RESUMEN
Aim: Superoxide dismutase (SOD) and catalase (CAT) immobilized on gold nanoparticles (AuNP) and silver nanoparticles (AgNP) nanoparticles were used to reduce UV radiation-induced oxidative stress in rat skin. Materials & methods: The antioxidant influence of the enzymes was investigated on level of malondialdehyde, 8-hydroksy-2'deoksyguanozine, myeloperoxidase, total antioxidant capacity, SOD2 and CAT activity and expression, and glutathione and glutathione peroxidase activity. Results: The application of immobilized SOD and CAT on UV-irradiated skin reduced malondialdehyde and 8-hydroksy-2'deoksyguanozine levels also SOD and CAT activity and expression increased. The tested enzymes influence glutathione peroxidase activity and level of total antioxidant capacity and glutathione. Conclusion: Immobilized enzymes increased the antioxidative potential of skin following UV irradiation.
Asunto(s)
Antioxidantes/farmacología , Enzimas Inmovilizadas/farmacología , Estrés Oxidativo/efectos de los fármacos , Protectores contra Radiación/farmacología , Piel/efectos de los fármacos , Animales , Antioxidantes/química , Catalasa/química , Catalasa/farmacología , Enzimas Inmovilizadas/química , Glutatión/química , Oro/química , Humanos , Malondialdehído/química , Nanopartículas del Metal/química , Protectores contra Radiación/química , Ratas , Especies Reactivas de Oxígeno/metabolismo , Piel/patología , Piel/efectos de la radiación , Superóxido Dismutasa/química , Superóxido Dismutasa/farmacología , Rayos Ultravioleta/efectos adversosRESUMEN
Superoxide dismutase (SOD) is one of the best characterized enzyme maintaining the redox state in the cell. A bacterial expression system was used to produce human recombinant manganese SOD with a His-tag on the C-end of the protein for better purification. In addition, gold and silver nanoparticles were chemically synthesized in a variety of sizes, and then mixed with the enzyme for immobilization. Analysis by dynamic light scattering and scanning transmission electron microscopy revealed no aggregates or agglomerates of the obtained colloids. After immobilization of the protein on AuNPs and AgNPs, the conjugates were analyzed by SDS-PAGE. It was determined that SOD was adsorbed only on the gold nanoparticles. Enzyme activity was analyzed in colloids of the gold and silver nanoparticles bearing SOD. The presence of a nanoparticle did not affect enzyme activity; however, the amount of protein and size of the gold nanoparticle did influence the enzymatic activity of the conjugate. Our findings confirm that active recombinant human superoxide dismutase can be produced using a bacterial expression system, and that the enzyme can be immobilized on metal nanoparticles. The interaction between enzymes and metal nanoparticles requires further investigation.
Asunto(s)
Oro/química , Oro/farmacología , Nanopartículas del Metal/química , Proteínas Recombinantes/metabolismo , Plata/química , Plata/farmacología , Superóxido Dismutasa/metabolismo , Humanos , Estrés Oxidativo/efectos de los fármacos , Tamaño de la Partícula , Transporte de Proteínas/efectos de los fármacosRESUMEN
Human catalase cDNA was cloned into a pEX-C-His vector. Purified recombinant catalase was immobilized on nanoparticles. Gold and silver nanoparticles were synthesized in a variety of sizes by chemical reduction; no agglomerates or aggregates were observed in any of the colloids during dynamic light scattering or scanning transmission electron microscopy analysis. After immobilization on gold nanoparticles, recombinant catalase activity was found to be lower than that of the same amount of enzyme in aqueous solution. However, after 10 days of storage at room temperature, the activity of catalase immobilized on gold nanoparticles (AuNPs) of 13 and 20 nm and coverage of 133% was 68 and 83% greater than catalase in aqueous solution, respectively. During 10 days of experiment, percentage activity of catalase immobilized on those gold nanoparticles was higher in comparison to CAT in aqueous solution. Catalase immobilized on silver nanoparticles did not lose activity as significantly as catalase immobilized on AuNPs. Those results confirm the ability to produce recombinant human enzymes in a bacterial expression system and its potential use while immobilized on silver or gold nanoparticles.