RESUMEN
The homogeneous reaction between glucose oxidase and osmium bipyridine-pyridine carboxylic acid in the presence of glucose has been studied in detail by cyclic voltammetry and digital simulation. Combination of the analytical equations that describe the dependence of the amperometric response on enzyme, substrate and co-substrate concentrations for the limiting cases with digital simulation of the coupled enzyme reaction diffusion problem allows us to extract kinetic parameters for the substrate-enzyme reaction: K(MS)=10.8 mM, k(cat)=254 s(-1) and for the redox mediator-enzyme reaction, k=2.2x10(5) M(-1) s(-1). The accurate determination of the kinetic parameters at low substrate concentrations (<7 mM) is limited by depletion of the substrate close to the electrode surface. At high substrate concentrations (>20 mM) inactivation of the reduced form of glucose oxidase in the bulk solution must be taken into account in the analysis of the results.
Asunto(s)
Electroquímica/métodos , Glucosa Oxidasa/química , Compuestos Organometálicos/química , Osmio/química , Activación Enzimática , Cinética , Oxidación-ReducciónRESUMEN
A description of the implementation of the relaxation method with automatic mesh point allocation for immobilized enzyme electrodes is presented. The advantages of this method for the solution of coupled reaction-diffusion problems are discussed. The relaxation numerical simulation technique is combined with the Simplex fitting algorithm to extract kinetic parameters from experimental data. The results of the simulations are compared to experimental data from self-assembled multilayered electrodes comprised of glucose oxidase (GOx) and an Os modified redox mediator and found to be in excellent agreement.
RESUMEN
Pyridine-based osmium complexes bearing either a carboxylate or aldehyde group were covalently attached to glucose oxidase and were shown to work as mediators for the reoxidation of the enzyme. For the complex containing the carboxylate group, the binding was made through carbodiimide coupling to the amine residues in the protein. For the complex containing the aldehyde group, the reductive coupling was carried out by condensation with the amino groups on the protein in the presence of sodium cyanoborohydride. Electrochemical studies show evidence for both intramolecular and intermolecular redox mediation for the electrochemical reoxidation of the modified glucose oxidases in the presence of glucose. The modified enzymes adsorbed on glassy carbon and platinum show different electrochemical responses for the two electrode materials, suggesting that orientation of the adsorbed enzyme is induced due to the interaction of the osmium complex with the different surfaces. Construction of enzyme switches based on these modified enzymes was carried out, and their responses were compared with those obtained using native glucose oxidase and a soluble redox mediator.
Asunto(s)
Glucosa Oxidasa/química , Glucosa/metabolismo , Compuestos de Osmio/química , Aspergillus niger/enzimología , Electroquímica , Transporte de Electrón , Glucosa Oxidasa/metabolismo , Especificidad por Sustrato , Transistores ElectrónicosRESUMEN
A serological survey for Brucella antibodies in sheep and goats was completed on Saint Croix, United States Virgin Islands (USVI). Seroprevalence (at suspect or reactor titer levels) for B. melitensis antibodies was 11.3% for sheep and 2.5% for goats. This is the first report, of which we are aware, of B. melitensis antibodies in sheep or goats in the Caribbean islands.
Asunto(s)
Anticuerpos Antibacterianos/análisis , Brucella/inmunología , Enfermedades de las Cabras/diagnóstico , Enfermedades de las Ovejas/diagnóstico , Animales , Cabras , Pruebas Serológicas , Ovinos , Islas Virgenes de los Estados UnidosRESUMEN
A serological survey of 16 serovars of Leptospira interrogans, previously reported in tropical small ruminants, was undertaken to determine the serovars involved and the prevalence of these antibodies in sheep and goats on St. Croix, U. S. Virgin Islands (USVI). Seven of eight goat herds (108 animals) had at least two seropositive animals in each herd with an individual animal seroprevalence of 26%. The 53 sheep tested (one flock only) showed a 32% seroprevalence. Antibodies against seven serovars were detected in goats (autumnalis, ballum, bataviae, bratislava, canicola, icterohemorrhagiae, and pyrogenes). In addition, hardjo antibodies were detected in sheep. Serovar autumnalis accounted for about 30% of seropositive animals in each species. Many animals showed titers against more than one serovar. The number of seropositive animals suggests Leptospira may be a factor in the health of small ruminants on St. Croix.