Fluorescence Changes Induced by Binding of 4-Pyridoxic Acid 5′-Phosphate to Proteins

The affinity constant (Ka) and stoichiometry of binding of 4-pyridoxic acid 5'-phosphate (4-pyridoxic-5'-P) complexed to the protein bovine serum albumin and aspartate aminotransferase were investigated by fluorometric methods. Whereas bovine serum albumin binds 1 mole of 4-pyridoxic-5'-P per mole of protein with a Ka = 4.6 x 105 m-1, the enzyme, aspartate aminotransferase, binds 2 moles of 4-pyridoxic-5'-P per mole of protein with a Ka = 1.3 x 106 m-1 at neutral pH. The spectroscopic properties (absorption and fluorescence spectra) of the complexes were examined over a wide range of pH values. At neutral pH, the formation of the 4-pyridoxic-5'-P bovine serum albumin complex causes the following spectroscopic changes in the ligand: (a) a red shift of 15 nm in the band position of the emission spectrum, (b) an increase in the degree of polarization of fluorescence (P = 0.32), (c) an enhancement of the fluorescence lifetime (τ = 12 ns), and (d) a small blue shift of approximately 2 nm in the band position of the absorption spectrum. In contrast to the protein bovine serum albumin, the apotransaminase causes a blue shift of approximately 10 nm in the band position of the emission spectrum and a concurrent decrease in the fluorescence quantum yield of the ligand (q = 0.05). No change in the band position of the absorption spectrum of the complexed ligand was detected. The fluorescence changes observed in 4-pyridoxic-5'-P bound to the aminotransferase can be correlated with the deprotonation of the pyridine nitrogen atom as a result of its interaction with an amino acid residue at the binding site.