of Fe(III) at controlled potential was investigated in the liquid phase over a Fe(III) concentration range 0.5-5 mM. OH radicals were generated by the reaction: Fe(II) + H202 r-Fe(III) + "OH + OH-The compounds studied were the linear alkane hydrocarbons from Cs to C10 and 3-methyl pentane. The results showed that the ketones are the only reaction products and that the yields decrease with increasing number of carbonium atoms of the hydrocarbon. Decreasing yields were also observed with increasing Fe(III) concentration.
Forty-one apple samples, representing eight of the most widely cultivated varieties in western Eu... more Forty-one apple samples, representing eight of the most widely cultivated varieties in western Europe, were collected in Trentino, Italy. Samples were extracted from fresh fruit with a mixture of acetone/ water to achieve a good extraction of polyphenols, including proanthocyanidin oligomers which were analyzed by normal-phase HPLC. Up to 20 compounds including catechin, epicatechin, B 2 procyanidin, hydroxycinnamates, flavonols, anthocyanins, and dihydrochalcones were analyzed by reversed-phase HPLC and LC-MS. Total polyphenol content was independently measured with an optimized Folin-Ciocalteu assay. The mean content of total polyphenols lay between 66.2 and 211.9 mg/100 g of FW depending on the variety. With chromatographic analysis, it was possible to explain the whole amount of total polyphenols measured by the FC assay. Flavanols (catechin and proanthocyanidins) are the major class of apple polyphenols (71-90%), followed by hydroxycinnamates (4-18%), flavonols (1-11%), dihydrochalcones (2-6%), and in red apples anthocyanins (1-3%).
Accurate ab initio CASSCF, CASFT2, and DFT computations have been performed on three different mo... more Accurate ab initio CASSCF, CASFT2, and DFT computations have been performed on three different model systems which emulate the oxygenated active site of hemocyanin (a Cu+-Cu+ dimer that binds oxygen as peroxide to form oxyhemocyanin). The three models differ in the number of the ammonia molecules (0,4, and 6 molecules, respectively) which emulate the real histidine metal ligands of the protein matrix. While the CASSCF computations indicate that the ground state wave function of the oxyhemocianin active site is in all cases a singlet, the CASPT2 and the DFT approaches provide a significantly different description and suggest that the greater stability of the singlet versus the triplet state (experimentally observed) is not an intrinsic property of the oxygenated form of the hemocyanin active site but depends on the presence of ligands on copper atoms. These results indicate that the dynamic correlation contributions (included in the CASPT2 and DFT methods) are essential to obtain a proper description of these systems that cannot be correctly emutated using models where metal ligands are not included.
Neutrophils are the first barrier against infections. Aged neutrophils display impaired oxidative... more Neutrophils are the first barrier against infections. Aged neutrophils display impaired oxidative burst and phagocytosis with subsequent less capability to destroy bacteria. In successful ageing (nonagenarians), neutrophil efficiency (phagocytosis) increases. After ingested microbes, aged neutrophils are less prone to undergo apoptosis favouring chronic inflammation. Moreover, the superoxide dismutase (SOD) activity, which is necessary in avoiding ROS produced by oxidative burst, is limited in ageing. The mechanisms of age-related changes in neutrophil function are not fully understood, taking also into account that nonagenarians escape infections in comparison with elderly. Zinc pool may be involved because it is pivotal for neutrophil efficiency and SOD activity. Since zinc also controls the inflammation, via IL-6 and soluble factor of gp130 (sgp130), we have assessed the possible interrelationship among oxidative burst, apoptosis, inflammation, SOD, adhesion molecule Mac-1 and zinc pool in elderly and in nonagenarians. The oxidative burst and the capacity to increase Mac-1 after PMA stimulation decrease both in elderly and nonagenarians, but the latter display a slight increased neutrophil induced apoptosis, decreased sgp130, increased SOD, and more neutrophil zinc content, as it occurs in young-adults. Significant correlation exists between sgp130 and zinc pool in very old age. These findings suggest lower chronic inflammation in nonagenarians, via more zinc available, with subsequent long-life survival. Therefore, a more correct interrelationship among neutrophil efficiency, inflammation, antioxidant activity and zinc pool exists in successful ageing with subsequent more effectiveness to control the inflammatory response to pathogens.
