Investigation of Spectra and Ionization Constants of 8-Alkyl Pterins by Fluorimetry

Abstract

SummarySpectrofluorimetric studies of the properties of 8-alkyl-pterins arc reported, together with preliminary results on the use of fluorimetry in studying the binding of these new mechanism-based substrates to dihydrofolate reductase (DHFR). The compounds fluoresce strongly in the 460-490 nm range with emission maxima for the cations being blue-shifted by ~5-20 nm. Quantum yields for the cations of ~0.14-0.23 have been measured. pKa values for the basic ionization of ~5.3-5.7 have been determined from fits to both the emission and excitation spectra using sample concentrations in the 1-2 µM range. Although the UV /vis spectra between pH 0 and 4 show little change. there were significant sigmoidal changes in fluorescence intensity which could he fitted to apparent ionization constant values of ~1.4-1.0 with fluorescence intensity decaying to almost zero at pH 0. Studies of the fluorescence for the cations and neutral forms in organic acids and polar organic solvents respectively indicate little solvent dependence. Spectral modelling using the CNDO/S-CI method confirms that the spectra are due to π→π* transitions. and that the long wavelength absorption hand of ~400 nm is due to transitions involving mostly the pyrazine ring and N1. Significant differences in emission wavelength and intensity between 6-methyl- and 7 -methyl-8-alkyl-pterins have been characterised with the latter exhibiting fluorescence stronger by a factor of ~2 and blue-shifted by ~20 nm which arc readily detectable visually: aqua-blue and green fluorescence respectively for the 7-methyl and 6-methyl isomers. From this understanding of the fluorescence properties of 8-alkyl-pterins and their dependence on charge state and pyrazine-ring substituents, and taking into account the fluorescence characteristics of DHFR and cofactor NADP. we have assessed the potential of fluorimetric experiments for studying both pterin dissociation constants and the form of enzyme-bound pterin