Hydrogen peroxide-induced deacetylation of acetyl resorufin as a novel indicator reaction for fluorometric detection of glucose using only glucose oxidase.
Hydrogen peroxide (H2O2)-induced deacetylation of non-fluorescent acetyl resorufin (1) to fluorescent resorufin (2) as a novel indicator reaction for fluorometric detection of glucose using only glucose oxidase (GOD) is described. When a 1:1:1 mixture of 1 (in CH3CN), glucose, and GOD (each in pH 7.4 phosphate buffer) was incubated at 25 degrees C under aerobic conditions, the resulting solution turned yellow to fluorescent pink due to 2. The formation of 2 was markedly retarded on incubation under anaerobic conditions. When a mixture of 1 and H2O2 was incubated under aerobic conditions, the formation of 2 was noted as in the case of the enzymatic reaction of 1. These results demonstrated that the observed color change is brought about through deacetylation of 1 to 2 induced by H2O2 generated in GOD-catalyzed oxidation of glucose. With regard to the fluorometric traces of the enzymatic reaction with 1 (0.2 mM), GOD (0.5 mg/ml), and glucose at 25 degrees C, fluorescence intensity exhibited a linear relationship against glucose concentration between 0.2 and 2.0 mm, with a correlation coefficient of 0.997. Neither ascorbic acid, uric acid, nor bilirubin significantly interfered with the transformation of 1 to 2 through GOD-catalyzed oxidation of glucose.
Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Japan. h-maeda@phs.osaka-u.ac.jp
H
H
Maeda
S
S
Matsu-ura
M
M
Nishida
T
T
Senba
Y
Y
Yamauchi
H
H
Ohmori
D001205:Ascorbic Acid; D001663:Bilirubin; D002384:Catalysis; D003640:Dealkylation; D005947:Glucose / Q000032:analysis*; D005949:Glucose Oxidase / Q000737:chemistry*; D006861:Hydrogen Peroxide / Q000737:chemistry*; D007202:Indicators and Reagents; D010078:Oxazines / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D014527:Uric Acid
D007202:Indicators and Reagents; D010078:Oxazines; D014527:Uric Acid; C014180:resorufin; D006861:Hydrogen Peroxide; D005949:Glucose Oxidase; D005947:Glucose; D001205:Ascorbic Acid; D001663:Bilirubin