LUMINESCENCE SPECTROSCOPY OF PYRIDOXIC ACID AND PYRIDOXIC ACID BOUND TO PROTEINS

Luminescence techniques, i.e. fluorescence and phosphorescence, have b een employed to study pyridoxic acid bound to proteins through a stabl e amide linkage. Proteins tagged with Lt-pyridoxic acid display the fo llowing fluorescence properties: (a) emission and excitation spectra c entered at around 430 and 320 nn, respectively; (b) fluorescence quant um 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 stu dy the dynamics of the protein in the nanosecond time scale. In the ab sence of molecular oxygen, free and bound 4-pyridoxic acid exhibit lon g-lived emission at room temperature. The long-lived emission is red-s hifted when compared to fluorescence and decays with average life time s ranging over 2.2-0.6 ms depending on the nature of the protein. The fluorophore pyridoxic acid covalently linked to proteins is suitable t o study the dynamics of proteins, i.e. fast and slow motions of the ma cromolecule in the nanosecond and millisecond time scales, respectivel y.