Journal
JOURNAL OF PHYSICAL CHEMISTRY B
Volume 108, Issue 26, Pages 9325-9332Publisher
AMER CHEMICAL SOC
DOI: 10.1021/jp038051g
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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 I 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) x 10(-3) s(-1) in solution and 8.8(+/-1.0) x 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, K-M, of 3 muM and a k(cat) of 0.67 muM/s per mg(enzyme). 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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