RESUMEN
The thermodynamical stability and remained activity of mushroom tyrosinase (MT) from Agaricus bisporus in 10 mM phosphate buffer, pH 6.8, stored at two temperatures of 4 and 40 degrees C were investigated in the presence of three different amino acids (His, Phe and Asp) and also trehalose as osmolytes, for comparing with the results obtained in the absence of any additive. Kinetics of inactivation obey the first order law. Inactivation rate constant (kinact) value is the best parameter describing effect of osmolytes on kinetic stability of the enzyme. Trehalose and His have the smallest value of kinact (0.7x10(-4) s-1) in comparison with their absence (2.5x10(-4) s-1). Moreover, to obtain effect of these four osmolytes on thermodynamical stability of the enzyme, protein denaturation by dodecyl trimethylammonium bromide (DTAB) and thermal scanning was investigated. Sigmoidal denaturation curves were analysed according to the two states model of Pace theory to find the Gibbs free energy change of denaturation process in aqueous solution at room temperature, as a very good thermodynamic criterion indicating stability of the protein. Although His, Phe and Asp induced constriction of MT tertiary structure, its secondary structure had not any change and the result was a chemical and thermal stabilization of MT. The enzyme shows a proper coincidence of thermodynamic and structural changes with the presence of trehalose. Thus, among the four osmolytes, trehalose is an exceptional protein stabilizer.
Asunto(s)
Agaricus/enzimología , Aminoácidos/farmacología , Ácido Aspártico/farmacología , Estabilidad de Enzimas/efectos de los fármacos , Proteínas Fúngicas/química , Histidina/farmacología , Cinética , Monofenol Monooxigenasa/química , Ósmosis , Fenilalanina/farmacología , Desnaturalización Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Temperatura , Trehalosa/farmacologíaRESUMEN
El cáncer indiferenciado de tiroides es un tumor muy agresivo, de muy mal pronóstico y sin tratamiento efectivo. La terapia por captura neutrónica de boro (BNCT) podría ser una alternativa para el tratamiento de esta enfermedad. Se basa en la captación selectiva de boro por el tumor y su activación por un haz de neutrones. El boro activado libera un núcleo de litio-7 y una partícula alfa, las cuales tienen una alta transmisión linear de energía (linear energy transfer, LET) y un alcance de 5-9 µm, destruyendo el tumor. En estudios previos hemos mostrado que la línea celular humana de cáncer indiferenciado de tiroides (ARO) tiene una captación selectiva de borofenilalanina (10BPA) tanto in vitro como después de ser implantada en ratones NIH nude. También demostramos en estos animales inyectados con BPA e irradiados con un haz de neutrones térmicos, un 100% de control sobre el crecimiento tumoral y un 50% de cura histológica. En trabajos posteriores mostramos que la porfirina 10BOPP tetrakis-carborane carboxylate ester de 2,4-bis-(a,b-dihydroxyethyl)-deutero-porphyrin IX) cuando es inyectada 5-7 días antes que el BPA se obtiene una concentración tumoral de boro de aproximadamente el doble que el BPA solo (45-38 ppm vs. 20 ppm). La posterior irradiación con neutrones mostró un 100% de remisión completa en animales con tumores cuyo volumen pre-tratamiento era de 50 mm3 o menor. Los perros padecen CIT espontáneo, con un comportamiento biológico similar al humano, y una captación selectiva de BPA, abriendo la posibilidad de su tratamiento por BNCT
Undifferentiated thyroid carcinoma (UTC) is an aggressive tumor with a poor prognosis due to the lack of an effective treatment. Boron neutron capture therapy (BNCT) is based on the selective uptake of boron by the tumor and its activation by a neutron beam, releasing lithium-7 and an alpha particle that will kill the tumor cells by their high linear energy transfer (LET). In previous studies we have shown a selective uptake of borophenylalanine (10BPA) in a human UTC cell line (ARO) and in NIH nude mice implanted with this cell line. When these animals were injected with BPA and irradiated with an appropriated neutron beam, we observed a 100% of tumor growth control and a 50 % of histological cure when the initial tumor volume was 50 mm3 or less. Further studies with BOPP (tetrakis-carborane carboxylate ester of 2,4-bis-(a, b-dihydroxyethyl)-deutero-porphyrin IX) showed that when this porphyrin was injected 5-7 days before BPA, and the animals were sacrificed 60 min after the i.p. injection of BPA, a significant increase in boron uptake by the tumor was found (45-38 ppm with both compounds vs. 20 ppm with BPA alone). The application of BNCT using the combination of boron compounds showed a 100% of complete remission in tumors with initial volumes under 50 mm3. Dogs suffer spontaneous UTC, with a similar biological behavior to the human tumor, and a selective uptake of BPA. These results open the possibility of applying BNCT to UTC