Photochemical Coenzyme Regeneration in an Enzymatically Active Optical Material

The photoinduced electron transfer between immobilized thionine and the dinucleotide enzyme cofactors NADH and NADPH in a SiO2 sol-gel matrix is reported. The electron-transfer quenching of thionine luminescence is used to monitor the rate of NADPH oxidation. Using Stern-Volmer quenching curves, the quenching rates in the silica matrix are 1 to 2 orders of magnitude smaller than those in solution. The rate constants for oxidation of NADPH by thionine were measured to be 9.8((2.9) 10-3 s-1 in solution and 8.8((1.0) 10-4 s-1 in the gel. Within the silica matrix, the photoinduced oxidation of NADPH is combined with the enzymatic reaction of isocitrate dehydrogenase, which uses the oxidized cofactor, NADP+, as an electron acceptor in the oxidation of isocitrate. The encapsulated isocitrate dehydrogenase is active with a Michaelis-Menten constant, KM, of 3 íM and a kcat of 0.67 íM/s per mgenzyme. Because optical sensors use NADPH fluorescence as an indicator of the presence and relative concentration of enzyme substrate, the successful demonstration of photoinduced regeneration of NADP+ makes possible continuous monitoring by the family of dehydrogenase enzymes. 1