Rosa Pia Ferrari

Oxidation of 2,4-dichlorophenol catalyzed by horseradish peroxidase: characterization of the reaction mechanism by UV-visible spectroscopy and mass spectrometry.

The hydrogen peroxide-oxidation of 2,4-dichlorophenol catalyzed by horseradish peroxidase has been studied by means of UV-visible spectroscopy and mass spectrometry in order to clarify the reaction mechanism. The dimerization of 2,4-dichlorophenol to 2,4-dichloro-6-(2,4-dichlorophenoxy)-phenol and its subsequent oxidation to 2-chloro-6-(2,4-dichlorophenoxy)-1,4-benzoquinone together with chloride release were observed. The reaction rate was found to be pH-dependent and to be influenced by the pK(a) value of 2,4-dichlorophenol. The dissociation constants of the 2,4-dichlorophenol/horseradish peroxidase (HRP) adduct at pH 5.5 and 8.5 were also determined: their values indicate the unusual stability of the adduct at pH 5.5 with respect to several adducts of HRP with substituted phenols.

Oxidative 4-dechlorination of 2,4,6-trichlorophenol catalyzed by horseradish peroxidase.

Abstract The well-known and easily available horseradish peroxidase (HRP) catalyzes the H2O2-dependent oxidative 4-dechlorination of the pollutant 2,4,6-trichlorophenol, which is recalcitrant to many organisms except those producing ligninases. UV-visible spectroscopy and gas chromatography-mass spectrometry identified the oxidized reaction product as 2,6-dichloro-1,4-benzoquinone. NMR and IR spectroscopic data further supported the above characterization. Experimental evidence for the elimination of HCl from the substrate was acquired by detecting the decrease in pH of the reaction mixture, and by observing the presence of the β-chlorocyclopentadienone cation fragment in the mass spectrum of 2,6-dichloro-1,4-benzoquinone. Consequently, nucleophilic attack by water on the 2,4,6-trichlorocyclohexadienone cation was proposed to give the final product. Our results indicate an oxidative dechlorination pathway catalyzed by HRP for 2,4,6-trichlorophenol, similar to that by extracellular lignin peroxidases. The relative catalytic efficiency of HRP seems higher than that of lignin peroxidases. The HRP-H2O2 catalytic system could be utilized in the degradation of polychlorinated phenols for industrial and biotechnological purposes.

Acetylenic derivatives of metal carbonyls XII. Diphenylacetylenic derivatives of dodecacarbonyltriosmium

The reaction of the complex (C6H5C2C6H5)2Os3(CO)8 (I) with CO has been investigated and a structure suggested for the compound, (C6H5C2C6H5)2Os3(CO)9, thus obtained. This consists basically of an Os3 cluster, in which one of the osmium atoms belongs to a osmacyclopentadiene ring, and to which only terminal carbonyl groups are bonded. The formation of this product from (I) and CO was found to be a second-order process. The reactions of complex (I) with halogens have also been investigated, and the resulting binuclear derivatives identified.

Effect of dimethyl sulfoxide on the structure and the functional properties of horseradish peroxidase as observed by spectroscopy and cyclic voltammetry.

Electrochemical biosensors have found wide application in food and clinical areas, as well as in environmental field. A large number of articles focused on horseradish peroxidase (HRP)-based biosensors have been published in the last decade, due to the capability of HRP to quantitatively detect the presence of hydrogen peroxide produced in a reaction. At present a large body of multi-enzymatic amperometric biosensors are realized by entrapping HRP together with other enzymes into a polymeric matrix; these systems represent a promising example of simple, low-cost electrochemical tools for the analysis of bioanalytes in solution, such as glucose, choline and cholesterol. Due to the fact that polymers used for HRP entrapping are soluble in organic solvents and that many solvents are strong denaturants of aquo-soluble proteins, in this paper we investigate (in particular, by circular dichroism and electron paramagnetic spectroscopies) the effect of dimethyl sulfoxide, one of the organic solvents employed for polymer solubilization, on the structure and the functionality of HRP, in order to determine the effect induced by the solvent concentration on the structure and activity of the hemoprotein. This is relevant for basic and applied biochemistry, HRP being largely employed in bioinorganic chemistry and sensor area.

Tyrosinase-catecholic substrates in Vitro model: kinetic studies on the o-quinone/o-semiquinone radical formation☆

Abstract The mechanism of o-semiquinone production was examined in the tyrosinase and peroxidase catalyzed oxidations of a series of catecholic compounds using the electron spin resonance (ESR) spin-stabilization approach and in the presence of 3-methyl-2-benzothiazolinone hydrazone (MBTH). In the tyrosinase process, the nonenzymatic o-semiquinone formation by inverse disproportion mechanism was clearly confirmed. Mechanistic and kinetic studies of o-semiquinone and o-quinone formation by mushroom tyrosinase were carried out by ESR spin stabilization and optical spectroscopy. Two different types of cyclizable catecholic substrates ( l -dopa and dopamine 3,4-dihydroxyphenylacetic acid and 3-(3,4-dihydroxyphenyl)propionic acid) together with an uncyclizable substrate (3,4-dihydroxybenzoic acid) were examined. The reactive quinones were monitored by measuring the apparent initial rates of the o-quinone-MBTH adducts. The transient behaviour of the o-semiquinone was studied by determining the pseudo first-order formation constants (k values in the range 0.226–0.035 s −1 ), the relative second-order decay kinetic constants (k = 3.3·10 2 M −1 s −1 for dopamine o-semiquinone) and the maximum concentrations of the o-semiquinone complexes formed in situ with Mg 2+ ions. The o-semiquinone formation constants are not directly correlated with their maximum concentrations; in fact, the o-semiquinone maximum concentration of the uncyclizable substrate is comparable with that derived from l -dopa. Furthermore, the secondary semiquinone formation is slow and not competitive with the primary semiquinone generation. Then, in our model the limiting factor for the o-semiquinone formation, is not simply the substrate ability to cyclize, and, therefore, the potential toxicity of the secondary semiquinone is questionable.

