Factors Contributing to the Absorption and Fluorescence Characteristics of Pyridoxal Phosphate in Glycogen Phosphorylase b

Abstract

AbstractThe Schiff base formed between salicylaldehyde and n‐hexylamine has absorption bands peaked at 255 and 317 nm in non‐polar solvents. In a polar solvent (ethanol) an additional band appears at 405 nm and a shoulder is resolved at 275 nm. The fluorescence band has a peak at 535 nm in non‐polar solvents (with the exception of CCl4) which is blue shifted to 505 nm in ethanol. These spectral data are similar to those of Schiff bases formed between pyridoxal phosphate and aliphatic amines. On the other hand, the spectral properties of the Schiff base of o‐methoxybenzaldehyde with n‐hexylamine are very different. It may thus be concluded that the nitrogen atom of the aromatic ring does not have to be invoked in explaining the spectral properties of pyridoxal phosphate Schiff bases and of pyridoxal phosphate in glycogen phosphorylase. The hydroxyl group seems however to play an important role in determining the spectral properties of Schiff bases of o‐hydroxyaldehydes. The optical rotatory power of the pyridoxal phosphate in phosphorylase is of comparable magnitude for the chromophore in the ground state or the electronically excited state, indicating similarity of mode of binding of the chromophore to the protein in the two electronic states. Measurements of circular dichroism indicate that the “deformer” imidazole citrate affects the interaction of the pyridoxal phosphate with the protein when the cofactor Schiff base is in the ketoenamine form but not when in the enolimine form.