Journal of Physical Chemistry B p. 9325 - 9332 (2004)
Update date:2022-08-29
Topics:
Rickus, Jenna L.
Chang, Pauline L.
Tobin, Allan J.
Zink, Jeffrey I.
Dunn, Bruce
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 ??M and a kcat of 0.67 ??M/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.
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