RESUMEN
Glucose oxidase immobilized in a transparent silicate gel prepared by a sol-gel method was used as a simple solid-state optical biosensor for glucose. The sensor was based upon the measurement of initial rates of reduction of the FAD prosthetic group of the enzyme in the presence of various concentrations of glucose. The analytical range of the sensor was 1-100 mM, and the measurement time was 2 min. The enzyme was considerably protected by the silicate matrix against leaching, thermal inactivation and even H2O2-dependent inactivation. The sensor was stable in daily use for 6 months.
Asunto(s)
Técnicas Biosensibles , Enzimas Inmovilizadas/química , Glucosa Oxidasa/química , Glucosa/análisis , Aspergillus niger/enzimología , Enzimas Inmovilizadas/antagonistas & inhibidores , Flavina-Adenina Dinucleótido/metabolismo , Glucosa Oxidasa/antagonistas & inhibidores , Concentración de Iones de Hidrógeno , Cinética , Óptica y Fotónica , Oxidación-Reducción , Gel de Sílice , Dióxido de Silicio , Especificidad por Sustrato , TemperaturaRESUMEN
Trypsin and acid phosphatase-containing silica sol-gel glasses were obtained by mixing a solution of an enzyme with polyethylene glycol (PEG) 6000 and tetramethoxy orthosilicate at room temperature, followed by gelation and drying. Activity of the immobilized trypsin toward small substrates, such as N-benzoyl-L-arginine-4-nitroanilide at its Km, for the best preparations equaled that of the soluble enzyme. Polylysine (M(r) less than or equal to 13,000) and aprotinin (M(r) = 6,500) inhibited this activity. Larger polylysines as well as soybean trypsin inhibitor (M(r) = 20,100) were ineffective. The sol-gel-entrapped trypsin activity was stable when sol-gel glasses were incubated at ambient temperature (pH 7.5) for several months. In comparison, trypsin, immobilized in sol-gel glass by surface adsorption and incubated under the same conditions overnight, was completely autodigested. The firm interaction between the protein molecules and the silica matrix stabilized the enzymes. Thus, the half-life of sol-gel-entrapped acid phosphatase at 70 degrees C (pH 8.0) was two orders of magnitude larger than that of the enzyme in solution. Transparent, mechanically and chemically stable bioactive sol-gel glasses may be used for the development of robust on-line biochemical photodetection sensors and for the purposes of chemical catalysis.
Asunto(s)
Fosfatasa Ácida , Enzimas Inmovilizadas , Tripsina , Biotecnología , Geles , VidrioRESUMEN
Ten derivatives of N-benzylphenanthrene 9,10-imine with different substituents on the phenyl ring were synthesized and subjected to mutagenicity tests in Salmonella typhimurium TA100. While electron donating groups were found to enhance the biological activity, electron attracting and bulky substituents lowered the mutagenic potency. A similar dependence on the electronic structure was observed in triethylamine/acetonitrile-promoted interaction of the title imines and 4-nitrothiophenol. This similarity suggests that both biochemical and chemical processes involve mechanisms in which protonation of the aziridine nitrogen is rate controlling, and the attack of the cellular or model nucleophile is a fast step. In contrast to these processes, the reaction of the imines with 4-nitrothiophenol in the presence of 1,5-diazabicyclo[4.3.0] non-5-ene proved to proceed by an SN2 mechanism and to be enhanced by electron attracting substituents.
Asunto(s)
Iminas/farmacología , Mutación , Fenantrenos/farmacología , Fenómenos Químicos , Química , Electrones , Cinética , Espectroscopía de Resonancia Magnética , Estructura Molecular , Pruebas de Mutagenicidad , Salmonella typhimurium/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
We previously showed that some (nonsubstituted) aziridines derived from polycyclic aromatic hydrocarbons (arene imines) elicit various mutagenic and genotoxic effects in bacteria and mammalian cells and that these arene imines are active at much lower concentrations than the corresponding epoxide analogues. In the present study, N-substituted derivatives of phenanthrene 9,10-imine were investigated. All 10 derivatives studied showed direct mutagenicity in Salmonella typhimurium TA100. Some of the compounds additionally exhibited weak effects in the strains TA98 and TA1537. Most N-substituted derivatives were weaker mutagens than unsubstituted phenanthrene 9,10-imine but stronger mutagens than phenanthrene 9,10-oxide. Bulky substituents reduced the mutagenicity more than did small substituents. In addition, the derivatives with electron-withdrawing substituents (with the exception of N-chlorophenanthrene 9,10-imine) were weaker mutagens than those with electron-donating substituents. Phenanthrene 9,10-imine and five N-substituted derivatives were investigated to determine whether they induce gene mutations at the hgprt locus in V79 cells. Four compounds, including the parent aziridine, were positive in the V79 test. The other two compounds were negative. The mutagenic potencies in the V79 cell system did not correlate well with those obtained with the Salmonella system. Overall, the study shows that in addition to unsubstituted arene imines, N-substituted derivatives are mutagenic. This finding is of interest, as metabolic pathways leading from aromatic compounds to N-substituted arene imines are conceivable.
Asunto(s)
Aziridinas/toxicidad , Azirinas/toxicidad , Mutágenos , Fenantrenos/toxicidad , Animales , Línea Celular , Cricetinae , Cricetulus , Pruebas de Mutagenicidad/métodos , Salmonella typhimurium/genética , Relación Estructura-ActividadRESUMEN
A series of K-region, N-substituted phenanthrene imines were tested for mutagenicity in Salmonella typhimurium TA100. All chemicals were mutagenic in the absence of an exogenous metabolic activation system. The apparent decay times of the mutagenic species in diffusion plates and their alkylating activities were also measured. The unsubstituted phenanthrene-9,10-imine was approximately 70-fold more mutagenic than the corresponding phenanthrene-9,10-oxide. N-substitution with electron-releasing groups resulted in chemicals that were more mutagenic than those substituted with electron-withdrawing groups. The mutagenic activity of the latter group of chemicals was comparable with that of phenanthrene-9,10-oxide. Except for N-chlorophenanthrene imine, both alkylation of p-nitrothiophenol and apparent decay times in diffusion plates were inversely correlated with mutagenicity. It is hypothesized that reactivity towards p-nitrothiophenol (alkylating activity) and mutagenicity reflect different reactions, in contrast to other chemical mutagens. The results suggest that the high potency of phenanthrene imines as mutagens is possibly due to DNA binding via an aziridinium ion rather than a carbonium ion.