Luminescence spectroscopy of pyridoxic acid and pyridoxic acid bound to proteins

Luminescence techniques, i.e. fluorescence and phosphorescence, have been employed to study pyridoxic acid bound to proteins through a stable amide linkage. Proteins tagged with 4‐pyridoxic acid display the following fluorescence properties: (a) emission and excitation spectra centered at around 430 and 320 nm, respectively; (b) fluorescence quantum yields of 0.3–0.4 and (c) average decay times covering the range 8–9.6 ns. The fluorescence properties of the probe have been used to study the dynamics of the protein in the nanosecond time scale. In the absence of molecular oxygen, free and bound 4‐pyridoxic acid exhibit long‐lived emission at room temperature. The long‐lived emission is red‐shifted when compared to fluorescence and decays with average life times ranging over 2.2–0.6 ms depending on the nature of the protein. The fluorophore pyridoxic acid covalently linked to proteins is suitable to study the dynamics of proteins, i.e. fast and slow motions of the macromolecule in the nanosecond and millisecond time scales, respectively.