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Resorufin (1) has been found to act as an electron acceptor in glucose oxidase (GOD)-catalyzed oxidation of glucose. When a 1: 1: 1 mixture of solutions of 1 (5.0 microM), glucose, and GOD (4.0 mg/ml) in phosphate buffer (pH 7.4, 0.1 M) was incubated at 36 degrees C under aerobic conditions and the reaction was followed by a measurement of changes in fluorescence intensity due to 1, only two types of fluorometric traces were observed: (1) when a glucose solution of less than 0.7 mM was subjected to the enzymatic reaction, no consumption of 1 was observed; (2) the reaction with glucose at more than 1.0 mM always consumed 1, affording a regression fluorometric curve, and yet the obtained fluorometric traces could be almost superimposed on one another with no dependence on the glucose concentration. The reasons for the observed phenomena are discussed.

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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Spectrophotometric or fluorometric methods with glucose oxidase (GOD)-peroxidase (POD)-chromogen(s) systems are well known as useful tools for determination of glucose. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] Indicator reactions in these methods are based on oxidative formation of a colored or fluorescent substance from a chromogen(s) with H 2 O 2 , generated through GODcatalyzed oxidation of glucose, in the presence of POD. These color reactions are well-characterized chemical processes. However, the POD-dependent indicator reactions are known to be inevitably disturbed by electron donors present in biological samples such as ascorbic acid, uric acid, and bilirubin.3) Major processes of interference by these compounds are as follows: 1) reduction of oxidatively formed colored or fluorescent substance to its original chromogen(s); and 2) competition with a chromogen in reduction of H 2 O 2 . Several methodologies have been developed for elimination of such interference in glucose determination with GOD-POD-chromogen(s) systems.3) However, the most straightforward approach to glucose determination free from such interference is design of a novel indicator reaction without recourse to redox reactions coupled with H 2 O 2 and POD. To our knowledge, only a few methods with non-redox color reactions for glucose determination using only GOD have been reported. These methods utilize transformation of a vanadium(V), 16) titanium(IV) 17) or dinuclear iron(III) 18) complex to its adduct with H 2 O 2 accompanied by a bathochromic shift as a spectrophotometric indicator reaction. As expected, it was demonstrated that glucose determination with vanadium(V) and titanium(IV) complexes was not affected by various substances usually present in serum or added to test solution. 16,17) However, spectrophotometric or fluorometric determination of glucose with high accuracy should use formation of a colored or fluorescent substance from a chromogen rather than a color change of a dye as an indicator reaction.Recently, resorufin (2) was shown to be able to reoxidize a reduced form of GOD at 37°C, being transformed to a colorless dihydro derivative, although the reductive bleaching of the dye is of no use for GOD-based determination of glucose as an indicator reaction. 19) This finding prompted us to examine how resorufin derivatives such as acetyl resorufin (1) would behave in GOD-catalyzed oxidation of glucose, and it was found that 1 behaves in a manner different from 2 in the enzymatic reaction. Here, we report that deacetylation of non-fluorescent 1 to fluorescent 2 is induced by H 2 O 2 generated in GOD-catalyzed oxidation of glucose, and the transformation is promising as an indicator reaction for fluorometric determination of glucose without significant effects of ascorbic acid, uric acid, or bilirubin. ExperimentalMaterials GOD from Aspergillus niger (EC 1.1.3.4) and glucose were used as supplied from Wako Pure Chemical Industries, Ltd. Acetyl resorufin (1) [20][21][22][23] was prepared by reaction of resorufin sod...