In stronger acid solution, the color becomes pink, with maximum absorbance at 540 nm, and both sensitivity and stability are improved. In some methods, the final mixture is acidified slightly to stop the reaction, and the intensity of the yellow chromophore is measured at 400 nm. Calibrators and unknowns should be analyzed simultaneously under conditions in which the rate of oxidation is proportional to the glucose concentration. Some glucose oxidase preparations contain catalase as a contaminant catalase activity decomposes peroxide and decreases the final color obtained. Most modern methods omit the preparation of protein-free filtrates to make the procedure faster and simpler. Acid filtrates cannot be used because peroxides, which cause falsely increased results, may be released. Most interfering substances can be eliminated by the use of a Somogyi filtrate. Incorporation of potassium ferrocyanide significantly decreases interference by bilirubin. Various substances, such as uric acid, ascorbic acid, bilirubin, hemoglobin, tetracycline, and glutathione, inhibit the reaction (presumably by competing with the chromogen for H 2O 2), producing lower values. The second step, which involves peroxidase, is much less specific than the glucose oxidase reaction. Otherwise, extended incubation time allows spontaneous conversion. Some commercial preparations of glucose oxidase contain an enzyme-mutarotase-that accelerates this reaction. Complete reaction of glucose therefore requires mutarotation of the α- to the β-form. As noted earlier, 36% and 64% of glucose in solution are in α- and β-forms, respectively. Glucose oxidase is highly specific for β-D-glucose. Addition of the enzyme peroxidase and a chromogenic oxygen acceptor, such as o-dianisidine, results in the formation of a colored compound that is measured:
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