SPECTROCHEMICAL AND STRUCTURAL STUDIES ON A ROMAN SAMPLE OF EGYPTIAN BLUE

Abstract A ball of Egyptian blue excavated on the archaeological site of Augusta Praetoria (Aosta, Italy) has been investigated by several techniques. Optical microscopy and X-ray diffraction gave proof of the identity of the pigment, while indicating that silica phases had also been formed because of sand excess in the reaction mixture. No evidence was found of the presence of tin compounds, thus excluding the use of bronze scraps in the preparation of the pigment. The crystal structure of the pigment was refined using single-crystal X-ray diffraction data; Fourier transform infrared spectroscopy, UV-vis-NIR reflectance spectroscopy and electron paramagnetic resonance spectroscopy were used to characterize further the pigment. Scanning electron microscopy, coupled with energy dispersive detection of emitted X-rays, was finally used to investigate morphology and determine the composition of representative areas of both pigment and impurities.

Acetylenic derivatives of metal carbonyls. Part XV. Reaction of dodecacarbonyltriosmium with unsymmetrically substituted alkynes

Abstract Dodecacarbonyltriosmium and phenylacetylene (L) in refluxing benzene give Os 3 (CO) 10 L, Os 3 (CO) 10 L 2 , Os 3 (CO) 9 L and HOs 3 (CO) 9 (LH) as primary products. Reactivity of these complexes is investigated and formation of 1,2,4-triphenylbenzene in a sequence starting from Os 3 (CO) 10 L is described. Corresponding reactions with propyne and 1-butyne exhibit a close analogy with those of phenylacetylene. A partially different behaviour is shown by methylphenylacetylene (L′), which reacts with dodecacarbonyltriosmium to give two isomers of molecular formula HOs 3 (CO) 8 (L′H)L′ as main products. These complexes are suggested to be isostructural with an analogous compound obtained from diphenylacetylene. The main factors affecting the reaction trends are considered.

Spectroscopic and binding studies on the interaction of inorganic anions with lactoperoxidase

The interaction of several inorganic species (SCN-, I-, Br-, Cl-, F-, NO2-, N3-, CN-) with bovine lactoperoxidase was investigated through kinetic and binding studies by using UV-Vis spectroscopy. The above ligands form 1:1 complexes with the protein and can be assigned to three different groups, on the basis of the dissociation constant values (KD) of the adducts: (1) SCN-, I-, Br-, and Cl- (KD increases along the series); (2) F- (which shows a singular behavior); (3) NO2-, N3-, and CN- (that bind at the iron site). KD values for the LPO/SCN- adduct appeared to be modified in the presence of other inorganic species; a strong competition between this substrate and all other anions (with the exception of F-) was evidentiated. Binding investigations on the natural substrates SCN- and I-, at varying pH and temperature, showed that their interaction with lactoperoxidase involves the protonation of a common site in proximity of the iron (possibly distal histidine). Michaelis-Menten constants for SCN-, I-, and Br- followed roughly the same trend as KD; KM for hydrogen peroxide is strongly dependent on the cosubstrate. Computer-assisted docking simulations showed that all ligands can penetrate inside the heme pocket.

A theoretical three-dimensional model for lactoperoxidase and eosinophil peroxidase, built on the scaffold of the myeloperoxidase X-ray structure

Abstract Lactoperoxidase (LPO), eosinophil peroxidase (EPO) and myeloperoxidase (MPO) belong to the class of haloperoxidases, a group of mammalian enzymes able to catalyze the peroxidative oxidation of halides and pseudohalides, such as thiocyanate. They all play a key role in the development of antibacterial activity. The homology in their functional role is emphasized by the striking similarity of their primary structures. A theoretical model for the three-dimensional structure of LPO and EPO has been developed on the basis of the X-ray structure of MPO, a high degree of similarity having been found in their sequences. Evidence supporting the hypothesis of an ester linkage between heme and apoprotein in LPO and EPO, originally proposed by Hultquist and Morrison is discussed.

Determination of the carbohydrate composition and the disulfide bond linkages of bovine lactoperoxidase by mass spectrometry.

The extent and distribution of N-glycosylation and the nature of most of the disulfide bond linkages were determined for bovine lactoperoxidase through proteolytic and glycolytic digestions combined with matrix-assisted laser desorption/ionization mass spectrometric analysis. In addition, 98% of the primary sequence of the protein was confirmed. All five of the asparagines present in sequons were found to be glycosylated, predominantly by high mannose and complex structures. Six disulfide bonds were assigned, including Cys 32-Cys 45, Cys 146-Cys 156, Cys 150-Cys 174, Cys 254-Cys 265, Cys 473-Cys 530 and Cys 571-Cys 596.