The Schiff base between pyridoxal-5′-phosphate and hexylamine. Equilibria in solution

Abstract

Electrochemical and spectroscopic studies of the pyridoxal-5′-phosphate-hexylamine Schiff base (PHSB) over a wide pH range have been carried out. This compound has been used as a simple model of the binding of pyridoxal-5′-phosphate (PLP) to protein. In this work different equilibria involved in the formation reaction have been considered and a quantitative distribution of the species in solution has been obtained. The method is based on the analysis of the reduction wave and absorption bands of the PHSB. Protonation equilibria constants (PHSB pK) have been obtained. Absorption spectra as a function of the medium composition have been resolved by a log normal distribution. Tautomeric equilibria have been considered and microscopic pK have been evaluated. Fluorescence results show that Schiff-base species bearing a protonated ring nitrogen are the most fluorescent. However, the fluorescence decreases in an acid medium due to an hydrolysis reaction. An important conclusion of this investigation is that the combined use of electrochemical and spectroscopic techniques is a valuable tool for the quantitative characterization of the pyridoxal-5′-phosphate Schiff bases.