ETUDE PAR FLUORESCENCE DE BASES DE SCHIFF DU PYRIDOXAL, COMPARAISON AVEC LA L ASPARTATE-AMINOTRANSFERASE

Abstract— –Absorption and emission spectra of Schiff bases of pyridoxal-HCI or pyridoxal-5-phosphate with L-valine, n -butylamine or N-α-acetyl-L-lysine-N-methylamide have been studied as a function of pH. We can write the complete ionization diagram and equilibria. The results of Martell6] are confirmed: the forms analogous to the coenzyme in aspartate aminotransferase, which absorb at 410 nm and 360 nm (or 340 nm for the Schiff base with n-butyl-amine) have the phenol OH ionized; the imine nitrogen is protonated for species absorbing at 410nm (in the enzyme: inactive form and complex with aminoacid) and unprotonated for species absorbing at 360 nm (in the enzyme, active form). Their fluorescence wavelengths are respectively 500 nm and 430 nm. Protonation of the pyridine nitrogen of these forms does not shift the absorption band; the fluorescence intensity is 20-fold greater for the N-protonated forms.
The real pK of the imine nitrogen is 8.5 ±0.8 for species with pyridine N-protonated or 10.4 for the non-protonated forms. The observed pK 6.3 in the enzyme can be explained if the imine nitrogen is hydrogen bonded to an amino-acid side chain of the protein: lysine, tyrosine, serine, sulfhydryl.
The quantum yield of the coenzyme fluorescence in the enzyme has been compared to that of the analogous Schiff base (absorbing at the same wavelength). According to the results, we cannot deduce whether the pyridine nitrogen is protonated in pyridoxal form of the enzyme.
If it is protonated, as in the pyridoxamine form, the coenzyme environment is not the same in the two forms. If the pyridine nitrogen is unprotonated in the pyridoxal form and protonated in the pyridoxamine form, the environment of the coenzyme is the same in these two forms of the enzyme.