Dynamical and structural properties of flavin adenine dinucleotide in aqueous solutions and bound to free and sol-gel immobilized glucose oxidase

摘要

Optical properties of flavin adenine dinucleotide moiety are nowadays widely used for biotechnological applications. In particular when flavin adenine dinucleotide is bound to glucose oxidase it is employed for developing optical biosensors for glucose concentration monitoring. In order to design such systems it can be extremely useful to have a deeper insight in structural properties and functional role of flavin adenine dinucleotide in experimental conditions usually employed in applicative field. For this purpose we investigated the behavior of this flavin cofactor in aqueous solutions at different pH values and bound to free and sol-gel immobilized glucose oxidase by means of complementary optical spectroscopic techniques (optical absorption, circular dichroism, infrared spectroscopy, steady-state and time-resolved fluorescence). The obtained results confirm that pH influences absorption and fluorescence spectra of flavin adenine dinucleotide and glucose oxidase while the sol-gel immobilization preserves optical properties of the enzyme. Circular dichroism spectra are also affected by pH changes but a predominance of beta-sheet component is evidenced for glucose oxidase in all cases. Infrared spectroscopy indicates that the immobilization procedure changes the secondary structure of the enzyme but still preserves the above-mentioned predominance. An innovative maximum entropy method was adopted for time-resolved fluorescence signal analysis showing a three-components decay for flavin adenine dinucleotide in aqueous solution with pH-depending lifetimes and relative amplitudes. For free and immobilized glucose oxidase a four-components decay is recovered and immobilization shortens all decay times and changes the relative amplitude of the observed lifetime components. All the results here reported give a more complete view of flavin adenine dinucleotide's properties that can be exploited in designing new biotechnological devices.