Resazurin as an Electron Acceptor in Glucose Oxidase-Catalyzed Oxidation of Glucose.

Abstract

Resazurin (1) (cf. Chart 1) is known to act as an electron acceptor in diaphorase-or N-methylphenazinium methosulfate (PMS ϩ )-catalyzed oxidation of NAD(P)H and to be reduced to resorufin (2). [1][2][3][4][5][6][7][8][9] The reductively deoxygenated product 2 exhibits strong emission (excitation maximum at 563 nm and emission maximum at 587 nm in pH 7.4 buffer) 10,11) at wavelengthsϾ550 nm, where potential interference in analysis of colored or turbid serum components can be avoided. Thus, the transformation of non-fluorescent 1 to fluorescent 2 has been utilized as a fluorometric indicator reaction for determination of activity [1][2][3] or substrates 6-8) of NAD(P) ϩ -specific dehydrogenases as well as NAD(P) : by enzymatic reaction with an NAD(P) ϩ -specific dehydrogenase, its substrate is oxidized, while NAD(P) ϩ is transformed to NAD(P)H; 1 is reduced to 2 by thus formed NAD(P)H in the presence of diaphorase or PMS ϩ , depending on dehydrogenase activity, or concentration of a substrate or NAD(P) ϩ . Based on the intriguing behavior of 1 in NAD(P)H-oxidation, it is speculated that 1 functions as an electron acceptor in enzymatic oxidation by other oxidoreductases such as glucose oxidase (GOD), being transformed to 2. However, there have been no studies of the possibility of transformation of 1 to 2 as a fluorometric indicator reaction for enzymatic analysis coupled with enzymatic redox reactions. Recently, 2 was found to function as an electron acceptor with a color change in GOD-catalyzed oxidation of glucose under certain conditions.12) This finding prompted us to examine how derivatives of 2 such as 1 would behave in GOD-catalyzed oxidation of glucose, and an interesting behavior of 1 in the enzymatic reaction was found. Here, we report that in GOD-catalyzed oxidation of glucose, 1 is transformed to 2 similarly to the case of diaphorase-or PMS ϩ -catalyzed oxidation of NAD(P)H (Chart 1), and is superior to 2 as an electron acceptor, although the observed behavior of 1 will not find direct application to enzymatic analysis of glucose unless the rate for 1 to reoxidize the reduced form of GOD can be improved. Results and DiscussionA solution of 1 in phosphate buffer (pH 7.4, 0.1 M) had a purple color (l max , 602 nm). When a mixture of 1 (20.0 nmol), glucose (5.0 mmol), and GOD (1.0 mg) in 3.0 ml phosphate buffer (pH 7.4, 0.1 M) was incubated at 25°C under anaerobic conditions, the mixture turned purple to fluorescent pink. In the absorption spectrum of the mixture, a new peak with l max at 571 nm appeared as soon as the incubation was started, as shown in Fig. 1. As incubation time was increased, the new peak became gradually larger, while the peak due to 1 became smaller and had almost disappeared after 15 min incubation (Fig. 1c). The new peak at 571 nm coincided with that of the deoxygenated product 2 in phosphate buffer. The color of 1 was not affected by treatment with GOD alone or with H 2 O 2 under these conditions. These results suggested that the color change observed in the incubation of 1, glu...

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