Kinetic and spectroscopic studies were carried out to study the role of hydrophobic effect on the... more Kinetic and spectroscopic studies were carried out to study the role of hydrophobic effect on the activity of bovine serum amine oxidase (BSAO). Increasing the chain length of the substrates (linear aliphatic primary monoamines), the affinity for the active site increases while the catalytic constant decreases in accordance with a relative low value of dielectric constant (about 10) estimated for the microenvironment of BSAO active site using a fluorescent probe sensitive to solvent polarity. The aliphatic chain of 1-aminononane induces a shift in the pK(a) of the product Schiff base, the hydrolysis of which appears to be a rate-determining step of the reaction. Furthermore, circular dichroism studies highlighted the "flexibility" of BSAO secondary structure that can explain the wide substrate specificity of this enzyme. These results should be useful to elucidate the substrate/inhibitor preferences of CuAOs, in particular of the human enzyme.
Bovine serum amine oxidase (BSAO) 2 is a copper en-The effect of incubation temperature on the ac... more Bovine serum amine oxidase (BSAO) 2 is a copper en-The effect of incubation temperature on the activity zyme which catalyzes the oxidative deamination of bioof bovine serum amine oxidase was studied using natu-genic spermine and spermidine according to the genral substrates of this enzyme, namely spermine and eral scheme [2] spermidine. The activity behavior was found to be rather complex and different from that observed using benzylamine as substrate. The enzyme is fully active RCH 2 NH 2 / O 2 / H 2 O r BSAO after 3 min incubation at temperatures up to 60ЊC, RCHO / NH 3 / H 2 O 2. [1] while at higher temperatures it shows an S-shaped irreversible decrease of the activity with a T 50 of about 70ЊC. The dependence of the kinetic parameters k cat This enzyme contains an organic cofactor, 6-hydroxyand K m on temperature was also studied in the range dopa (3), characterized by the presence of carbonyl 5-66ЊC, measuring the initial rates of oxidation of spergroups which have been demonstrated to be directly midine. The Arrhenius plot of k cat shows a continuous involved in the formation of a Schiff base. Reaction bend in the thermal stability range of the enzyme. This [1] can be tentatively written as a sequence of partial nonlinearity is due to the dramatic change of k cat actireactions involving the following steps: (a) formation vation entropy (from /11 eu at 8ЊC, to 031 eu at 50ЊC) of a molecular complex between the enzyme and the and indicates that k cat is a composite constant, involvpolyamine, (b) condensation between a terminal amino ing two or more successive steps characterized by difgroup of the polyamine and a carbonyl group of the ferent kinetic parameters. In the case of the ratio k cat / cofactor with the generation of an imine (Schiff base), K m , which for natural substrates of bovine serum (c) isomerization of the imine with the parallel reducamine oxidase was shown that its dependence on ionic tion of the cofactor, (d) hydrolysis of the isomerized strength gives information on the influence of electroimine, and (e) oxidation of the cofactor by molecular static field on k 1 (Stevanato et al. (1994) Biochem. J. oxygen (1, 4). 299, 317-320), where k 1 is the kinetic constant of inter-In a previous paper on the dependence of benzylaction between the polyamine and the enzyme, the Aramine oxidase activity of BSAO on temperature, it has rhenius plot is linear in the range 12-45ЊC, and an activation entropy of 18.8 eu was calculated at 37ЊC. This been found that the enzyme shows a nonstrict Arrhenvalue is in accord with a mechanism involving the for-ius behavior (5). Furthermore, conflicting hypotheses mation of a polyamine-enzyme complex which is acabout the protonation state of the substrate in the first companied by neutralization of charges between the steps of catalytic process have been reported (2). In polycationic substrate and negatively charged groups fact, at physiological pH values, the natural substrates of the enzyme.
A rotating disk electrode coated with a thin mercury film is described. It is suitable for electr... more A rotating disk electrode coated with a thin mercury film is described. It is suitable for electrochemical determinations of the catalytic constant of superoxide dismutase in a much wider pH range than that accessible to polarographic methods. In particular this is the only direct method, except for pulse radiolysis, that allows mechanistic studies of the enzyme in the physiological pH range. Furthermore, the continuous response of this electrode allows enzyme activity changes characterized by half-lives in the range from a few to 10(3) s. to be followed.
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Papers by Adelio Rigo