RESUMO
Two histidines were introduced by site-directed mutagenesis into the structure of Enhanced Green Fluorescent Protein, replacing the serine at position 202 and the glutamine at position 204 for increasing the sensitivity of the protein towards different metal ions by creating possible metal binding sites near the chromophore group. There is no appreciable difference between the absorbance and fluorescence spectra of the two proteins (wild type and the double-histidine mutant) indicating that the mutation does not change the environment of the fluorophore. Fluorescence quenching was measured at different pH (6.5-8) and temperatures (20-45 °C) varying the concentration of metal ions. Under optimal conditions (pH = 7.5, 20 °C) the mutant's Kd is 16 nM, it binds copper more than 200fold stronger than the wild type EGFP.
Assuntos
Cobre/química , Proteínas de Fluorescência Verde/química , Histidina/química , Espectrometria de Fluorescência/métodos , Eletroforese em Gel de Poliacrilamida , Proteínas de Fluorescência Verde/genética , Concentração de Íons de Hidrogênio , Modelos Moleculares , Mutagênese Sítio-Dirigida , Plasmídeos , TemperaturaRESUMO
Human serum transferrin has a potential for drug-delivery systems. Oxalate and aziridine-carboxylate was conjugated to serum transferrin in order to transport into the targeted cancer cells via transferrin-receptor mediated endocytosis. Capillary zone electrophoresis and capillary isoelectric focusing were used to analyze the effectiveness of complexation reactions. The electropherograms show the differences between iron-free- and iron-complexed molecular forms of human serum transferrin. The iron-complexed transferrin sample containing the different anions as synergistic complexing agents were characterized by different electrophoretic parameters.
