Selective fluorescent and fluorogenic substrates for purine-nucleoside phosphorylases from various sources, and direct fluorimetric determination of enzyme levels in human and animal blood

Abstract

Abstract The decrease in fluorescence of 7-methylguanosine (m7Guo) accompanying its phosphorolysis is readily applicable to monitoring the level of purine nucleoside phosphorylase (PNP) in mammalian whole blood lysates, without prepurification of the latter. A 10-fold higher sensitivity is attained with the use of 8-azaguanine (8-azaGua), a non-reversible, highly fluorogenic, substrate in the reverse, synthetic pathway. Although its affinity for human PNP is low (Km=250 μM at pH 7), its high reaction rate and fluorescence quantum yield of the product, 8-azaguanosine (ϕ=0.55 at pH 9), permit direct fluorometric monitoring of nucleoside synthesis in 1000-fold diluted hemolysates. It is also quite selective for mammalian enzymes, 8-azaGua being a poor substrate for bacterial (E. coli) PNP. Specificity of the assay was confirmed by the competitive effects of the natural purines hypoxanthine and adenine, with IC50 values comparable to their Km values. PNP levels in animal blood, assayed with both substrates, gave comparable results. With the exception of mice, PNP blood levels in animals were lower than in humans. We have also characterized specific fluorescent/fluorogenic substrates for bacterial (E. coli) PNP. The best of these is 2,6-diaminopurine riboside. Kinetic parameters for all the foregoing substrates are listed, and shown to be favorable for analytical applications.