Abstract
The structure of the coenzyme-binding site of several pyridoxal 5'-phosphate containing enzymes has recently been determined after fixation of the cofactor by sodium borohydride reduction. In all instances, pyridoxal 5'-phosphate was found to be linked to a unique lysyl residue of the protein. This work describes the synthesis, separation and characterization of several pyridoxyl-lysine derivatives, namely N α-pyridoxyl-, N ε-pyridoxyl-, N ε-phosphopyridoxyl-, and N α, N ε-dipyridoxyl-lysine that should allow for their unambiguous identification and determination. Their fluorescence properties, quantum yields and differential responses to 2,6-dichloroquinone chloroimide and diazotized p-amino acetophenone are also described together with a number of solvent and buffer systems enabling their separation on thin layer chromatography, paper chromatography and electrophoresis and various automated methods of analysis. Absorption spectra of all four derivatives were determined as a function of pH from which ionization constants were calculated. Sharp isosbestic points were observed with N ε-pyridoxyl- and phosphopyridoxyl-lysine but not with the unphosphorylated N α-substituted derivatives, probably because of the slow ionization of their phenolic hydroxyl groups. This effect was particularly pronounced with N α-pyridoxyl-lysine which showed a marked hysteresis during titration in the alkaline range.
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