Estrella Núñez-Delicado

Triton X-114-aided purification of latent tyrosinase.

Mushroom tyrosinase was partially purified using an aqueous two-phase system with Triton X-114. The purification achieved was 5.5-fold from a crude extract of mushroom pileus, with a high recovery of 84%. The phenols were reduced to 8% of the original content, avoiding pre- and post-purification tanning of the enzyme. The enzyme obtained was latent and was activated 3-fold by trypsin, 2.7-fold by changes in the pH and to different extents by cationic and anionic detergents, the latter being the more effective. There was also a synergistic effect between trypsin and detergent, at low detergent concentrations. When kinetically characterized, latent enzyme showed both monophenolase and diphenolase activities, the latter activity displaying an unexpected lag period before reaching the steady-state rate. This behaviour is characteristic of a hysteretic enzyme, and has not been previously described for this enzyme. In addition, inhibition studies with substrate analogues were carried out, tropolone being found to be the most effective inhibitor.

HYDROPEROXIDASE ACTIVITY OF LIPOXYGENASE : A KINETIC STUDY OF ISOPROTERENOL OXIDATION

The hydroperoxidase activity of soybean lipoxygenase, a non-heme protein, oxidized isoproterenol using H2O2 at pH 6.0. This oxidation was enzymatic, since neither heat-denaturated enzyme or iron ions in the presence of H2O2 produced an increase in absorbance. The initial rate was not linear and showed a characteristic lag period whose length depended on the enzyme and substrate concentration. The lag was decreased if the enzyme and isoproterenol concentration were increased, whereas it increased if the H2O2 concentration was increased. Lipoxygenase showed the typical low specificity for electron donor characteristic of this hydroperoxidase activity (26 mM), but a high affinity for H2O2 (94 microM), although with substrate inhibition (ksi = 3.6 mM). The chemical intermediates produced during the oxidation of isoproterenol were characterized in order to determine the origin of the lag period. A plausible kinetic mechanism is proposed to explain the observed lag period and inhibition by high concentrations of H2O2.

Cyclodextrins as hosts for poorly water-soluble compounds in enzyme catalysis

The capability of cyclodextrins to enhance greatly the solubility in water of poorly water-soluble substances makes them an ideal alter native for investigating the expression of enzyme activity with such substrates in aqueous solution. This capability is demonstrated by using soybean lipoxygenase with linoleic acid/β-cyclodextrin and di ethylstilbestrol/γ-cyclodextrin, and cholesterol oxidase with choles terol/methyl-β-cyclodextrin.

Cyclodextrins as Diethylstilbestrol Carrier System: Characterization of Diethylstilbestrol-Cyclodextrins Complexes

AbstractPurpose. The in vitro formation of DES-cyclodextrins inclusion complexes was characterized using lipoxygenase as enzymatic system.nMethods. DES-cyclodextrins complexes were obtained in aqueous solution.nResults. The addition of cyclodextrins to the reaction medium had an inhibitory effect on DES oxidation by lipoxygenase due to the drugs complexation into the cyclodextrin cavity. This inhibitory effect depends on the complexation constant between DES and the cyclodextrins type used. In this case, β-, 2-hydroxypropyl-β- and γ-cyclodextrins have similar complexation constants and therefore produce the same inhibitory effect. Moreover, depending on the type of cyclodextrins used, the solubility of DES can be enhanced up to 956 times, while the lipoxygenase activity remains constant.nConclusions. These results suggest that the system described may be used as a controlled-release delivery system for DES, since it may diminish the local and systemic adverse side effects caused by high concentrations of the drug.

Simultaneous Determination of Nine Sulphonamides by LC-MS for Routine Control of Raw Honey Samples

A liquid chromatography-mass spectrometry method for the simultaneous determination of nine sulphonamides (sulphadiazine, sulphathiazole, sulphapyridine, sulphamerazine, sulphamethizole, sulphasoxazole, sulphamethoxypyridazine, sulphamethoxazole and sulphadimethoxine) in honey samples was developed. Sample pretreatment included acidic hydrolysis, followed by solid-phase extraction on a Sep-Pack® C18. Separation is carried out in a C18 column and mobile phases consisting of acetic acid/methanol mixtures. Detection is performed by mass spectrometry with ion trap, using electrospray interface in the positive ionization mode. Method validation has been performed according to European Commission Decision 2002/657/EC criteria, in terms of linearity, trueness, precision, specificity, decision limit (CCα) and detection capability (CCβ). Linearity was obtained with an average coefficient of determination (R2) higher than 0.9995 in matrix. Typical recoveries in fortified honey samples ranged between 89 and 118xa0% at all levels tested (5, 10, 50 and 100xa0μg/kg). The method demonstrated good intra- and interbatch precision and accuracy. No interferences with the peaks of interest were observed throughout the chromatographic run. The method has been successfully applied to the analysis of raw honey samples. Forty-five (21xa0%) of 215 analysed were positive (range 6.7–140.8xa0μg/kg) for sulphonamides.

Anomalous oxidation of MDL 73,404 by horseradish peroxidase

3,4-Dihydro-6-hydroxy-N,N,N-2,5,7,8-heptamethyl-2H-1-benzopyran-2-ethanaminium-4-methylbenzene sulfonate (MDL 73,404) is a cardioselective water-soluble quaternary ammonium analogue of Vitamin E which is synthesized to augment the antioxidant defence in situations of free radical injury such as myocardial infarction/reperfusion. Its oxidation by any peroxidative enzyme has not been studied kinetically. This paper describes its enzymatic oxidation by horseradish peroxidase (HRP). The activity was followed spectrophotometrically at 255nm, and the experimental results were simulated using the program KINETIC 3.1 for Windows 3.x. The MDL 73,404 was oxidized by horseradish peroxidase in the presence of H2O2 to its corresponding MDL 73,404 quinone. During this oxidation, the horseradish peroxidase showed an unexpectedly slow kinetic response with time, which contrast with the linear product accumulation curve measured with 2,2-azino-bis-(3-estilbenzotiazol-6-sulfonic acid) (ABTS). This response was dependent on the respective concentrations of enzyme, MDL 73,404 and H2O2. However, when the enzyme was incubated with H2O2, the slow kinetic response disappeared and a lag period was observed. Furthermore, when p-coumaric acid (PCA) was added, the activity increased and the slow kinetic response became a straight line. In order to explain this anomalous behaviour, a kinetic model has been proposed and its differential equations simulated. From the correlation between experimental and simulated results it is concluded that MDL 73,404 can act as a slow response substrate for peroxidase, probably due to the presence of a quaternary ammonium side chain that confers on it a slow capacity to convert compound III into ferriperoxidase.

Oxidation of Hydrophobic O-Diphenols by Lipoxygenase in the Presence of Cyclodextrins

Cyclodextrins (CDs) are cyclic oligosaccharides composed of six to eight glucopyranose residues linked by α (l→4) glicosidic bonds in a cylinder-shaped structure. The most important feature of cyclodextrins is their ability to complex a variety of guest molecules into their hydrophobic cavities in aqueous solutions [2]. The physicochemical properties of these inclusion complexes have been extensively studied and several analytical techniques have been used to determine their formation constants [3]: spectrophotometry, fluorimetiy, calorimetry, circular dichroism, phase solubility, etc.